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Structure-Guided Design of Cell Wall Biosynthesis Inhibitors That Overcome ??-Lactam Resistance in Staphylococcus aureus (MRSA)
Abstract
β-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering β-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low β-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing β-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens.
Supplementary resource (1)
... Boronic acids have been investigated as PBP inhibitors by several research groups over the past decades 18,[31][32][33][34][35][36][37] , and there have been a few recent publications on the subject 38,39 . In general, boronic acids can covalently attack serine residues. ...
... Compound 1 is a boronic acid where the boron atom connects to an aromatic group. The PBP1b inhibitory potency of these types of compounds was investigated in Contreras-Martel et al. 31 Compound 2 is an aliphatic boronic acid and we decided to investigate how the structural variation of aliphatic boronic acids affects PBP1b inhibitory activity. Compounds 3-6 contain variations in the amide bonding moiety, and they did not show improved activity. ...
... We note that the highly similar compound 9 exhibits an order of magnitude lower IC 50 . This latter compound is part of a linear boronic acid series studied in Contreras-Martel et al. 31 The synthesis of the boroproline derivatives is shown in Scheme 1. To obtain 6, 1-methyl-1H-1,2,3-triazole-4-carboxylic acid (10) and pyrrolidine-3-boronic acid pinacol ester hydrochloride (11) were coupled in the presence of HATU and DIPEA. ...
Penicillin-binding proteins (PBPs) contribute to bacterial cell wall biosynthesis and are targets of antibacterial agents. Here, we investigated PBP1b inhibition by boronic acid derivatives. Chemical starting points were identified by structure-based virtual screening and aliphatic boronic acids were selected for further investigations. Structure–activity relationship studies focusing on the branching of the boron-connecting carbon and quantum mechanical/molecular mechanical simulations showed that reaction barrier free energies are compatible with fast reversible covalent binding and small or missing reaction free energies limit the inhibitory activity of the investigated boronic acid derivatives. Therefore, covalent labelling of the lysine residue of the catalytic dyad was also investigated. Compounds with a carbonyl warhead and an appropriately positioned boronic acid moiety were shown to inhibit and covalently label PBP1b. Reversible covalent labelling of the catalytic lysine by imine formation and the stabilisation of the imine by dative N–B bond is a new strategy for PBP1b inhibition.
... Pocket 2 is close to this loop region and harbors residues that stabilize aromatic rings in different inhibitors. 25 Despite this structural and functional homology, the expression, spatiotemporal localization, and mechanisms of the individual PBPs are not fully understood. While the different functions of several PBPs have long been studied using methods such as deletion strains or fusion constructs (e.g., fluorescent or affinity tags), those strategies provide an incomplete picture, as they cannot specifically report on PBP catalytic activities. ...
... We have reported that β-lactones, based on a 3-amino-4-methyl-2oxetanone scaffold, can specifically target the PBPs in a full proteome. 25,31 This result was not anticipated, as this scaffold lacks a negatively charged moiety to mimic the carboxy terminus of the native substrate (within 3−3.6 Å of carbonyl carbon) that is thought to be essential for active-site recognition and is found in all clinically approved PBP inhibitors (highlighted in blue, Figure 2a). 32 This striking result, together with the fact that alteration of a single stereocenter in several of the β-lactone analogues drastically changes their PBP-isoform selectivity, prompted us to further explore the binding mode(s) of this novel PBP inhibitor class. ...
... The binding modes of 5Az, 6Az, 7Az, and 8Az within the PBP1b* active site are broadly similar to that in which previously published boronic acid inhibitors (Figure 1) bind to pocket I of PBP1b*, where the hydrophobic regions are located proximally to the β3/β4 loop and the highly flexible azide moieties point to the exterior of the active site. 25 Notably, the phenyl group of 8Az is also stabilized by a groove formed by the side chains of Met556, Ile519, and Phe490 (Figure 3d). Molecular Dynamics Simulations of PBP1b* X-ray Structures. ...
β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four β-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the β-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.
... By contrast with the work on SBLs and MBLs, there are relatively limited reports on the interactions of boronbased inhibitors with PBPs. 22−33 Although there are reports of boronic acids reacting with PBPs, 23,24,26−31,34−36 and with antibacterial activity, 25 there are no reports of potent bicyclic boron-based PBP inhibitors/antibacterials in the peer-reviewed literature. The development of dual-action PBP/BL boronbased inhibitors is also of interest. ...
... While the inhibition is weak, these results are consistent with other investigations of boronate binding in HMM PBPs, which typically show IC 50 s in the μM range. [23][24][25]32,33 Selected compounds were screened against E. coli, P. aeruginosa, H. influenzae, A. baumannii, and N. gonorrhoeae and a P. aeruginosa strain engineered to remove the outer membrane permeability barrier to investigate their antimicrobial activity (Table S2). 60 All were ineffective (MICs ≥64 μg/ mL). ...
... The observed binding mode depends on the nature of the boron compound, with vaborbactam reacting monocovalently, benzoxaboroles (3−15) reacting dicovalently, and phenyl boronates (1 and 2) reacting tricovalently. Tricovalent 27 and monocovalent 22,[25][26][27]29,30 bonding of boron compounds with PBPs are known, but to our knowledge, this is the first time that benzoxaborole compounds have been shown to bind to a PBP in a dicovalent manner. Although we cannot rule out the possibility that some of our structures are a consequence of crystallization, analogous data were collected at pH 6 and pH 8; the observed covalent binding mode thus does not appear to correlate with the crystallization conditions but is probably related to the form of the warhead (Table S3). ...
The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.
... The major involves Zn(II) complexation via one of the exocyclic boronate oxygens (binding mode A, shown); in the minor (not shown) the inhibitor complexes via its endocyclic boronate oxygen and one of its exocyclic boronate oxygens [68]; (e) AmpC (E. coli) with an acyclic boronic acid [69]; (f) class A SBL CTX-M-15 with Vaborbactam [36]; (g) penicillin-binding protein 1b (PBP-1B) with an acyclic boronic acid [48]; (h) DD-transpeptidase (Actinomadura sp. R39) with an acyclic boronic acid showcasing an unusual tricovalent binding mode of the boronate [70]; (i) class D SBL OXA-10 with a benzoxaborole analogue [71]. ...
... Acyclic boronic acids have also been developed as transpeptidase inhibitors, as exemplified in work on methicillin-resistant Staphylococcus aureus (MRSA) acting compounds [48]. Multiple structures are reported for alkyl boronic acids bound to PBP-1B [48]. ...
... Acyclic boronic acids have also been developed as transpeptidase inhibitors, as exemplified in work on methicillin-resistant Staphylococcus aureus (MRSA) acting compounds [48]. Multiple structures are reported for alkyl boronic acids bound to PBP-1B [48]. Subsequent work has defined boronic acid inhibitors that may more directly mimic the deacylation tetrahedral intermediate in class C SBLs [49 ]. ...
The β-lactams remain the most important antibacterials, but their use is increasingly compromised by resistance, importantly by β-lactamases. Although β-lactam and non-β-lactam inhibitors forming stable acyl-enzyme complexes with nucleophilic serine β-lactamases (SBLs) are widely used, these are increasingly susceptible to evolved SBLs and do not inhibit metallo-β-lactamases (MBLs). Boronic acids and boronate esters, especially cyclic ones, can potently inhibit both SBLs and MBLs. Vaborbactam, a monocyclic boronate, is approved for clinical use, but its β-lactamase coverage is limited. Bicyclic boronates rapidly react with SBLs and MBLs forming stable enzyme-inhibitor complexes that mimic the common anionic high-energy tetrahedral intermediates in SBL/MBL catalysis, as revealed by crystallography. The ability of boronic acids to 'morph' between sp2 and sp3 hybridisation states may help enable potent inhibition. There is limited structure-activity relationship information on the (bi)cyclic boronate inhibitors compared to β-lactams, hence scope for creativity towards new boron-based β-lactamase inhibitors/antibacterials.
... The development of S. aureusresistant strain is attributed to the production of an additional PBP responsible for cross-linking peptidoglycan, known as PBP2a. PBP2a is encoded by the gene MecA, and when overexpressed, it results in low binding affinity of PBPs, leading to resistance against these drugs (Contreras-Martel et al., 2011;Yocum et al., 1979;Zapun et al., 2008). The survival and growth of bacterial cells depend on the stability of peptidoglycan, an essential component of bacterial cell walls. ...
... The survival and growth of bacterial cells depend on the stability of peptidoglycan, an essential component of bacterial cell walls. Disrupting the biosynthesis of these proteins can lead to bacterial lysis and death (Contreras-Martel et al., 2011). ...
Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging nosocomial pathogen among hospitalized patients, with high morbidity and mortality rates. The discovery of a novel antibacterial is urgently needed to address this resistance problem. The present study aims to explore the antibacterial potential of three depsidone compounds: 2-clorounguinol (1), unguinol (2), and nidulin (3), isolated from the marine sponge-derived fungus Aspergillus unguis IB1, both in vitro and in silico. The antibacterial activity of all compounds was evaluated by calculating the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) against MRSA using agar diffusion and total plate count methods, respectively. Bacterial cell morphology changes were studied for the first time using scanning electron microscopy (SEM). Molecular docking, pharmacokinetics analysis, and molecular dynamics simulation were performed to determine possible protein-ligand interactions and the stability of the targeting penicillin-binding protein 2a (PBP2a) against 2-clorounguinol (1). The research findings indicated that compounds 1 to 3 exhibited MIC and MBC values of 2 µg/mL and 16 µg/mL against MRSA, respectively. MRSA cells displayed a distinct shape after the addition of the depsidone compound, as observed in SEM. According to the in silico study, 2-chlorounguinol exhibited the highest binding-free energy (BFE) with PBP2a (-6.7 kcal/mol). For comparison, (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl) benzoic acid inhibits PBP2a with a BFE less than -6.6 kcal/mol. Based on the Lipinski's rule of 5, depsidone compounds constitute a class of compounds with good pharmacokinetic properties, being easily absorbed and permeable. These findings suggest that 2-chlorounguinol possesses potential antibacterial activity and could be developed as an antibiotic adjuvant to reduce antimicrobial resistance.
... Casi todos los antibióticos β-lactámicos inhiben a 15 estas enzimas (Kaman et al., 2014). Sin embargo, la aparición de la resistencia bacteriana en S. aureus, tras su exposición a los β-lactámicos, ha impulsado el desarrollo de nuevos inhibidores (Contreras- Martel et al., 2011;Turk et al., 2011). ...
... La búsqueda de compuestos líderes para el desarrollo de candidatos a medicamentos contra enfermedades infecciosas humanas, causadas por microorganismos, se ha convertido en una de las prioridades para las investigaciones biomédicas en los últimos años a nivel mundial, debido al alarmante incremento de la resistencia microbiana a los antibióticos convencionales (Wilke, 2010;Silver et al., 2011;Scholz et al., 2012;Pathak et al., 2012;Dusano, 2016;Paterson, 2016). Entre estos esfuerzos, ocupan un lugar destacado los proyectos encaminados a identificar inhibidores de proteasas con potencialidades como agentes antimicrobianos (Cathcart et al., 2011;Contreras-Martel et al., 2011;Newman et al., 2011;Tuk et al., 2011;Singh et al., 2012;Zindel et al., 2013), en virtud de las cruciales funciones fisiológicas que desempeñan estas enzimas para las bacterias y los hongos, y su importancia para la patogénesis (Madigan et al., 2014;Kaman et al., 2014). Las APs de tipo metalo neutras de varios microorganismos patógenos se encuentran entre las nuevas dianas moleculares reconocidas en la última década, con posibilidades para constituir el punto de partida de novedosas terapias efectivas (Florent et al., 1998;Cadavid-Restrepo et al., 2011;McGowan et al., 2017). ...
