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Mechanism of inhibition by e29. A) Relative to NADPH, e29 is an uncompetitive inhibitor of the purified Y. pestis MEP synthase. B) Relative to DXP, e29 is a noncompetitive inhibitor. C) A model of e29 inhibition. MEP synthase (E) undergoes a conformational change (E*) upon binding of NADPH (N), exposing an allosteric site to which the inhibitor (I) binds. As the inhibitor is noncompetitive with respect to DXP (D), I may bind the E*N or E*ND complex, thereby inhibiting the enzyme. doi:10.1371/journal.pone.0106243.g017
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The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated b...
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... The resulting mevalonate pyrophosphate undergoes decarboxylation to get IPP by an enzyme called mevalonate Essential Oil-Derived Monoterpenes in Drug Discovery and Development 56 57 58 59 60 pyrophosphate decarboxylate. The conversion of IPP to DMAPP is enabled by isopentyl pyrophosphate isomerase (Baser and Demirci 2007;Lange and Ahkami 2013;Zuzarte and Salgueiro 2015;Haymond et al. 2014;Zebec et al. 2016). ...
... Finally, the enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase or isoprenoid synthesis H (IspH) converts (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) to both IPP and DMAPP. A head-to-tail condensation of IPP with DMAPP in the presence of prenyltransferase forms geranyl diphosphate or geranyl pyrophosphate (GPP, C 10 ), the common precursor for monoterpenes (Baser and Demirci 2007;Lange and Ahkami 2013;Zuzarte and Salgueiro 2015;Haymond et al. 2014;Zebec et al. 2016). ...
Essential oils are complex mixtures of plant secondary metabolites composed mostly of terpenoids, aliphatic and aromatic hydrocarbons, and their derivatives such as aldehydes, ketones, alcohols, and esters. They play important roles as defense compounds against microbes, herbivores, and other ecological stress factors according to their structural designs. Among the secondary metabolites, monoterpenes (C10) form the major group. Several reports have shown that both natural monoterpenes and their synthetic derivatives exhibit a wide range of biological activities. In this chapter, a review of the anti-inflammatory, analgesic, antitumor, anticonvulsant, cardioprotective, gastroprotective, wound-healing, antifungal, antibacterial, and antiviral properties of different classes of monoterpenes is discussed.KeywordsPlant secondary metabolitesEssential oilsMEP pathwayMVA pathwayMonoterpenoidsAcyclic monoterpenes
... The disease mostly manifests in two forms: the bubonic and pneumonic plague [55]. Both fosmidomycin (IC 50 = 0.71 µM) and FR900098 (IC 50 = 0.23 µM) showed submicromolar inhibitory activity [56]. Both agents lacked the ability to inhibit the growth of Y. pestis, even though uptake of fosmidomycin was likely mediated by a transport protein homologous to the E. coli GlpT transporter [57]. ...
... The synthesized derivatives are structurally diverse. These modifications encompass alterations of the linker length (38)(39)(40)(41), insertion of a double bond (42)(43)(44)(45)(46) or hetero atoms (47)(48)(49)(50)(51)(52)(53)(54), restriction of the linker flexibility (55)(56)(57)(58)(59)(60) and substitution of the linker in the α-, β-and γ-position. ...
... The synthe derivatives are structurally diverse. These modifications encompass alterations o linker length (38)(39)(40)(41), insertion of a double bond (42)(43)(44)(45)(46) or hetero atoms (47 restriction of the linker flexibility (55)(56)(57)(58)(59)(60) and substitution of the linker in the α-, β-a position. ...
To address the continued rise of multi-drug-resistant microorganisms, the development of novel drugs with new modes of action is urgently required. While humans biosynthesize the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the established mevalonate pathway, pathogenic protozoa and certain pathogenic eubacteria use the less well-known methylerythritol phosphate pathway for this purpose. Important pathogens using the MEP pathway are, for example, Plasmodium falciparum, Mycobacterium tuberculosis, Pseudomonas aeruginosa and Escherichia coli. The enzymes of that pathway are targets for antiinfective drugs that are exempt from target-related toxicity. 2C-Methyl-D-erythritol 4-phosphate (MEP), the second enzyme of the non-mevalonate pathway, has been established as the molecular target of fosmidomycin, an antibiotic that has so far failed to be approved as an anti-infective drug. This review describes the development and anti-infective properties of a wide range of fosmidomycin derivatives synthesized over the last four decades. Here we discuss the DXR inhibitor pharmacophore, which comprises a metal-binding group, a phosphate or phosphonate moiety and a connecting linker. Furthermore, non-fosmidomycin-based DXRi, bisubstrate inhibitors and several prodrug concepts are described. A comprehensive structure–activity relationship (SAR) of nearly all inhibitor types is presented and some novel opportunities for further drug development of DXR inhibitors are discussed.
