No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Probing the dual inhibitory mechanisms of novel thiophenecarboxamide derivatives against Mycobacterium tuberculosis PyrG and PanK: an insight from biomolecular modeling study
Abstract
The growing occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (Mtb) strains underscores an urgent need for new antibiotics. The development of more bioactive antibiotics against drug-resistant organisms with a different mode of action could be a game-changer for the cure and eradication of tuberculosis (TB). Pantothenate Kinase (PanK) and CTP synthetase (PyrG) are both essential for RNA, DNA, and Lipids biosynthesis pathways. Given the extensive knowledge on these biosynthesis pathways inhibition of Mtb growth and survival, these enzymes present a fascinating opportunity for anti-mycobacterial drug discovery. Recently, it was experimentally established that the active metabolite 11426026 of compound 7947882 (a prodrug activated by EthA monooxygenase, 5-methyl-N-(4-nitrophenyl) thiophene-2-carboxamide) inhibits the activities of PyrG and PanK to indicate novel multitarget therapy aimed at discontinuing Mtb growth. However, the molecular mechanisms of their selective inhibition remain subtle. In this work, molecular dynamics simulations were employed to investigate the inhibitory mechanism as well as the selectivity impact of the active metabolite inhibitor of these enzymes. Computational modeling of the studied protein-ligand systems reveals that the active metabolite can potentially inhibit both PanK and PyrG, thereby creating a pathway as a double target approach in tuberculosis treatment.
Communicated by Ramaswamy H. Sarma
... Earlier TB remedy involves medicines such as isoniazid, rifampicin, ethambutol, and pyrazinamide. Unfortunately, the emergence of M. tuberculosis resistance to the available therapies has hindered TB management [3,5,6]. ...
... Although the binding affinity of ZINC53995076 is lower than 11426026, it does not override the possibility of ZINC53995076 as a potential inhibitor of the PanK and PyrG enzymes. Recall that 11426026 is a metabolite of 7947882 [6,19,53] that requires activation by monooxygenase EthA through the Baeyer-Villiger mechanism. However, the emergence of mutations in the EthA gene led to the loss of function in EthA (mutations) [6,19,53], which in turn becomes less efficient to activate the prodrug (7947882) to the active metabolite (11426026). ...
... Recall that 11426026 is a metabolite of 7947882 [6,19,53] that requires activation by monooxygenase EthA through the Baeyer-Villiger mechanism. However, the emergence of mutations in the EthA gene led to the loss of function in EthA (mutations) [6,19,53], which in turn becomes less efficient to activate the prodrug (7947882) to the active metabolite (11426026). This challenge underscores the need for a new direct-acting dual inhibitor for PanK and PyrG enzymes without the mediation of an activator. ...
Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis, responsible for 1.66 million documented deaths in 2020. The increase in reported cases of M. tuberculosis resistance to the main drugs shows the need to develop new and efficient drugs for better TB control. A strategy to overcome the resistance problem could be the polypharmacology approach, whereby researchers develop a single molecule that acts on different targets. Polypharmacology could have features that make it more effective than classical polypharmacy, in which investigators pull various drugs with high affinity for one target together. Based on the effectiveness of multitargeting, this work aimed to use combined in silico techniques to identify potential dual inhibitors for Pantothenate Kinases (PanK) and CTP synthetase PyrG. In silico drug design allows for screening of potential leads, thus decreasing time and resource consumption. This study demonstrates an optimised and proven screening technique to discover a potential small-molecule inhibitor of Mycobacterium tuberculosis PanK and PyrG. The favourable binding free energy values and interaction profiles show ZINC53995076 as a potent dual inhibitor of the Mycobacterium tuberculosis PanK and PyrG.
... Nucleotide synthesis is a vital process, and inhibiting the respective key enzyme may perturb the synthesis of DNA, RNA and other metabolic pathways, which requires nucleotides. In this study, a pyrG gene-encoded enzyme, cytidine triphosphate (CTP) synthase-PyrG, is selected as a therapeutic target for developing anti-tuberculosis drug due to its catalytic activity, which is required for the conversion of uridine triphosphate (UTP) into cytidine triphosphate (CTP) for the biosynthesis of pyrimidine nucleotide (Ejalonibu et al., 2022). As the Mtb is evolving continuously and developing drug resistance strains due to genetic modifications, the strategies and approaches to combat this notorious pathogen also needs improvision. ...
... Therefore, selecting a new drug target and identifying novel antitubercular agents would be promising because Mtb has developed resistance against almost all the available drugs used in its treatment. Recently, CTP synthase -PyrG was targeted by the 5-methyl-N-(4-nitrophenyl) thiophene-2-carboxamide, a prodrug activated by EthA monooxygenase (Ejalonibu et al., 2022;Chiarelli et al., 2018). Therefore, targeting CTP synthase can be a good strategy for combating Mtb infection. ...