La aminopeptidasa de tipo metalo neutra de la familia M1 (APN), presente en la bacteria Escherichia coli (ePepN), es una de las nuevas dianas identificadas para el desarrollo de antimicrobianos, con el objetivo de combatir el incremento de la resistencia de los microorganismos a diferentes antibióticos. Su alto grado de conservación en los organismos procariontes, las cruciales funciones biológicas que desempeña y la facilidad de obtenerla por vía recombinante, determinan que esta enzima se considere un buen modelo de otras APNs de bacterias patógenas, para la identificación de inhibidores. Un inhibidor bien conocido de las APNs es el compuesto no selectivo bestatina, que combina un grupo de unión al Zn2+ en el centro activo con sustituyentes que reconocen los subsitios S1 y S1’. A partir de estos antecedentes, el objetivo del presente trabajo fue la identificación de inhibidores potentes y selectivos de la ePepN recombinante (ePepNr) en dos series de compuestos sintéticos. Las características cinéticas determinadas para esta enzima (actividad a pH 7,0 y 8,0 y valor de KM) indican que la misma es un buen modelo de su contraparte natural para la identificación de inhibidores. Por otra parte, la inhibición no competitiva o mixta observada para la bestatina sugiere que es posible lograr la inhibición potente de la ePepN de esta manera. Mediante estudios de dosis-efecto, se identificaron los tetrazoles sintéticos YTE003, YTE007 y YTE008 como inhibidores potentes y selectivos de la ePepN, respecto a su ortóloga porcina (APNp). Además, se obtuvieron datos valiosos sobre los determinantes estructurales de la inhibición de la ePepN por la biblioteca de tetrazoles y la serie de peptidomiméticos derivados de la bestatina. Al contrario de lo esperado, el tipo de inhibición de la enzima bacteriana por los representantes de ambas series de compuestos, no es competitivo. Este conocimiento puede facilitar el estudio del modo de unión de los inhibidores a la enzima in silico, guiar la futura optimización de estas estructuras y contribuir al diseño de nuevos inhibidores de la ePepN. De modo interesante, el tetrazol YTE003 presenta actividad antibacteriana in vitro, frente a la bacteria E. coli, lo que refuerza sus potencialidades como compuesto líder para el desarrollo de agentes antibacterianos.
... At the N-terminus there is a transglycosylase domain involved in the formation of linear glycan strands. And at the C-terminus there is a transpeptidase domain involved in the cross-linking of peptide subunits and drug binding, which is also responsible of the penicillin-sensitivity (Macheboeuf et al., 2005;Sauvage et al., 2008;Contreras-Martel et al., 2011). ...
... On the other hand, many common pathogenic bacteria are acquiring antibiotic resistance in all regions of the world (e.g., urinary tract infections, pneumonia, bloodstream infections; WorldHealthOrganisation, WHO). These bacteria cause many hospital-acquired infections, such as the methicillin-resistant S. aureus, with an associated high mortality rate (Contreras-Martel et al., 2011;WorldHealthOrganisation, WHO). ...
Prion proteins were initially associated with diseases such as Creutzfeldt Jakob and transmissible spongiform encephalopathies. However, deeper research revealed them as versatile tools, exploited by the cells to execute fascinating functions, acting as epigenetic elements or building membrane free compartments in eukaryotes. One of the most intriguing properties of prion proteins is their ability to propagate a conformational assembly, even across species. In this context, it has been observed that bacterial amyloids can trigger the formation of protein aggregates by interacting with host proteins. As our life is closely linked to bacteria, either through a parasitic or symbiotic relationship, prion-like proteins produced by bacterial cells might play a role in this association. Bioinformatics is helping us to understand the factors that determine conformational conversion and infectivity in prion-like proteins. We have used PrionScan to detect prion domains in 839 different bacteria proteomes, detecting 2200 putative prions in these organisms. We studied this set of proteins in order to try to understand their functional role and structural properties. Our results suggest that these bacterial polypeptides are associated to peripheral rearrangement, macromolecular assembly, cell adaptability, and invasion. Overall, these data could reveal new threats and therapeutic targets associated to infectious diseases.
... Therefore, to predict the possible mechanism by which the chalcone derivatives can induce antibacterial activity, molecular docking of the potent antibacterial compound IIIf was performed on the binding model based on PBP-1b of S. aureus (2Y2H.pdb) and the docking result of IIIf was compared with the interaction of N-alkyl boronic acid analogues, the PBP-1b inhibitors of S. aureus (Contreras-Martel et al., 2011). The docking results show (Fig. S6, Supplementary data) that compound IIIf binds to the same active pocket of the PBP-1b receptor as that of endogenous boronate ligands. ...
... pocket 1 surrounded by ALA459, SER457, ASN518 and MET556 amino acid residues and pocket 2 surrounded by ALA499, VAL628, ASP658, and GLN686 amino acid residues. However, only the alkyl boronic acid analogues binding to pocket 2 show antibacterial activity (Contreras-Martel et al., 2011). Fig. 4 shows that compound IIIf significantly interacts with ALA499, VAL628, and GLN686 including other amino acid residues in pocket 2 of PBP-1b receptor. ...
In the present study, a series of chalcone derivatives including 17 new compounds were synthesised; their antibacterial activities against eleven bacteria, and their free radical-scavenging activities using DPPH were evaluated. All compounds showed significant antibacterial activities against both Gram-positive and Gram-negative bacteria. In particular, compound IIIf strongly inhibited Staphylococcus aureus (JMC 2151) and Enterococcus faecalis (CARS 2011-012) with MIC values of 6.25 µg mL −1 and 12.5 µg mL −1 , respectively, which are comparable to that of the standard antibiotic, nalidixic acid. Compound IIIg also inhibited S. aureus with a MIC value similar to that of nalidixic acid (6.25 µg mL −1). Furthermore, like nalidixic acid (MIC value of 25 µg mL −1), compounds IIIa, IIIc and IIId inhibited Listeria monocytogenes (ATCC 43256) with MIC values of 25 µg mL −1 , 12.5 µg mL −1 and 25 µg mL −1 , respectively. Quantitative structure–activity relationship (Q-SAR) studies using physicochemical calculations indicated that the antibacterial activities of chalcone derivatives correlated well with predicted physicochemical parameters (logP and PSA). Docking simulation by positioning the most active compound IIIf in the active site of the penicillin-binding protein (PBP-1b) of S. aureus was performed to explore the feasible binding mode. Furthermore, most of the compounds synthesised exhibited significant DPPH radical-scavenging activity, although compounds IIc and IIIc exhibited the greatest antioxidant activity with IC50 values of 1.68 µM and 1.44 µM, respectively, comparable to that of the standard antioxidant, ascorbic acid (1.03 µM).
... For this reason, proteases involved in PGN synthesis are potential targets for antimicrobial therapy. Previously, studies towards the development of antimicrobial therapies targeting PGN synthesis have predominantly focused on inhibition of the PBP cross-linking proteases [49][50][51]. These D-alanine-D-alanine carboxy peptidases are essential for bacterial growth and are present in all PGNcontaining bacteria. ...
... The effectiveness of PBP inhibitors in the treatment of bacterial infections has already been proven, as beta-lactam antibiotics are widely used to treat a broad spectrum of bacterial infections. However, novel PBP inhibitors are currently being developed in response to the rapid emergence of extendedspectrum beta-lactamase (ESBL) resistance [49,52]. ...
Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.
... Inhibition of the synthesis of PBP2a leads to cell wall lysis and the bacteria's death. PBPs have a catalytic site on the outside portion of the bacterial cell wall, which allows to access the ligand without crossing the lipid bilayer 76 .It provides a rigid mechanism for stability due to its highly cross-linked lattice structure. Two sugar units generate the lattice structure, one is N-acetyl glucosamine, and the another one is N-acetyl muramic acid. ...
Staphylococcus aureus is a common pathogen in human. Methicillin resistant Staphylococcus aureus (MRSA) infection poses a big and perplexing difficulty in terms of therapy. The acquisition of the non-native gene PBP2a, which has a decreased tolerance for β-lactam antibiotics, frequently confers resistance. PBP2a has a less attraction for methicillin and it helps bacteria to continue peptidoglycan biosynthesis, and it is cell wall’s core component in bacteria. So, even in the presence of methicillin or any other antibiotic, bacteria develop resistance. Due to resistance-causing genes, S. aureus becomes MRSA. The main premise of the resistance mechanism is well understood. The current demand for novel antibiotics is legitimate in the face of therapeutic concerns posed by resistant micro-organisms. The emphasis of this review is on PBP2a scaffolds and the different screening approaches used to find PBP2a inhibitors. Penicillin, Cephalosporins, Pyrazole-Benzimidazole based derivatives, Oxadiazole containing derivatives, non-β-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycle (β-lactam antibiotics with 1, 3-Bridges), Macrocycle-embedded β-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-β-lactam antibiotics Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a are also represented as well as with their biological activity is discussed. The penicillin-binding protein is also discussed, which is the crucial target for the cell wall of MRSA. Various aspects of PBP2a, the cell wall of bacteria, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives of MRSA inhibitors are also enumerated.
... of penicillin-binding protein 2 by Streptococcus pneumoniae is a typical example of this mechanism. Resistance was developed among Streptococcus species by horizontal transfer of the pbp2b and pbp2x genes.23,32 As for the Gram-negative pathogens, the main causes of drug resistance are 1) ...
Antimicrobial resistance (AMR) emerged rapidly after the introduction of the penicillins, the first generation of β-lactam antibiotics, in 1946. Resistance to antibiotics of last resort has highlighted AMR in bacterial pathogens as a pressing therapeutic issue. Gram-negative bacteria manifest high-level resistance to most classes of antibiotics and are the leading cause of severe infectious disease globally. Therefore, reversing their resistant status is of our interest. Among many mechanisms discovered, the expression of drug-inactivating enzymes is the major cause that leads to Gram-negative bacterial AMR. We aim to probe the chemical biology of two proteins associated with drug resistance: Klebsiella pneumoniae carbapenemase (KPC-2) which hydrolyses β-lactam antibiotics and a bacterial glutathione transferase, glutathione transferase (GST-A) which plays roles in antibiotic conjugation and inactivation. Based on the known crystal structures of the proteins (KPC-2: PDB id 3RXX, GST-A: PDB id 1A0F), small molecules were designed and synthesised and tested as inhibitors of the purified enzymes. Promising inhibitors for KPC-2 have been developed with a scaffold containing a 1,4-disubstituted 1,2,3-triazole. In vitro tests indicated that the compounds have a clear SAR and the best inhibitors have nanomolar Ki values. Antibiotic susceptibility tests were used to validate boronic acid KPC-2 inhibitors as potentiators of β-lactam antibiotic activity in cellulo. The compounds showed the successful reversal of resistance to cefotaxime (CTX) and meropenem (MEM) in cellulo in KPC-2 producing Escherichia coli (over 512-fold more sensitive). A small library of glutathione (GSH) analogues was synthesised and tested against GST-A. Binding assays and enzyme kinetics studies suggested that the Gly moiety of GSH is less important than Glu in protein G-site binding, and π-stacking is a critical factor in GST-A H-site binding. We also used susceptibility tests to explore whether GST-A plays a role in antibiotic detoxification and may serve as a target to combat AMR. However, target validation work suggested that GST-A is not essential for E. coli survival and inhibiting the protein may not be a promising approach for drug discovery.