... Generally, the recombinant A. baumannii IspC and K. pneumoniae IspC have K M to those reported for homologous enzymes from other organisms (Table 2). The apparent specificity constant (K cat DXP /K M DXP ) of both A. baumannii and K. pneumoniae IspC are approximately 2-fold higher than that reported for Yersinia pestis IspC, approximately 30-fold lower than that reported for the E. coli enzyme, and approximately 2fold lower than those reported for the Francisella tularensis and Mycobacterium tuberculosis enzymes(Table 2).IspC requires a divalent cation−generally Mg 2+ or Mn 2+ − for catalysis.35,57,73 Like many other IspC enzymes,35,57,73 evaluation of enzyme activity in the presence of various divalent cations reveals that recombinant AbIspC and KpIspC preferentially use Mg 2+ ...
... The apparent specificity constant (K cat DXP /K M DXP ) of both A. baumannii and K. pneumoniae IspC are approximately 2-fold higher than that reported for Yersinia pestis IspC, approximately 30-fold lower than that reported for the E. coli enzyme, and approximately 2fold lower than those reported for the Francisella tularensis and Mycobacterium tuberculosis enzymes(Table 2).IspC requires a divalent cation−generally Mg 2+ or Mn 2+ − for catalysis.35,57,73 Like many other IspC enzymes,35,57,73 evaluation of enzyme activity in the presence of various divalent cations reveals that recombinant AbIspC and KpIspC preferentially use Mg 2+ ...
The ESKAPE pathogens comprise a group of multidrug-resistant bacteria that are the leading cause of nosocomial infections worldwide. The prevalence of antibiotic resistant strains and the relative ease by which bacteria acquire resistance genes highlight the continual need for the development of novel antibiotics against new drug targets. The methylerythritol phosphate (MEP) pathway is an attractive target for the development of new antibiotics. The MEP pathway governs the synthesis of isoprenoids, which are key lipid precursors for vital cell components such as ubiquinone and bacterial hopanoids. Additionally, the MEP pathway is entirely distinct from the corresponding mammalian pathway, the mevalonic acid (MVA) pathway, making the first committed enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), an attractive target for antibiotic development. To facilitate drug development against two of the ESKAPE pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, we cloned, expressed, purified, and characterized IspC from these two Gram-negative bacteria. Enzyme inhibition assays using IspC from these two pathogens, and compounds fosmidomycin and FR900098, indicate IC50 values ranging from 19.5-45.5 nM. Antimicrobial susceptibility tests with these inhibitors reveal that A. baumannii is susceptible to FR900098, whereas K. pneumoniae is susceptible to both compounds. Finally, to facilitate structure-based drug design of inhibitors targeting A. baumannii IspC, we determined the 2.5 Å crystal structure of IspC from A. baumannii in complex with inhibitor FR900098, and cofactors NADPH and magnesium.
... The synthesis of compounds 4a, 1c, 2c, 3c, and 1d was performed, as described previously. 12,13,17,18 The synthesis of compound 3a is shown in Scheme 1. Briefly, acylation of compound 5 10 was achieved using valeryl chloride and TEA to afford compound 6. ...
... The cloning, expression, and purification of P. falciparum IspC were performed, as described previously. 10,12,17 Briefly, the P. falciparum IspC gene was cloned into a pET100/D-TOPO vector to facilitate the expression of an N-terminal His 6 -tagged protein. The recombinant protein was expressed in Escherichia coli Rosetta2(DE3) cells obtained from Novagen (San Diego, CA). ...
... The IspC activity was assayed at 37°C by spectrophotometrically monitoring the enzyme-catalyzed oxidation of NADPH, as described previously. 12,13,17 Briefly, the assay mixture contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , and 0.86 μM P. falciparum IspC. To determine the half-maximal inhibition (IC 50 ) concentrations, the enzyme was preincubated with the inhibitor for 10 min at 37°C ...
Malaria is a global health threat that requires immediate attention. Malaria is caused by the protozoan parasite Plasmodium, the most severe form of which is Plasmodium falciparum. The methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential to the survival of many human pathogens, including P. falciparum, but is absent in humans, and thus shows promise as a new antimalarial drug target. The enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the MEP pathway. In addition to a divalent cation (Mg²⁺), the enzyme requires the substrates 1-deoxy-D-xylulose 5-phosphate (DXP) and NADPH to catalyze its reaction. We designed N-alkoxy and N-acyl fosmidomycin analogs to inhibit the activity of P. falciparum IspC in a bisubstrate manner. Enzyme assays reveal that the N-alkoxy fosmidomycin analogs have a competitive mode of inhibition relative to both the DXP- and NADPH-binding sites, confirming a bisubstrate mode of inhibition. In contrast, the N-acyl fosmidomycin analogs demonstrate competitive inhibition with respect to DXP but uncompetitive inhibition with respect to NADPH, indicating monosubstrate inhibitory activity. Our results will have a positive impact on the discovery of novel antimalarial drugs.