... For example, point mutations in the Penicillin-Binding Proteins confer resistance to blactam antibiotics by making the active site amenable to hydrolysis, or reducing binding affinity for the antibiotic [73]. Structure guided design demonstrated the potential of boronate-based PBP inhibitors to overcome b-lactam resistance in Gram positive organisms [74]. Similarly, missense mutations in the Mtb gidB gene (target for the antibiotic streptomycin) are responsible for drug resistance through distortion of the binding pocket affecting SAM (co-factor) binding [71]. ...
Antimicrobials against bacterial, viral and parasitic pathogens have transformed human and animal health. Nevertheless, their widespread use (and misuse) has led to the emergence of antimicrobial resistance (AMR) which poses a potentially catastrophic threat to public health and animal husbandry. There are several routes, both intrinsic and acquired, by which AMR can develop. One major route is through non-synonymous single nucleotide polymorphisms (nsSNPs) in coding regions. Large scale genomic studies using high-throughput sequencing data have provided powerful new ways to rapidly detect and respond to such genetic mutations linked to AMR. However, these studies are limited in their mechanistic insight. Computational tools can rapidly and inexpensively evaluate the effect of mutations on protein function and evolution. Subsequent insights can then inform experimental studies, and direct existing or new computational methods. Here we review a range of sequence and structure-based computational tools, focussing on tools successfully used to investigate mutational effect on drug targets in clinically important pathogens, particularly Mycobacterium tuberculosis. Combining genomic results with the biophysical effects of mutations can help reveal the molecular basis and consequences of resistance development. Furthermore, we summarise how the application of such a mechanistic understanding of drug resistance can be applied to limit the impact of AMR.
... Contreras-Martel and coworkers used a crystallography-guided approach to identify boronic acid analogs that bound reversibly to the active site of the PBP and showed antibacterial activity. One of the analogs was shown to specifically bind to the low affinity PBP2a, isolated from the membranes of S. aureus, and to inhibit growth of MRSA (Contreras-Martel et al., 2011). Notably, D-Ala-D-Ala ligases, which participate at an early, cytoplasmic step of peptidoglycan biosynthesis, have also been shown to be the targets of d-boro-Ala analogs that show some specificity towards MRSA (Putty et al., 2011). ...
The widespread resistance to antibiotics developed by bacterial pathogens calls for the characterization of original, yet unexplored potential targets in bacteria. Alpha2-macroglobulins (α2Ms) are broad-spectrum protease inhibitors that play key roles in eukaryotic immunity. They are multi-domain molecules that carry approximately 1,800 residues and harbor a central amino acid sequence, the ‘bait site’, which is recognized and cleaved by a large number of proteases. Upon cleavage, the resulting conformational change exposes a buried thioester bond between a cysteine and a glutamine, which is readily hydrolyzed, allowing the resulting glutamate to associate covalently to the target protease, trapping it within the α2M cage-like structure. Recently, α2M homologs from pathogenic and colonizing bacteria have also started to be characterized. These findings suggest that bacteria possess a rudimentary immune system that mimics initial key steps of the eukaryotic immune pathway and that could represent a yet unexplored target in pathogen biology.The genes for two types of α2M are present in bacterial genomes: type 1, which contains the thioester bond, and type 2 that does not harbor it. Type 1 bacterial A2Ms persistently co-occur within the same operon with a gene that encodes a cell wall biosynthesis enzyme, Penicillin-Binding Protein 1c (PBP1c). This suggests that the association between the two proteins could be highly advantageous for the cell during infection/colonization, when the outer cell wall is targeted by host defenses. In this situation, A2M and PBP1c could exert the role of ‘guardians of the periplasm’, with PBP1c repairing damaged peptidoglycan, and A2M trapping invading proteases.The aim of this work was to demonstrate the existence of such complex and characterize this interaction structurally and functionally. For this purpose, α2M and PBP1c from E. coli were studied. The proteins were expressed and purified separately. α2M (also called ECAM in E. coli) is highly soluble, monomeric protein with a mass of 182 kDa, monodisperse and stable during the course of time. PBP1c is a membrane-bound, 87 kDa protein, predominantly present as a dimer. The complex reconstitution in vitro by mixing and incubating the proteins for 2 hours resulted in formation of a complex, demonstrated by appearance of a new peak in size exclusion chromatography. This result was further confirmed by SDS-PAGE, analytical centrifugation and small-angle x-ray scattering (SAXS) experiments. ECAM and PBP1c associated in 2:2 and 4:4 stoichiometries. The activity test confirmed that PBP1c performs polymerization of glycan chains and that its activity is enhanced in the presence of ECAM.Crystallization trials yielded crystals of PBP1c in several conditions. The study of ECAM by electron microscopy proved that this technique could be used for structural studies of the complex. Both approaches are under optimization, and combined, they could be employed for structural characterization of the ECAM-PBP1c complex.
... The bacteria consist of various enzymes/proteins like pencillin binding protein, topoisomerase, cell adhesion protein, beta-lactamase, arylsulfatase and UDP-3-O- [3-hydroxymyristoyl] N-acetylglucosamine deacetylase for normal biological functions. The X-ray crystal structures of pencillin binding protein 1B (2Y2M, 1.62 Å) [13], topoisomerase (3TTZ, 1.63 Å) [14], cell adhesion protein (4QRK (1.95 Å) [15], beta-lactamase (5CTN, (1.35 Å) [16] and Gram negative bacterial proteins like arylsulfatase (1HDH, [18]. The extracted structures have been prepared by the removal of cofactors and water molecules and assigning of bonds, bond orders and hybridization, hydrogen atoms, and charges [19]. ...
Aim
The aim of the study was to find out the role of auranofin as a promising broad spectrum antibacterial agent.
Methods
In-vitro assays (Percentage growth retardation, Bacterial growth kinetics, Biofilm formation assay) and In-silico study (Molegro virtual docker (MVD) version 6.0 and Molecular operating environment (MOE) version 2008.10 software).
Results
The in vitro assays have shown that auranofin has good antibacterial activity against Gram positive and Gram negative bacterial strains. Further, auranofin has shown synergistic activity in combination with ampicillin against S. aureus and B. subtilis whereas in combination with neomycin has just shown additive effect against E. coli, P. aeruginosa and B. pumilus. In vivo results have revealed that auranofin alone and in combination with standard drugs significantly decreased the bioburden in zebrafish infection model as compared to control. The molecular docking study have shown good interaction of auranofin with penicillin binding protein (2Y2M), topoisomerase (3TTZ), UDP-3-O-[3- hydroxymyristoyl] N-acetylglucosaminedeacetylase (3UHM), cell adhesion protein (4QRK), β-lactamase (5CTN) and arylsulphatase (1HDH) enzyme as that of reference ligand which indicate multimodal mechanism of action of auranofin. Finally, MTT assay has shown non-cytotoxic effect of auranofin.
Conclusion
In conclusion, auranofin in combination with existing antibiotics could be developed as a broad spectrum antibacterial agent; however, further studies are required to confirm its safety and efficacy. This study provides possibility of use of auranofin apart from its established therapeutic indication in combination with existing antibiotics to tackle the problem of resistance.
... The transpeptidase (TP) domain is composed of a central, five-stranded b-sheet (b1-b5) surrounded by a-helices (green and brown in Fig. 8.3), and has been the target of many studies viewing the development of novel b-lactam-related or non-b-lactam inhibitors (Macheboeuf et al. 2005(Macheboeuf et al. , 2007Fedarovich et al. 2012;Contreras-Martel et al. 2011;Zervosen et al. 2011;van der Akker and Bonomo 2018;King et al. 2017). The fold of this domain is conserved within most enzymes that bind b-lactam antibiotics, including most b-lactamases (Nikolaidis et al. 2014). ...
... The search for leading compounds for the development of drug candidates against human infectious diseases, caused by microorganisms, has become one of the worldwide priorities for biomedical research in the last years, due to the alarming increase of the microbial resistance to conventional antibiotics (Wilke, 2010;Silver, 2011;Pathak et al., 2012;Scholz et al., 2012;Dusano, 2016;Paterson, 2016). Among these efforts, the projects directed to identify protease inhibitors with potentialities as antimicrobial agents have a relevant place (Cathcart et al., 2011;Contreras-Martel et al., 2011;Newman et al., 2011;Turk et al., 2011;Singh et al., 2012;Zindel et al., 2013), since the crucial physiological functions of these enzymes for bacteria and fungi, and their relevance for pathogenesis (Kaman et al., 2014;Madigan et al., 2014). The neutral metallo-APs from various pathogen microorganisms are among the novel molecular targets recognized in the last decade, with possibilities to constitute the starting point of novel effective therapies (Florent et al., 1998;Cadavid-Restrepo et al., 2011;Drinkwater et al., 2017). ...
The enzymes ePepN and PfA-M1 are two M1 alanyl-aminopeptidases which are targets in the potential treatment of microbial infections and malaria, respectively. The classical inhibitor of M1-aminopeptidases is bestatin. The synthetic bestatin-derived peptidomimetic KBE009 was previously identified as a PfA-M1 inhibitor with in vitro antimalarial activity. The objective of this work was to test a synthetic library of 10 bestatin (KBE009)-derived peptidomimetics in the inhibition of recombinant ePepN (rePepN) and PfA-M1 (rPfA-M1), and to study the resultant structure-activity relationship. In the first place, the main kinetic characteristics of rePepN and of the inhibition of both enzymes by bestatin were assessed. The same rePepN activity at pH 7.0 and 8.0, as well as a K M = 72 µM toward the Leu-p-nitroanilide substrate were determined. A 5-min preincu-bation between bestatin and rePepN is enough to establish the inhibition equilibrium and bestatin is a non-competitive inhibitor of the enzyme with a K i = 2.31 µM. On the other hand, for rPfA-M1 a 15-min preincubation time is enough, the K i of bestatin is 1.29 µM and this inhibitor is competitive. Although none compound is a potent inhibitor, the structural characteristics of the bestatin (KBE009)-derived peptidomimetics that are favorable for the inhibition are: the central isopropyl and terminal 3-phenylpropyl groups in the branch (for both aminopeptidases), the central isopropyl and terminal cyclohexyl groups (for rePepN), and the central 2-furyl and terminal benzyl groups (for rPfA-M1). The compound KBE053, as representative of this series, is an uncompetitive inhibitor of rePepN with a K i = 10.13 µM. This knowledge could contribute to the design of novel ePepN and PfA-M1 inhibitors.
... Although there are multiple crystal structures of boronates complexed to both SBLs 22 and the related penicillin binding proteins, 23 there are few with MBLs. 11,18 To investigate the possible structural basis of vaborbactam interaction with the MBLs, a model of vaborbactam bound to the B1 MBL VIM-2, based upon the binding mode of a bicyclic boronate (Fig. 1, PDB ID: 5FQC), 18 was constructed (Fig. 2C). ...
β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20-100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.
... and the inaccuracy of the obtained data if we try to use the same data for studying boron in some developed drugs; or the lack of data diminishing the theoretical performance of ligand-protein simulations and requiring boron substitution in theoretical assays [67]. But also, the difficulty to reproduce the ligand-protein data found in the Protein Data Bank [68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85]. ...
In the last few decades, research into boron-containing compounds (BCCs) has notably increased in medicinal chemistry. Multiple maladies are now targeted by means of BCCs. Since bioinformatics tools have become a common and efficient methodology for drug design, their application to the study of BCCs is expected to intensify. This review compiles the use of computational technology to elucidate the chemical-biological effects of BCCs, whether coming from natural sources or drug development strategies. A broad range of computational approaches facilitate pharmacochemical analysis of BCCs, focusing on the essential parameters of a boron atom, the reasons for an experimental event, and the shared pharmacodynamics, pharmacokinetics or toxic effects of components of a group of BCCs. Some studies have examined the best quantitative structure-activity relationship for a specific target. A final remark is made as to the potential impact on BCC research that could result from advances in bioinformatics.
... Contreras-Martel and coworkers used a crystallography-guided approach to identify boronic acid analogs that bound reversibly to the active site of the PBP and showed antibacterial activity. One of the analogs was shown to specifically bind to the low affinity PBP2a, isolated from the membranes of S. aureus, and to inhibit growth of MRSA (Contreras-Martel et al., 2011). Notably, D-Ala-D-Ala ligases, which participate at an early, cytoplasmic step of peptidoglycan biosynthesis, have also been shown to be the targets of d-boro-Ala analogs that show some specificity towards MRSA (Putty et al., 2011). ...
Staphylococcus aureus is a major cause of bacterial infection in humans, and has been notoriously able to acquire resistance to a variety of antibiotics. An example is methicillin-resistant S. aureus (MRSA), which despite having been initially associated with clinical settings, now is one of the key causative agents of community-acquired infections. Antibiotic resistance in S. aureus involves mechanisms ranging from drug efflux to increased expression or mutation of target proteins, and this has required innovative approaches to develop novel treatment methodologies. This review provides an overview of the major mechanisms of antibiotic resistance developed by S. aureus, and describes the emerging alternatives being sought to circumvent infection and proliferation, including new generations of classic antibiotics, synergistic approaches, antibodies, and targeting of virulence factors.
... A boronic acid and a trifluoroketone bearing an appropriate peptidoglycan mimetic side-chain could form covalent adducts with the Actinomadura R39 DD-peptidase [109,110]. Other boronic acids were also designed and shown to bind to S. pneumoniae PBP1b [111]. Strikingly, the peptidoglycan mimetic boronic acid could not bind to HMW-PBPs [112], demonstrating the poor affinity of HMW-PBPs for their natural substrate; however, a ceftazidime or cefotaxime such as boronic acid might possibly do so. ...
Peptidoglycan (PG) is an essential macromolecular sacculus surrounding most bacteria. It is assembled by the glycosyltransferase (GT) and transpeptidase (TP) activities of multimodular penicillin-binding proteins (PBPs) within multiprotein complex machineries. Both activities are essential for the synthesis of a functional stress-bearing PG shell. Although good progress has been made in terms of the functional and structural understanding of GT, finding a clinically useful antibiotic against them has been challenging until now. In contrast, the TP/PBP module has been successfully targeted by β-lactam derivatives, but the extensive use of these antibiotics has selected resistant bacterial strains that employ a wide variety of mechanisms to escape the lethal action of these antibiotics. In addition to traditional β-lactams, other classes of molecules (non-β-lactams) that inhibit PBPs are now emerging, opening new perspectives for tackling the resistance problem while taking advantage of these valuable targets, for which a wealth of structural and functional knowledge has been accumulated. The overall evidence shows that PBPs are part of multiprotein machineries whose activities are modulated by cofactors. Perturbation of these systems could lead to lethal effects. Developing screening strategies to take advantage of these mechanisms could lead to new inhibitors of PG assembly. In this paper, we present a general background on the GTs and TPs/PBPs, a survey of recent issues of bacterial resistance and a review of recent works describing new inhibitors of these enzymes.
... Most of the β-lactam antibiotics inhibit the function of PBPs by binding to them irreversibly [42]. Four PBPs (PBP 1-4) have been found to be essential for S. aureus cell wall function, their X-ray structures being available in the Protein data bank (PDB): PBP1b (PDB ID 2Y2P), PBP4 (PDB ID 3HUN), PBP2a (PDB ID 3ZG0), and PBP3 (PDB ID 3VSL) [43][44][45][46]. The source organism for the first two is S. aureus, while the latter are related to MRSA. ...
We have shown that novel silver salts of poly (propyl ether) imine (PETIM) dendron and dendrimers developed in our group exhibit preferential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus. This led us to examine whether molecular modeling methods could be used to identify the key structural design principles for a bioactive lead molecule, explore the mechanism of binding with biological targets, and explain their preferential antibacterial activity. The current article reports the conformational landscape as well as mechanism of binding of generation 1 PETIM dendron and dendrimers to penicillin-binding proteins (PBPs) in order to understand the antibacterial activity profiles of their silver salts. Molecular dynamics at different simulation protocols and conformational analysis were performed to elaborate on the conformational features of the studied dendrimers, as well as to create the initial structure for further binding studies. The results showed that for all compounds, there were no significant conformational changes due to variation in simulation conditions. Molecular docking calculations were performed to investigate the binding theme between the studied dendrimers and PBPs. Interestingly, in significant accordance with the experimental data, dendron and dendrimer with aliphatic cores were found to show higher activity against S. aureus than the dendrimer with an aromatic core. The latter showed higher activity against MRSA. The findings from this computational and molecular modeling report together with the experimental results serve as a road map toward designing more potent antibacterial dendrimers against resistant bacterial strains.
... Staphylococci are generally sensitive to ßlactam antibiotics [82]; however, a large proportion of staphylococcal strains are developing antibiotic resistance to penicillin [83]. This antibiotic resistance is based on the formation of penicillinases [84,85]. Therefore, various penicillin antibiotics are administered in mixtures with lactamase inhibitors, such as sulbactam and tazobactam [86]. ...
Background:
Staphylococci can cause wound infections and community- and nosocomial-acquired pneumonia, among a range of illnesses. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) have been rapidly increasing as a cause of infections worldwide in recent decades. Numerous reports indicate that S. aureus and MRSA are becoming resistant to many antibiotics, which makes them very dangerous. Therefore, this study retrospectively investigated the resistance to antimicrobial agents in all hospitalized patients suffering from community- or nosocomial-acquired pneumonia due to S. aureus and MRSA.
Methods:
Information from the study groups suffering from either community- or nosocomial-acquired pneumonia caused by S. aureus or MRSA was gathered by searching records from 2004 to 2014 at the HELIOS Clinic Wuppertal, Witten/Herdecke University, Germany. The findings of antibiotic resistance were analyzed after the evaluation of susceptibility testing for S. aureus and MRSA.
Results:
Total of 147 patients (63.9%, 95% CI 57.5%-69.8%), mean age 67.9 ± 18.5 years, with pneumonia triggered by S. aureus, and 83 patients (36.1%, 95% CI 30.2%-42.5%), mean age 72.3 ± 13.8 years, with pneumonia due to MRSA. S. aureus and MRSA developed no resistance to vancomycin (P = 0.019 vs. < 0.0001, respectively) or linezolid (P = 0.342 vs. < 0.0001, respectively). MRSA (95.3%) and S. aureus (56.3%) showed a high resistance to penicillin. MRSA (87.7%) was also found to have a high antibiotic resistance against ß-lactam antibiotics, compared to S. aureus (9.6%). Furthermore, MRSA compared to S. aureus, respectively, had increased antibiotic resistance to ciprofloxacin (90.1% vs. 17.0%), cefazolin (89.7% vs. 10.2%), cefuroxime (89.0% vs. 9.1%), levofloxacin (88.2% vs. 18.4%), clindamycin (78.0% vs. 14.7%), and erythromycin (76.5% vs. 20.8%).
Conclusion:
No development of resistance was found to vancomycin and linezolid in patients with pneumonia caused by S. aureus and MRSA.
... It is also reported that 15 does not inhibit Streptococcus pneumoniae PBP1b. 31 As would be anticipated from the above discussion, analogues of 16, unsubstituted α to the boronic acid, are more likely to be DD-peptidase inhibitors. 32−36 The results of Table 1 show that the new boronic acids 11 and 12 at micromolar concentrations do inhibit class C β-lactamases, the P99 and AmpC enzymes. ...
Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.
... The persistence of MRSA as a pathogen, the continuing proliferation of its antibiotic-resistance mechanisms, and the extraordinary difficulty of empirical structural optimization collectively demand new strategies for antibiotic discovery, which are exemplified by the discoveries of non-β-lactams targeting PBP2a (35,36) and unique targets synergistically lethal with existing β-lactams (37)(38)(39)(40)(41)(42)(43)(44)(45). Binding of an allosteric effector can influence protein function, and thus, allosteric binding sites can be targets for new drugs. ...
Significance
Penicillin binding protein 2a imparts to the human pathogen Staphylococcus aureus resistance to β-lactam antibiotics. Our structural characterization of the allosteric basis governing its resistance mechanism identifies a basis for the design of new antibacterials that can both activate and inhibit this key resistance enzyme.
... A boronic acid and a trifluoroketone bearing an appropriate peptidoglycan mimetic side-chain could form covalent adducts with the Actinomadura R39 DD-peptidase [109,110]. Other boronic acids were also designed and shown to bind to S. pneumoniae PBP1b [111]. Strikingly, the peptidoglycan mimetic boronic acid could not bind to HMW-PBPs [112], demonstrating the poor affinity of HMW-PBPs for their natural substrate; however, a ceftazidime or cefotaxime such as boronic acid might possibly do so. ...
Peptidoglycan (PG) is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to bacterial cell lysis, making it an important target for many antibiotics. The final two reactions in PG synthesis are performed by penicillin-binding proteins (PBPs). Their glycosyltransferase (GT) activity uses the lipid II precursor to synthesize glycan chains and their transpeptidase (TP) activity catalyzes the cross-linking of two glycan chains via the peptide side chains. Inhibition of either of these two reactions leads to bacterial cell death. β-lactam antibiotics target the transpeptidation reaction while antibiotic therapy based on inhibition of the GTs remains to be developed. Ongoing research is trying to fill this gap by studying the interactions of GTs with inhibitors and substrate mimics and utilizing the latter as templates for the design of new antibiotics. In this review we present an updated overview on the GTs and describe the structure-activity relationship of recently developed synthetic ligands.
Developing novel catalysts with potent activity is of great importance in organocatalysis. In this study, we designed and prepared a new class of benzotetramisole Lewis base catalysts (AxBTM) that have both central and axial chirality. This unique feature of these catalysts results in a three‐dimensional microenvironment with multi‐layers of chirality. The performance of the developed catalysts was tested in a series of cycloaddition reactions. These included the AxBTM‐catalyzed (2 + 2) cycloaddition between α‐fluoro‐α‐aryl anhydride with imines or oxindoles, and the sequential gold/AxBTM‐catalyzed (4 + 2) cycloaddition of enynamides with pentafluorophenyl esters. The interplay between axial and central chirality had a collaborative effect in regulating the stereochemistry in these cycloadditions, leading to high levels of stereoselectivity that would otherwise be challenging to achieve using conventional BTM catalysts. However, the (2 + 2) and (4 + 2) cycloadditions have different predilections for axial and central chirality combinations.
Developing novel catalysts with potent activity is of great importance in organocatalysis. In this study, we designed and prepared a new class of benzotetramisole Lewis base catalysts (AxBTM) that have both central and axial chirality. This unique feature of these catalysts results in a three‐dimensional microenvironment with multi‐layers of chirality. The performance of the developed catalysts was tested in a series of cycloaddition reactions. These included the AxBTM‐catalyzed (2+2) cycloaddition between α‐fluoro‐α‐aryl anhydride with imines or oxindoles, and the sequential gold/AxBTM‐catalyzed (4+2) cycloaddition of enynamides with pentafluorophenyl esters. The interplay between axial and central chirality had a collaborative effect in regulating the stereochemistry in these cycloadditions, leading to high levels of stereoselectivity that would otherwise be challenging to achieve using conventional BTM catalysts. However, the (2+2) and (4+2) cycloadditions have different predilections for axial and central chirality combinations.