... 15 Furthermore, we demonstrated the effectiveness of inhibiting both the purified Y. pestis enzyme (YpIspC) and liquid cultures of Y. pestis using the known IspC inhibitor, fosmidomycin ( Figure 1). 14 Previous studies have shown that α-phenyl substitutions of reverse derivatives of fosmidomycin are efficacious. 16−18 Furthermore, crystal structures of IspC-and α-phenylsubstituted reverse derivatives of fosmidomycin have been resolved. ...
Fosmidomycin inhibits IspC (1-deoxy-d-xylulose 5-phosphate reductoisomerase), the first committed enzyme in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. The MEP pathway of isoprenoid biosynthesis is essential to the causative agent of the plague, Yersinia pestis, and is entirely distinct from the corresponding mammalian pathway. To further drug development, we established structure–activity relationships of fosmidomycin analogues by assessing a suite of 17 α-phenyl-substituted reverse derivatives of fosmidomycin against Y. pestis IspC. Several of these compounds showed increased potency over fosmidomycin with IC50 values in the nanomolar range. Additionally, we performed antimicrobial susceptibility testing with Y. pestis A1122 (YpA1122). The bacteria were susceptible to several compounds with minimal inhibitory concentration (MIC) values ranging from 128 to 512 μg/mL; a correlation between the IC50 and MIC values was observed.
... P. falciparum DXR activity was assayed at 37°C by spectrophotometrically monitoring the enzyme catalyzed oxidation of NADPH upon addition of 1-deoxy-D-xylulose 5-phosphate (DOXP; Echelon Biosciences, Salt Lake City, UT) to the assay mixture, as described previously. 47,48 Briefly, the assay system contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 0.86 μM Pf DXR, and 150 μM NADPH. The reaction was initiated by adding 144 μM DOXP to the complete assay mixture. ...
Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality, as well as its absence in humans. In this study, we designed and synthesized a series of α,β-unsaturated analogs of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC50 = 13 nM) without significant inhibition of HepG2 cells (IC50 > 50 μM). 18a was also tested in a luciferase-based P. berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.
... In this report, we describe a bench scale high throughput screening (HTS) campaign using purified, recombinant IspC and IspD, a commercially available 1280 compound molecular library, and a 150 sample, in-house prepared, natural product extract library [16]. We report a screening protocol for both IspC and IspD that minimizes the number of assays required while effectively identifying false positive compounds. ...
... Subsequently, 45 μL of assay master mix, containing 100 mM Tris pH 7.8, 25 mM MgCl 2 , 150 μM NADPH, and 0.89 μM YpIspC was added to each well and the plate was allowed to incubate at room temperature for 10 minutes to facilitate inhibitor binding. The reaction was initiated with the addition of the K m value of DXP (252 μM [16]), and was subsequently quenched after 1 minute with the addition of EDTA to a final concentration of 100 mM. Decrease in A 340 as compared to wells without DXP was measured using a Beckman Coulter DTX800 plate reader. ...
The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.
... 3A, the insert) with an IC 50 of 0.27 μ M. These data are in good agreement with the previously published results5,15 . ...
1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 μM) and theaflavin-3,3′-digallate exhibiting the highest (IC50 14.9 μM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments.
... Our work has recently focused on two organisms, Mycobacterium tuberculosis (Mtb), causing tuberculosis (TB), and Yersinia pestis (Yp), causing plague (or black death). (6)(7)(8)(9)(10) TB is still responsible for nearly 2 million deaths each year and threatens public health in both developed and developing countries. (11)(12)(13) Gram-negative Yersinia pestis, a bacterial cousin of Mtb, continues to infect individuals worldwide. ...
... (8) We refer to these compounds collectively as MEPicides (inhibitors of the MEP pathway displaying antimicrobial activity). While a small set of compounds from these series has been reported, (8,9,34) we describe here an expanded set of compounds from both chemical classes, SAR of these compounds against Dxr from Mtb and Yp, and antibacterial activities of these compounds against Mtb, Yp, and E. coli. ...
... In other words, the longer/ more flexible phenpropyl substituent of 8h may adopt an alternate binding pattern compared with the smaller CH 2 -2-naphthyl (16j) and phenyl (8e) substituents, which are more constrained. (9) suggesting that the enhanced potency of the inhibitors towards the Yp Dxr predominantly reflects an enhanced ability of the inhibitor to associate with its NADPH binding site. ...
Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. Mycobacterium tuberculosis, the causative agent of tuberculosis, and Yersinia pestis, resulting in the plague or "black death," both rely on the MEP pathway for isoprene production. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended N-acyl or O-linked alkyl/aryl group, and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homolog, with the best analogs displaying nM IC50 values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and diPOM prodrug esters of these compounds were made. While the added lipophilicity did not enhance Yersinia activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3-12 ?g/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologs from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development.
... Another approach has been tried by Haymond et al. (2014), where several compounds from resolved crystal structures of M. tuberculosis MEP synthase in complex with Fos were designed, containing an amide-linked or O-linked functional group. The strategy was to target two major binding sites in MEP synthase; the Fos/DXP site and the NADPH site, bridging these adjacent sites to yield a highly specific inhibitor ligand. ...