β-Lactams are the most widely prescribed class of antibiotics that inhibit penicillin-binding proteins (PBPs), particularly transpeptidases that function in peptidoglycan synthesis. A major mechanism of antibiotic resistance is the production of β-lactamase enzymes, which are capable of hydrolyzing β-lactam antibiotics. There have been many efforts to counter increasing bacterial resistance against β-lactams. These studies have mainly focused on three areas: discovering novel inhibitors against β-lactamases, developing new β-lactams less susceptible to existing resistance mechanisms, and identifying non-β-lactam inhibitors against cell wall transpeptidases. Drug discovery in the β-lactam field has afforded a range of research opportunities for academia. In this review, we summarize the recent new findings on both β-lactamases and cell wall transpeptidases because these two groups of enzymes are evolutionarily and functionally connected. Many efforts to develop new β-lactams have aimed to inhibit both transpeptidases and β-lactamases, while several promising novel β-lactamase inhibitors have shown the potential to be further developed into transpeptidase inhibitors. In addition, the drug discovery progress against each group of enzymes is presented in three aspects: understanding the targets, screening methodology, and new inhibitor chemotypes. This is to offer insights into not only the advancement in this field but also the challenges, opportunities, and resources for future research. In particular, cyclic boronate compounds are now capable of inhibiting all classes of β-lactamases, while the diazabicyclooctane (DBO) series of small molecules has led to not only new β-lactamase inhibitors but potentially a new class of antibiotics by directly targeting PBPs. With the cautiously optimistic successes of a number of new β-lactamase inhibitor chemotypes and many questions remaining to be answered about the structure and function of cell wall transpeptidases, non-β-lactam transpeptidase inhibitors may usher in the next exciting phase of drug discovery in this field.
Bacterial cell wall formation is essential for cellular survival and morphogenesis. The peptidoglycan (PG), a heteropolymer that surrounds the bacterial membrane, is a key component of the cell wall, and its multistep biosynthetic process is an attractive antibacterial development target. Penicillin-binding proteins (PBPs) are responsible for cross-linking PG stem peptides, and their central role in bacterial cell wall synthesis has made them the target of successful antibiotics, including β-lactams, that have been used worldwide for decades. Following the discovery of penicillin, several other compounds with antibiotic activity have been discovered and, since then, have saved millions of lives. However, since pathogens inevitably become resistant to antibiotics, the search for new active compounds is continuous. The present review highlights the ongoing development of inhibitors acting mainly in the transpeptidase domain of PBPs with potential therapeutic applications for the development of new antibiotic agents. Both the critical aspects of the strategy, design, and structure–activity relationships (SAR) are discussed, covering the main published articles over the last 10 years. Some of the molecules described display activities against main bacterial pathogens and could open avenues toward the development of new, efficient antibacterial drugs.
Staphylococcus aureus is a common human pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose significant and challenging therapeutic difficulties. MRSA often acquires the non-native gene PBP2a, which results in reduced susceptibility to β-lactam antibiotics, thus conferring resistance. PBP2a has a lower affinity for methicillin, allowing bacteria to maintain peptidoglycan biosynthesis, a core component of the bacterial cell wall. Consequently, even in the presence of methicillin or other antibiotics, bacteria can develop resistance. Due to genes responsible for resistance, S. aureus becomes MRSA. The fundamental premise of this resistance mechanism is well-understood. Given the therapeutic concerns posed by resistant microorganisms, there is a legitimate demand for novel antibiotics. This review primarily focuses on PBP2a scaffolds and the various screening approaches used to identify PBP2a inhibitors. The following classes of compounds and their biological activities are discussed: Penicillin, Cephalosporins, Pyrazole-Benzimidazole-based derivatives, Oxadiazole-containing derivatives, non-β-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (β-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded β-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-β-lactam antibiotics, Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a. Additionally, we discuss the penicillin-binding protein, a crucial target in the MRSA cell wall. Various aspects of PBP2a, bacterial cell walls, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives on MRSA inhibitors are also explored.
The present study was designed to appraise the photoprotective, antioxidant, and antibacterial bioactivities of Ruellia tuberosa leaves extracts (RtPE, RtChl, RtEA, RtAc, RtMe, and RtHMe). The results showed that the RtHMe extract of leaves of R. tuberosa was rich in total phenolic content i.e., 1.60 mgGAE/g dry extract, while TFC highest total flavonoid content was found in RtAc extract, i.e., 0.40 mgQE/g. RtMe showed effective antioxidant activity (%RSA: 58.16) at the concentration of 120 µL. RtMe, RtEA and RtHMe exhibited effective in vitro antibacterial activity against Gram-negative bacteria (E. coli). In silico docking studies revealed that paucifloside (-11.743 kcal/mol), indole-3-carboxaldehyde (-7.519 kcal/mol), nuomioside (-7.275 kcal/mol), isocassifolioside (-6.992 kcal/mol) showed best docking score against PDB ID 2EX8 [penicillin binding protein 4 (dacB) from Escherichia coli, complexed with penicillin-G], PDB ID 6CQA (E. coli dihydrofolate reductase protein complexed with inhibitor AMPQD), PDB ID 2Y2I [Penicillin-binding protein 1B in complex with an alkyl boronate (ZA3)] and PDB ID 2OLV (from S. aureus), respectively. Docked phytochemicals also showed good drug likeness properties.
Lactam antibiotics are critical antibacterial agents and have rapidly become active ingredients in modern medicine. On the other hand, in the face of increasing rate of antibacterial resistance, there are many concerns requiring the design and synthesis of new antibacterial agents. Herein, we synthesized some monocyclic β‐lactams with various substituents through the Staudinger cycloaddition reaction. The formation of cycloadducts was confirmed by elemental analysis and different spectral data, including NMR, FTIR, and Mass spectroscopy. The investigation of in vitro antibacterial activity of synthesized β‐lactams was performed against some broad‐spectrum strains, including Escherichia coli (E. coli), streptococcus aureus (S. aureus), Salmonella Typhi (S. Typhi), Enterococcus faecalis (E. faecalis), and Candida albicans (C. albicans) and also some nosocomial multidrug‐resistant pathogens such as methicillin‐resistant Staphylococcus aureus (MRSA), and vancomycin‐resistant enterococci (VRE). Penicillin Binding Proteins (PBPs) are responsible for the cell wall synthesis process as an enzymatic target for β‐lactam antibiotics. The molecular docking study exhibited a good correlation between the calculated binding affinity to PBP and the experimental data. Based on the obtained results, compound 4 d was successfully fitted in the BPB active site, and could potentially serve as a promising lead compound for the treatment of infectious diseases.
Antimicrobial resistance (AMR) mediated by β-lactamases is the major and leading cause of resistance to penicillins and cephalosporins among Gram-negative bacteria. β-Lactamases, periplasmic enzymes that are widely distributed in the bacterial world, protect penicillin-binding proteins (PBPs), the major cell wall synthesizing enzymes, from inactivation by β-lactam antibiotics. Developing novel PBP inhibitors with a non-β-lactam scaffold could potentially evade this resistance mechanism. Based on the structural similarities between the evolutionary related serine β-lactamases and PBPs, we investigated whether the potent β-lactamase inhibitor, vaborbactam, could also form an acyl-enzyme complex with Pseudomonas aeruginosa PBP3. We found that this cyclic boronate, vaborbactam, inhibited PBP3 (IC 50 of 262 μM), and its binding to PBP3 increased the protein thermal stability by about 2°C. Crystallographic analysis of the PBP3:vaborbactam complex reveals that vaborbactam forms a covalent bond with the catalytic S294. The amide moiety of vaborbactam hydrogen bonds with N351 and the backbone oxygen of T487. The carboxyl group of vaborbactam hydrogen bonds with T487, S485, and S349. The thiophene ring and cyclic boronate ring of vaborbactam form hydrophobic interactions, including with V333 and Y503. The active site of the vaborbactam-bound PBP3 harbors the often observed ligand-induced formation of the aromatic wall and hydrophobic bridge, yet the residues involved in this wall and bridge display much higher temperature factors compared to PBP3 structures bound to high-affinity β-lactams. These insights could form the basis for developing more potent novel cyclic boronate-based PBP inhibitors to inhibit these targets and overcome β-lactamases-mediated resistance mechanisms.
Antimicrobial resistance is an imminent threat worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the “superbug” family, manifesting resistance through the production of a penicillin binding protein, PBP2a, an enzyme that provides its transpeptidase activity to allow cell wall biosynthesis. PBP2a's low affinity to most β-lactams, confers resistance to MRSA against numerous members of this class of antibiotics. An Achilles' heel of MRSA, PBP2a represents a substantial target to design novel antibiotics to tackle MRSA threat via inhibition of the bacterial cell wall biosynthesis.
In this review we bring into focus the PBP2a enzyme and examine the various aspects related to its role in conferring resistance to MRSA strains. Moreover, we discuss several antibiotics and antimicrobial agents designed to target PBP2a and their therapeutic potential to meet such a grave threat. In conclusion, we consider future perspectives for targeting MRSA infections.
Background
The search for new antimicrobial drugs is a never ending task due to microbial resistance to the existing drugs. Antioxidants are essential to prevent free radical reactions which lead to chronic diseases to human kind.
Objective
The present studies were aimed to synthesis, characterization, antimicrobial and antioxidant activities of pyridine and benzoisothiazole decorated chalcones.
Materials and Methods
FTIR spectra were recorded using KBr pellets on Shimadzu FT-IR spectrophotometer. 1H and 13C NMR spectra were recorded on Bruker 400 MHz spectrometer. Antimicrobial activity of the synthesized chalcones was found to be good against diffenet bacterial and fungal strains. Antioxidant activity was studied in terms of 2,2-diphenyl-1-picrylhydrazyl, hydroxyI and superoxide radical scavenging activities. Molecular docking was studied using Discovery Studio Visualizer Software, version 16 whereas Autodock Vina program was used to predict toxicity profile of the compounds using FAFDrugs2 predictor.
Results
The compounds 5c, 5d & 6c showed good antioxidant activities. The insilico molecular docking study supports the experimental results and demonstrated that the chalcones 5d, 6a and 7a are the most active among the synthesized derivatives.
Conclusion
Prediction of pharmacokinetic parameters and molecular docking studies suggest that the synthesized chalcones have good pharmacokinetic properties to act as lead molecules in the drug discovery process.
The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other β-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles’ heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.
Resistance to β-lactams has arisen in a variety of ways. In this chapter, we consider those cases where resistance results from the expression of targets, the penicillin-binding proteins, which have a “low affinity” for the drugs. In the last three decades, different resistance mechanisms that involve the PBPs have been uncovered in important human pathogens such as Staphylococcus aureus, Enterococci, Streptococcus pneumoniae, and Neisseria.
The synthesis of the enantiopure thioester (R)-2-(2-benzamidopropanoylthio)acetic acid was developed. After the exploration of several activation methods, reaction conditions were found for the formation of the thioester bond in the presence of propylphosphonic anhydride with high enantioselectivity (ee > 99%). The thioester activity of Penicillin Binding Proteins is helpful in research programs looking for new lead structures to overcome the problem of bacterial resistance.
The extensive use of antimicrobials is leaving medicine with few effective therapeutic options to treat many infections due to the fact that many organisms developed resistance to commonly used drugs. It is therefore pertinent to search not only for new antimicrobials but also for compounds able to restore or potentiate the activity of existing antibiotics. We have screened a library consisting of 40 (thio)xanthone derivatives for antibacterial activity and possible synergistic effects when used in combination with antibiotics. Nine out of the 40 compounds exhibited antibacterial activity against Gram-positive bacteria. Two xanthone derivatives, 1-formyl-4-hydroxy-3-methoxy (7), 2-formyl-3-hydroxy-4-methoxyxanthone (8) and the thioxanthone derivative 1-((2-(diethylamino)ethyl)amino)-4-propoxythioxanthone (10) and its hydrochloride form 13, showed activity against a methicillin-resistant Staphylococcus aureus (MRSA) isolate with minimum inhibitory concentration (MIC) values lower than 256 μg/ml. Thioxanthone 10 demonstrated antibacterial activity and also synergy when combined with ampicillin and oxacillin against MRSA. Additionally, thioxanthone 1-(piperidin-1-yl)-4-propoxythioxanthone (9), despite not having antibacterial activity presented remarkable synergy with oxacillin against MRSA; the MIC of tioxanthone 9 and oxacillin when both were in combination were 128 and 8 μg/ml, respectively. Thioxanthones 9 and 10 were also found to be synergistic when both were combined. Subsequently, docking simulations between thioxanthones 9 and 10 and the allosteric domain of penicillin-binding protein 2A (PBP2A) were undertaken in AutoDock Vina. Both compounds had the ability to bind with an allosteric domain of PBP2A, which may explain their synergy with oxacillin. These two thioxanthone derivatives with different profiles may be promising tools for restoring the activity of oxacillin against MRSA.
In the present study, a series of chalcone derivatives including 17 new compounds were synthesised; their antibacterial activities against eleven bacteria, and their free radical- scavenging activities using DPPH were evaluated. All compounds showed significant antibacterial activities against both Gram-positive and Gram-negative bacteria. In particular, compound IIIf strongly inhibited Staphylococcus aureus (JMC 2151) and Enterococcus faecalis (CARS 2011-012) with MIC values of 6.25 µg mL−1 and 12.5 µg mL−1, respectively, which are comparable to that of the standard antibiotic, nalidixic acid. Compound IIIg also inhibited S. aureus with a MIC value similar to that of nalidixic acid (6.25 µg mL−1). Furthermore, like nalidixic acid (MIC value of 25 µg mL−1), compounds IIIa, IIIc and IIId inhibited Listeria monocytogenes (ATCC 43256) with MIC values of 25 µg mL−1, 12.5 µg mL−1 and 25 µg mL−1, respectively. Quantitative structure-activity relationship (Q-SAR) studies using physicochemical calculations indicated that the antibacterial activities of chalcone derivatives correlated well with predicted physicochemical parameters (logP and PSA). Docking simulation by positioning the most active compound IIIf in the active site of the penicillin-binding protein (PBP-1b) of S. aureus was performed to explore the feasible binding mode. Furthermore, most of the compounds synthesised exhibited significant DPPH radical- scavenging activity, although compounds IIc and IIIc exhibited the greatest antioxidant activity with IC50 values of 1.68 µM and 1.44 µM, respectively, comparable to that of the standard antioxidant, ascorbic acid (1.03µM).
The penicillin-binding proteins (PBPs) are well known targets for the β-lactam antibiotics. They continue to be a focus of interest for pharmaceutical design, as exemplified by the number of new agents under clinical investigation as well as novel experimental molecules. Considerable advances have been made in understanding the structure and function of this family of enzymes, through high-resolution structural studies and mechanistic studies in solution. These studies have thrown light on role of the high molecular mass PBPs in mediating β-lactam resistance, although much work remains to be done to enable a full description of the mechanisms by which these proteins modulate their sensitivity towards β-lactams while retaining their essential activity in cell wall biosynthesis.
The transformation proceeds smoothly at room temperature providing the desired esters (III) with enantioselectivities up to 84%.
Proper understanding of the mechanisms of binding to Gram-positive bacteria cell wall layers-especially to the peptidoglycan (PG) layer, seems to be crucial for proper development of new drug candidates which are effective against these bacteria. In this work we have constructed two different models of the Gram-positive bacteria PG layer: the layered and the scaffold models. PG conformational changes during geometry optimization, models relaxation, and molecular dynamics were described and discussed. We have found that the border surface of both PG layer models differs from the surface located away from the edge of models and the chains formed by disaccharide units prefer helix-like conformation. This curling of PG chains significantly affects the shape of antibiotic-accessible surface and the process is thus crucial for new drug development. Glycopeptide antibiotics effective against Gram-positive bacteria, such as vancomycin and its semisynthetic derivatives-oritavancin and telavancin, bind to d-alanyl-d-alanine stem termini on the peptidoglycan precursors of the cell wall. This binding inhibits cross-linking between the peptides and subsequently prevents cell wall synthesis. In this study some of the aspects of conformational freedom of vancomycin and restrictions from the modifications of vancomycin structure introduced into oritavancin and telavancin and five other vancomycin derivatives (with addition of 2-acetamido-2-deoxy-β-d-galactopyranosylamine, 2-acetamido-2-deoxy-β-d-glucopyranosylamine, 1-amine-1-deoxy-d-glucitol, 2-amino-2-deoxy-d-galactitol, or 2-amino-2-deoxy-d-glucitol to the C-terminal amino acid group in the vancomycin) are presented and discussed. The resulting molecular dynamics trajectories, root mean square deviation changes of aglycon and saccharide moieties as well as a comparative study of possible interactions with cyclic and chain forms of modified groups have been carried out, measured, and analyzed. Energetically advantageous conformations show close similarity to the structures known from the experimental data, but the diversity of others suggest very high conformational freedom of all modeled antibiotics and vancomycin derivatives. Alditol derivatives move closer to the peptidoglycan chain more easily but they also form intramolecular interactions more frequently than their homologous cyclic forms. One of the proposed derivatives seems to be a promising agent which is efficient in treatment of infections caused by Gram-positive bacteria.
Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units cross-linked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review aims at providing an overview of resistance mechanisms developed towards antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies towards the development of novel inhibitors that could overcome resistance are also discussed.
A highly diastereoselective cross-coupling reaction of an α-bromo-α-fluoro-β-lactam with a wide range of aryl Grignard reagents was catalyzed by Ni/bis(oxazoline) in yields of up to 98%. The product was obtained diastereoselectively as an anti-isomer. This is the first successful α-arylation of an α-fluoro-β-lactam to produce diverse α-aryl-α-fluoro-β-lactams.
Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to β-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-α-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics.
Centre d'Ingeiere des Proteínes, Universitee Liè ge, B-4000 Sart Tilman, Liè ge, Belgium ABSTRACT: Inhibitors of bacterial DD-peptidases represent potential antibiotics. In the search for alternatives to β-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with DD-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C DD-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [D-α-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 DD-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective DD-peptidase inhibitors and therefore, perhaps, antibiotics. T he bacterial DD-peptidases are of considerable impor-tance in medical practice because they are the targets of β-lactam antibiotics. 1 These enzymes catalyze the final transpeptidation reaction in the biosynthesis of bacterial cell walls and are essential to bacterial survival. The β-lactams, acting as mechanism-based, transition state analogue inhibitors, 2−4 are precisely structured to inactivate DD-peptidases in a manner that these enzymes have been unable to escape through evolution of a hydrolytic pathway. Bacteria have, however, been able to achieve resistance to β-lactams in a number of ways unrelated to DD-peptidase active site structure and, in particular, through evolution of β-lactamases from DD-peptidases. 2,4 The β-lactamases, unlike DD-peptidases, are able to catalyze rapid β-lactam hydrolysis and thus destruction of their antibiotic activity. 5 The rapid evolution of β-lactamases in response to new β-lactam antibiotics and also to β-lactam-based β-lactamase inhibitors 6−8 emphasizes the need for and stimulates the search for DD-peptidase inhibitors that are not β-lactam-based. 9−15 One obvious approach is through transition state analogues, because such molecules should, in principle, inhibit any enzyme. 16−18 Because the reactions catalyzed by DD-peptidases in vivo are acyl transfer reactions with a covalent acyl(serine)− enzyme intermediate (Scheme 1), substrate-based tetrahedral anions covalently bound to the active site serine should be good analogues of the transition states of both acylation and deacyla-tion steps. In principle, therefore, molecule 1, in which "peptidoglycan" is a specific peptidoglycan or peptidoglycan-mimetic fragment and X is a reactive moiety that generates a tetrahedral anion upon reaction with the active site serine, would be the inhibitor of choice.
The synthesis of the bacterial peptidoglycan has been recognized for over 50 years as fertile ground for antibacterial discovery. Initially, empirical screening of natural products for inhibition of bacterial growth detected many chemical classes of antibiotics whose specific mechanisms of action were eventually dissected and defined. Of the nontoxic antibiotics discovered, most were found to be inhibitors of either protein synthesis or cell wall synthesis, which led to more directed screening for inhibitors of these pathways. Directed screening and design programs for cell wall inhibitors have been undertaken since the 1960s. In that time it has become clear that, while certain steps and intermediates have yielded selective inhibitors and are established targets, other potential targets have not yielded inhibitors whose antibacterial activity is proven to be solely due to that inhibition. Why has this search been so problematic? Are the established targets still worth pursuing? This review will attempt to answer these and other questions and evaluate the viability of targets related to peptidoglycan synthesis.
Transition state analogue boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates for the TEM-1 beta-lactamase of Escherchia coli were designed using graphic analyses based on the enzyme's 1.7 Angstrom crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethlboronic acid (1) and (1R)-1-acetamido2-(3-carboxy-2-hydroxyphenyl) ethylboronic acid (2), is reported. Kinetic measurements show that, as designed, compounds 1 and 2 are highly effective deacylation transition state analogue inhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent competitive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1, k(1) = 5.9 nM), which resembles most closely the best known substrate of TEM-1, benzylpenicillin (penicillin G). The high-resolution crystallographic structures of these two inhibitors covalently bound to TEM-1 are also described. In addition to verifying the design features, these two structures show interesting and unanticipated changes in the active site area, including strong hydrogen bond formation, water displacement, and rearrangement of side chains. The structures provide new insights into the further design of this patent class of beta-lactamase inhibitors.
β-Lactamases are the major resistance mechanism to β-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to β-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-β-lactam inhibitors of β-lactamases were investigated for complementarity to the structure of AmpC β-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo (b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a Kit of 27 nM. This inhibitor is chemically dissimilar to β-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 Å resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-β-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against β-lactams in conjunction with β-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many β-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-β-lactam inhibitors.
Penicillin-binding proteins (PBPs), the main targets of β-lactam antibiotics, are membrane-associated enzymes that catalyze the two last steps in the biosynthesis of peptidoglycan. In Streptococcus pneumoniae, a major human pathogen, the surge in resistance to such antibiotics is a direct consequence of the proliferation of mosaic PBP-encoding genes, which give rise to proteins containing tens of mutations. PBP2b is a major drug resistance target, and its modification is essential for the development of high levels of resistance to piperacillin. In this work, we have solved the crystal structures of PBP2b from a wild-type pneumococcal strain, as well as from a highly drug-resistant clinical isolate displaying 58 mutations. Although mutations are present throughout the entire PBP structure, those surrounding the active site influence the total charge and the polar character of the region, while those in close proximity to the catalytic nucleophile impart flexibility onto the β3/β4 loop area, which encapsulates the cleft. The wealth of structural data on pneumococcal PBPs now underlines the importance of high malleability in active site regions of drug-resistant strains, suggesting that active site “breathing” could be a common mechanism employed by this pathogen to prevent targeting by β-lactams.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
Boronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.
Penicillin-binding protein 6 (PBP6) is one of the two main DD-carboxypeptidases in Escherichia coli, which are implicated in maturation of bacterial cell wall and formation of cell shape. Here, we report the first X-ray crystal structures of PBP6, capturing its apo state (2.1 A), an acyl-enzyme intermediate with the antibiotic ampicillin (1.8 A), and for the first time for a PBP, a preacylation complex (a "Michaelis complex", determined at 1.8 A) with a peptidoglycan substrate fragment containing the full pentapeptide, NAM-(L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala). These structures illuminate the molecular interactions essential for ligand recognition and catalysis by DD-carboxypeptidases, and suggest a coupling of conformational flexibility of active site loops to the reaction coordinate. The substrate fragment complex structure, in particular, provides templates for models of cell wall recognition by PBPs, as well as substantiating evidence for the molecular mimicry by beta-lactam antibiotics of the peptidoglycan acyl-D-Ala-D-Ala moiety.
Penicillin binding proteins (PBPs) catalyze steps in the biosynthesis of bacterial cell walls and are the targets for the beta-lactam antibiotics. Non-beta-lactam based antibiotics that target PBPs are of interest because bacteria have evolved resistance to the beta-lactam antibiotics. Boronic acids have been developed as inhibitors of the mechanistically related serine beta-lactamases and serine proteases; however, they have not been explored extensively as PBP inhibitors. Here we report aromatic boronic acid inhibitors of the D,D-carboxypeptidase R39 from Actinomadura sp. strain. Analogues of an initially identified inhibitor [3-(dihydroxyboryl)benzoic acid 1, IC(50) 400 microM] were prepared via routes involving pinacol boronate esters, which were deprotected via a two-stage procedure involving intermediate trifluorborate salts that were hydrolyzed to provide the free boronic acids. 3-(Dihydroxyboryl)benzoic acid analogues containing an amide substituent in the meta, but not ortho position were up to 17-fold more potent inhibitors of the R39 PBP and displayed some activity against other PBPs. These compounds may be useful for the development of even more potent boronic acid based PBP inhibitors with a broad spectrum of antibacterial activity.
Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, beta-lactams show activity against intracellular methicillin (methicillin)-susceptible S. aureus (MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistant S. aureus (MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these beta-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (E(max)), relative potency (the concentration causing a reduction of the inoculum halfway between E(0) and E(max) [EC(50)]), and static concentration (C(s))} were measured. All strains showed sigmoidal dose-responses, with E(max) being about a 1 log(10) CFU decrease from the postphagocytosis inoculum, and EC(50) and C(s) being 0.2 to 0.3x and 0.6 to 0.9x the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.
The synthesis and properties of six fluorescein-labelled penicillins are reported. The two isomers of fluoresceyl-glycyl-6-amino-penicillanic acid are probably the best compounds to use for detection of all the penicillin-binding proteins (PBPs) present in a bacterial membrane preparation. However, the derivatives of ampicillin were much more efficient against Enterobacter aerogenes PBP3. The two isomers obtained when a commercial mixture of the two isomers of carboxyfluorescein was used most often exhibited similar properties, but the Streptomyces R61 extracellular DD-peptidase was only efficiently acylated by the 5'-carboxyfluorescein derivative of glycyl-6-aminopenicillanic acid.
The gene pbpC from Staphylococcus aureuswas sequenced: it encodes a 691-amino-acid protein with all of the conserved motifs of a class B high-molecular-weight penicillin-binding
protein (PBP), including the transpeptidase conserved motifs SXXK, SXN, and KTG. Insertional inactivation of pbpC and introduction of the intact gene in a laboratory mutant missing PBP 3 showed that thepbpC gene encodes the staphylococcal PBP 3. Inactivation ofpbpC caused no detectable change in the muropeptide composition of cell wall peptidoglycan and had only minimum, if any, effect
on growth rates, but caused a small but significant decrease in rates of autolysis. Cells of abnormal size and shape and disoriented
septa were produced when bacteria with inactivated pbpCwere grown in the presence of a sub-MIC of methicillin.
Transition state analogue boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates for the TEM-1 beta-lactamase of Escherchia coli were designed using graphic analyses based on the enzyme's 1.7 A crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1) and (1R)-1-acetamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (2), is reported. Kinetic measurements show that, as designed, compounds 1 and 2 are highly effective deacylation transition state analogue inhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent competitive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1, K(I) = 5.9 nM), which resembles most closely the best known substrate of TEM-1, benzylpenicillin (penicillin G). The high-resolution crystallographic structures of these two inhibitors covalently bound to TEM-1 are also described. In addition to verifying the design features, these two structures show interesting and unanticipated changes in the active site area, including strong hydrogen bond formation, water displacement, and rearrangement of side chains. The structures provide new insights into the further design of this potent class of beta-lactamase inhibitors.
Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.
The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control of the inducible Pspac promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by
inhibition of transcription of the pbp2gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism
of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated
for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.
Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.
The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome
this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics
is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase
(TP) activity is irreversibly inhibited by β-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential
target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble
extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry
and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed
an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited
by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified
functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif
of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.
Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single d-Ala,d-Ala (d,d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 A resolution. PBP3 folds into an NH(2)-terminal, d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated beta-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel beta-sheet surrounded by alpha-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (k(cat)/K(m) = 50,500 M(-1) x s(-1)), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to "skid" on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.
Bacterial cell division is a complex, multimolecular process that requires biosynthesis of new peptidoglycan by penicillin-binding proteins (PBPs) during cell wall elongation and septum formation steps. Streptococcus pneumoniae has three bifunctional (class A) PBPs that catalyze both polymerization of glycan chains (glycosyltransfer) and cross-linking of pentapeptidic bridges (transpeptidation) during the peptidoglycan biosynthetic process. In addition to playing important roles in cell division, PBPs are also the targets for β-lactam antibiotics and thus play key roles in drug-resistance mechanisms. The crystal structure of a soluble form of pneumococcal PBP1b (PBP1b*) has been solved to 1.9 Å, thus providing previously undescribed structural information regarding a class A PBP from any organism. PBP1b* is a three-domain molecule harboring a short peptide from the glycosyltransferase domain bound to an interdomain linker region, the transpeptidase domain, and a C-terminal region. The structure of PBP1b* complexed with β-lactam antibiotics reveals that ligand recognition requires a conformational modification involving conserved elements within the cleft. The open and closed structures of PBP1b* suggest how class A PBPs may become activated as novel peptidoglycan synthesis becomes necessary during the cell division process. In addition, this structure provides an initial framework for the understanding of the role of class A PBPs in the development of antibiotic resistance.
• antibiotic
• cell wall
• transpeptidase
• glycosyltransferase
Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.
Manual intervention is usually required in the multiple rounds of refinement of protein crystal structures, including linking and/or extending the fragments of the initial model and rebuilding (fitting) ill-matched residues using computer-graphics software. Such manual modification is both time-consuming and requires a great deal of expertise in crystallography. Consequently, the refinement process becomes the bottleneck for high-throughput structure analysis. A program, Local correlation coefficient-based Automatic FItting for REfinement (LAFIRE), has been developed to achieve manual intervention-free refinement. This program was designed to perform the entire process of protein structural refinement automatically using the refinement programs CNS1.1 (CNS v.1.1) or REFMAC5. The automatic process begins from an initial model, which can be approximate, fragmentary or even only main-chain, and refines it to the final model including water molecules, controlled by monitoring the R(free) factor. More than 30 structures have now been refined successfully in a fully or semi-automatic manner within a few hours or days using LAFIRE.
We constructed a conditional mutant of pbpA in which transcription of the gene was placed under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter in order to explore the role of PBP1 in growth, cell wall structure, and cell division.
A methicillin-resistant strain and an isogenic methicillin-susceptible strain, each carrying the pbpA mutation, were unable to grow in the absence of the inducer. Conditional mutants of pbpA transferred into IPTG-free medium underwent a four- to fivefold increase in cell mass, which was not accompanied by a proportional
increase in viable titer. Examination of thin sections of such cells by transmission electron microscopy or fluorescence microscopy
of intact cells with Nile red-stained membranes showed a morphologically heterogeneous population of bacteria with abnormally
increased sizes, distorted axial ratios, and a deficit in the number of cells with completed septa. Immunofluorescence with
an antibody specific for PBP1 localized the protein to sites of cell division. No alteration in the composition of peptidoglycan
was detectable in pbpA conditional mutants grown in the presence of a suboptimal concentration of IPTG, which severely restricted the rate of growth,
and the essential function of PBP1 could not be replaced by PBP2A present in methicillin-resistant cells. These observations
suggest that PBP1 is not a major contributor to the cross-linking of peptidoglycan and that its essential function must be
intimately integrated into the mechanism of cell division.
Beta-lactam antibiotics, including penicillins and cephalosporins, inhibit penicillin-binding proteins (PBPs), which are essential for bacterial cell wall biogenesis. Pathogenic bacteria have evolved efficient antibiotic resistance mechanisms that, in Gram-positive bacteria, include mutations to PBPs that enable them to avoid beta-lactam inhibition. Lactivicin (LTV; 1) contains separate cycloserine and gamma-lactone rings and is the only known natural PBP inhibitor that does not contain a beta-lactam. Here we show that LTV and a more potent analog, phenoxyacetyl-LTV (PLTV; 2), are active against clinically isolated, penicillin-resistant Streptococcus pneumoniae strains. Crystallographic analyses of S. pneumoniae PBP1b reveal that LTV and PLTV inhibition involves opening of both monocyclic cycloserine and gamma-lactone rings. In PBP1b complexes, the ring-derived atoms from LTV and PLTV show a notable structural convergence with those derived from a complexed cephalosporin (cefotaxime; 3). The structures imply that derivatives of LTV will be useful in the search for new antibiotics with activity against beta-lactam-resistant bacteria.
Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information.
To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a co-valently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for-growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of ct multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.
Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as β-lactams.Keywords (keywords): Penicillin binding proteins; boronic acids; antibiotics; antibiotic-resistance; β-lactams; transpeptidase-inhibition
For a successful analysis of the relation between amino acid sequence and protein structure, an unambiguous and physically meaningful definition of secondary structure is essential. We have developed a set of simple and physically motivated criteria for secondary structure, programmed as a pattern-recognition process of hydrogen-bonded and geometrical features extracted from x-ray coordinates. Cooperative secondary structure is recognized as repeats of the elementary hydrogen-bonding patterns “turn” and “bridge.” Repeating turns are “helices,” repeating bridges are “ladders,” connected ladders are “sheets.” Geometric structure is defined in terms of the concepts torsion and curvature of differential geometry. Local chain “chirality” is the torsional handedness of four consecutive Cα positions and is positive for right-handed helices and negative for ideal twisted β-sheets. Curved pieces are defined as “bends.” Solvent “exposure” is given as the number of water molecules in possible contact with a residue. The end result is a compilation of the primary structure, including SS bonds, secondary structure, and solvent exposure of 62 different globular proteins. The presentation is in linear form: strip graphs for an overall view and strip tables for the details of each of 10.925 residues. The dictionary is also available in computer-readable form for protein structure prediction work.
PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 Å resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the “x” of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between β5 and α11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or β-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.
The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.
To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.
The bacterial cell wall is a complex three-dimensional structure that protects the cell from environmental stress and ensures its shape. The biosynthesis of its main component, the peptidoglycan, involves the coordination of activities of proteins present in the cytoplasm, the membrane, and the periplasm, some of which also interact with the bacterial cytoskeleton. The sheer complexity of the cell wall elongation process, which is the main focus of this review, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. The availability of new structural and biochemical data on a number of components of peptidoglycan assembly machineries, including a complex between MreB and RodZ as well as structures of penicillin-binding proteins (PBPs) from a number of pathogenic species, now provide novel insight into the underpinnings of an intricate molecular machinery.
Lactivicin (LTV) is a natural non-beta-lactam antibiotic that inhibits penicillin-binding proteins and serine beta-lactamases. A crystal structure of a BS3-LTV complex reveals that, as for its reaction with PBPs, LTV reacts with the nucleophilic serine and that cycloserine and lactone rings of LTV are opened. This structure, together with reported structures of PBP1b with lactivicins, provides a basis for developing improved lactivicin-based gamma-lactam antibiotics.
The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of <20 microM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C(6) hydroxyethyl group. To improve our understanding of the interactions between the beta-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the beta-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADC beta-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R(2) side group and also suggest that elimination of the C(6) hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new beta-lactamase inhibitors against these critical drug resistance targets.
The LIGPLOT program automatically generates schematic 2-D representations of protein-ligand complexes from standard Protein
Data Bank file input The output is a colour, or black-and-white, PostScript file giving a simple and informative representation
of the intermolecular interactions and their strengths, including hydrogen bonds, hydrophobic interactions and atom accessibilities.
The program is completely general for any ligand and can also be used to show other types of interaction in proteins and nucleic
acids. It was designed to facilitate the rapid inspection of many enzyme complexes, but has found many other applications
An algorithm has been developed for the automatic interpretation of a given set of observed reciprocal-lattice points. It extracts a reduced cell and assigns indices to each reflection by a graph-theoretical implementation of the local indexing method. All possible symmetries of the observed lattice compatible with the metric of the reduced cell are recognized and reported, together with the unit-cell constants and the linear index transformation relating the conventional to the reduced cell. This algorithm has been incorporated into the program XDS [Kabsch (1988). J. Appl. Cryst.21, 916–924], which is now able to process single-crystal area-detector data without prior knowledge of the symmetry and the unit-cell constants.
Several new antimicrobials demonstrate in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and other Gram-positive bacteria. Data from large surveys indicate that linezolid, daptomycin, and tigecycline are almost universally active against MRSA. Linezolid and tigecycline inhibit both Enterococcus faecium and Enterococcus faecalis at low concentrations; daptomycin is somewhat more potent against the latter. The investigational agents dalbavancin and telavancin are more potent than vancomycin against vancomycin-susceptible organisms. Dalbavancin inhibits vanB type VRE at low concentrations, but is not active against vanA type VRE. Telavancin is less active against VRE than against vancomycin-susceptible enterococci, but minimum inhibitory concentrations are lower than those of vancomycin against VRE. With continued careful use of available antimicrobials, the vast majority of these organisms should remain susceptible to 1 or more of the agents discussed for the foreseeable future.
The continued evolution of antimicrobial resistance in the hospital and more recently in the community threatens to seriously compromise our ability to treat serious infections. The major success of the seven-valent Streptococcus pneumoniae vaccine at reducing both infection and resistance has been followed by the emergence of previously minor serotypes that express multiresistance. The almost universal activity of cephalosporins and fluoroquinolones against community Escherichia coli strains has been compromised by the spread of CTX-M beta-lactamase-producing, fluoroquinolone-resistant strains, and the emergence of community-onset methicillin-resistant Staphylococcus aureus, particularly in the United States, has forced us to re-think our empirical treatment guidelines for skin and soft-tissue infections. Finally, our most potent and reliable class of antibiotics, the carbapenems, is compromised by the growth, primarily in intensive care units, of multiresistant Klebsiella pneumoniae, Acinetobacter baumanni, and Pseudomonas aeruginosa. The lack of a robust pipeline of new agents, particularly against resistant Gram-negative bacteria, emphasizes the importance of optimizing our use of current antimicrobials and promoting strict adherence to established infection control practices.
Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent in hospitals and the community. New antibiotics
are needed to combat these bacterial pathogens, but progress in developing them has been slow. Historically, most antibiotics
have come from a small set of molecular scaffolds whose functional lifetimes have been extended by generations of synthetic
tailoring. The emergence of multidrug resistance among the latest generation of pathogens suggests that the discovery of new
scaffolds should be a priority. Promising approaches to scaffold discovery are emerging; they include mining underexplored
microbial niches for natural products, designing screens that avoid rediscovering old scaffolds, and repurposing libraries
of synthetic molecules for use as antibiotics.
Penicillin-binding proteins (PBPs) catalyse the synthesis of cell wall peptidoglycan. PBP1 of Staphylococcus aureus is a high-molecular-weight monofunctional transpeptidase (TPase) and previous studies with a conditional mutant showed that this protein was essential for bacterial growth and survival: cells in which PBP1 was depleted stopped dividing but continued to enlarge in size, accompanied by rapid loss of viability. Also, cell walls produced under PBP1 depletion appeared to have normal composition. We describe here construction of a second PBP1 mutant in which the active site of the TPase domain was inactivated. Cells in which the wild-type PBP1 was replaced by the mutant protein were able to initiate and complete septa and undergo at least one or two cell divisions after which growth stopped accompanied by inhibition of cell separation, downregulation in the transcription of the autolytic system and production of cell walls with increased proportion of monomeric and dimeric muropeptides and decrease in oligomeric muropeptides. PBP1 seems to perform a dual role in the cell cycle of S. aureus: as a protein required for septation and also as a transpeptidase that generates a critical signal for cell separation at the end of cell division.
Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.
In the search for new beta-lactam antibiotics of natural origin, the discoveries of cephamycins and sulfazecins (monobactams) were important turning points in that they accelerated many screening efforts aimed at other new compounds. In our target-directed screening for beta-lactam antibiotics using beta-lactam hypersensitive mutants, we have examined Gram-negative bacteria isolated from natural habitats and have recently reported several types of beta-lactam antibiotics such as cephabacins and formadicins. Here we report a novel antibiotic, lactivicin, found using this system. Although lactivicin has various biological activities commonly observed in beta-lactam antibiotics, it does not possess a beta-lactam ring in its molecule, but has the unique structure of a dicyclic dipeptide.
The expression of beta-lactamases is the most common form of bacterial resistance to beta-lactam antibiotics. To combat these enzymes, agents that inhibit (e.g. clavulanic acid) or evade (e.g. aztreonam) beta-lactamases have been developed. Both the beta-lactamase inhibitors and the beta-lactamase-resistant antibiotics are themselves beta-lactams, and bacteria have responded to these compounds by expressing variant enzymes resistant to inhibition (e.g. IRT-3) or that inactivate the beta-lactamase-resistant antibiotic (e.g. TEM-10). Moreover, these compounds have increased the frequency of bacteria with intrinsically resistant beta-lactamases (e.g. AmpC). In an effort to identify non-beta-lactam-based beta-lactamase inhibitors, we used the crystallographic structure of the m-aminophenylboronic acid-Escherichia coli AmpC beta-lactamase complex to suggest modifications that might enhance the affinity of boronic acid-based inhibitors for class C beta-lactamases. Several types of compounds were modeled into the AmpC binding site, and a total of 37 boronic acids were ultimately tested for beta-lactamase inhibition. The most potent of these compounds, benzo[b]thiophene-2-boronic acid (36), has an affinity for E. coli AmpC of 27 nM. The wide range of functionality represented by these compounds allows for the steric and chemical "mapping" of the AmpC active site in the region of the catalytic Ser64 residue, which may be useful in subsequent inhibitor discovery efforts. Also, the new boronic acid-based inhibitors were found to potentiate the activity of beta-lactam antibiotics, such as amoxicillin and ceftazidime, against bacteria expressing class C beta-lactamases. This suggests that boronic acid-based compounds may serve as leads for the development of therapeutic agents for the treatment of beta-lactam-resistant infections.
Background:
Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these beta-lactams, most often through bacterial expression of beta-lactamases, threatens public health. To understand how beta-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because beta-lactams form covalent adducts with beta-lactamases. This has complicated functional analyses and inhibitor design.
Results:
To investigate the contribution to interaction energy of the key amide (R1) side chain of beta-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine beta-lactamases. Therefore, binding energies can be calculated directly from K(i) values. The K(i) values measured span four orders of magnitude against the Group I beta-lactamase AmpC and three orders of magnitude against the Group II beta-lactamase TEM-1. The acylglycineboronic acids have K(i) values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of beta-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of beta-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to beta-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 A and 1.75 A resolution; these structures suggest interactions that are important to the affinity of the inhibitors.
Conclusions:
Acylglycineboronic acids allow us to begin to dissect interaction energies between beta-lactam side chains and beta-lactamases. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in beta-lactam inhibitors or beta-lactam substrates of serine beta-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series.
The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.
This paper reviews the mathematical basis of maximum likelihood. The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estimating sigma(A) using 'free' reflections is described and its effects analysed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial alpha-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement.
Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.
High-throughput screening (HTS) campaigns can be dominated by hits that ultimately turn out to be non-drug-like. These "nuisance" compounds often behave strangely, with steep dose-response curves, absence of clear structure-activity relationships, and high sensitivity to assay conditions. Several mechanisms contribute to these artifacts, including chemically reactive molecules, those that absorb light in assays and those that affect redox conditions. One of the most common mechanisms behind artifactual inhibition is discussed in this review: at micromolar concentrations organic molecules can aggregate to form particles in aqueous buffers, and these aggregates can sequester and thereby inhibit protein targets. Aggregation-based inhibition is baffling from a chemical perspective, but viewed biophysically such behavior is expected. The range of molecules that behave this way, their rapid detection in a screening environment and their possible biological implications will be considered here.
At micromolar concentrations, many small molecules self-associate into colloidal aggregates that non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor inhibition of beta-lactamase in the absence and presence of detergent. Inhibition that is attenuated in the presence of detergent is characteristic of an aggregate-based mechanism. In the 96-well-format assay described here, about 200 molecules can be tested, in duplicate, per hour for detergent-dependent sensitivity. Furthermore, we also describe simple experiments that can offer additional confirmation of aggregate-based inhibition.
A phase 2 trial was performed to study the combination of bortezomib (VELCADE) with intermediate-dose dexamethasone (DEX), and continuous low-dose oral cyclophosphamide (CY) in patients with relapsed multiple myeloma (MM). Fifty-four patients with advanced MM were enroled to receive eight 3-week treatment cycles with bortezomib 1.3 mg/m(2) on days 1, 4, 8, and 11, followed by three 5-week cycles with bortezomib 1.3 mg/m(2) on days 1, 8, 15, and 22. Within all cycles, DEX 20 mg/d was given orally on the day of bortezomib injection and the day thereafter. In addition, patients received CY continuous oral treatment at a dose of 50 mg/d p.o. once daily. Fifty patients completing at least one treatment cycle were evaluable for response. Complete, partial, and minor responses occurred in 16%, 66% and 8% of patients, respectively; overall response rate 90% (efficacy analysis). Median event-free survival was 12 months, with a median overall survival of 22 months. Adverse events (AE) of grades 3 or 4 occurring in at least 10% of patients comprised leucopenia, infection, herpes zoster, thrombocytopenia, neuropathy and fatigue. Bortezomib combined with DEX and CY is a highly effective treatment for relapsed MM at an acceptable rate of grade 3/4 AE. Antiviral prophylaxis appears to be mandatory.
Experiences with the molecular-replacement program Beast have shown that maximum-likelihood rotation targets are more sensitive to the correct orientation than traditional targets. However, this comes at a high computational cost: brute-force rotation searches can take hours or even days of computation time on current desktop computers. Series approximations to the full likelihood target have been developed that can be computed by fast Fourier transforms in minutes. These likelihood-enhanced targets are more sensitive to the correct orientation than the Crowther fast rotation function and they take advantage of information from partial solutions. The likelihood-enhanced rotation targets have been implemented in the program Phaser.
Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of beta-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a "trapped" covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-D-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the D-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of beta-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of beta-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.
Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.