Genome evolution of Mycobacterium tuberculosis (Mtb) produces new strains resistant to various pre-existing anti-tubercular drugs. Hence, there is an urgent need to explore potent compounds with the most negligible side effects and effective Mtb inhibition. Mtb PyrG (CTP synthase) is a crucial enzyme for the conversion of the uridine triphosphate (UTP) into cytidine triphosphate (CTP) and is essential for the growth of Mtb. Thus, in this study, phytochemicals of Withania somnifera (W. somnifera) were screened to find the potential inhibitors against Mtb PyrG. Molecular docking resulted in the identification of quercetin 3-rutinoside-7-glucoside, rutin, chlorogenic acid and isochlorogenic acid C with a substantial docking score (from −12.6 to −10.8 kcal/mol) contributed by significant intermolecular interactions. Furthermore, 100 ns molecular dynamics simulation, ADME analysis and free binding energy calculations support the stability of docked complexes and drug-likeness for selected compounds, respectively. Collectively, these findings suggest that phytochemicals present in W. somnifera can be considered for further evaluation against Mtb in a series of in vitro and in vivo models.
Communicated by Ramaswamy H. Sarma
... An additional full minimization of 1000 steps was carried out by unrestrained conjugate gradient. Gradual heating of the systems from 0 to 300 K with a 5 kcal/mol Å harmonic restraint potential and a Langevin thermostat of collision frequency of 1/ps using a canonical ensemble (NVT) molecular dynamics simulation were then carried out [46]. The systems were equilibrated at 300 K in an NPT ensemble for 500 ps without restraint. ...
In March 2013, a novel avian influenza A (H7N9) virus emerged in China. By March 2021, it had infected more than 1500 people, raising concerns regarding its epidemic potential. Similar to the highly pathogenic H5N1 virus, the H7N9 virus causes severe pneumonia and acute respiratory distress syndrome in most patients. Moreover, genetic analysis showed that this avian H7N9 virus carries human adaptation markers in the hemagglutinin and polymerase basic 2 (PB2) genes associated with cross-species transmissibility. Clinical studies showed that a single mutation, neuraminidase (NA) R292K (N2 numbering), induces resistance to peramivir in the highly pathogenic H7N9 influenza A viruses. Therefore, to evaluate the risk for human public health and understand the possible source of drug resistance, we assessed the impact of the NA-R292K mutation on avian H7N9 virus resistance towards peramivir using various molecular dynamics approaches. We observed that the single point mutation led to a distorted peramivir orientation in the enzyme active site which, in turn, perturbed the inhibitor’s binding. The R292K mutation induced a decrease in the interaction among neighboring amino acid residues when compared to its wild-type counterpart, as shown by the high degree of fluctuations in the radius of gyration. MM/GBSA calculations revealed that the mutation caused a decrease in the drug binding affinity by 17.28 kcal/mol when compared to the that for the wild-type enzyme. The mutation caused a distortion of hydrogen bond-mediated interactions with peramivir and increased the accessibility of water molecules around the K292 mutated residue.
Background
The prevalence of drug-resistant organisms has steadily increased over the past few decades worldwide. Especially in tuberculosis (TB) disease, the problems of co-morbidity and the rapid emergence of multidrug resistance have necessitated the development of multitarget-based therapeutic regimens. Several multitargeting compounds against Mycobacterium tuberculosis (Mtb) have been studied through novel in silico tools but these have rendered reduced efficacy in clinical trials. The authors have focussed on many exotic targets belonging to crucial Mtb survival pathways whose molecular structures and functions are underexplored. Likewise, insights into the hidden possibilities of promiscuous compounds from natural products or repurposed drugs to inhibit other cellular proteins apart from their validated targets are also depicted in this review. In addition to the existing line of drugs currently recommended for multidrug-resistant TB, newer host-directed therapies could also be fruitful. Furthermore, several challenges, including safety/efficacy ratios of multitarget compounds highlighted here, can also be circumnavigated by researchers to design “smart drugs” for improved tuberculosis therapeutics.
Conclusion
A holistic approach towards alleviating the existing drawbacks of drug discovery in drug-resistant TB has been outlined. Finally, considering the current needs, the authors have put forward an overall summary of possible trends in multitargeting that are significant for futuristic therapeutic solutions.
Hybrid inorganic/organic compounds opened up the window of opportunities to a wide variety of fields. The carbon-based nanocomposites (Fe3O4/MWCNT-COOH/Extract/[email protected]/p-CRISPR/Extract) have been prepared using tangerine or/and egg white extract as a second layer of nanocomposites. Then, doxorubicin (DOX) and PdC16H10N4O3 (PdL) were added to the mixture simultaneously; based on the last studies, L (carboxamide-based ligand) has a potent desire for connecting and then blocking the HER-2, and σ2 (tumour's overexpressed receptors) and PdL could enhance the sustainability of the DOX by hydrogen bonding and π-π interaction. The effect of biotin on site-specific delivery has been investigated by utilizing two different types of extracts that naturally has a different amount of biotin. Based on this study's results, settling natural extracts (tangerine and egg white), bioactive complexes, and Fe3O4 on the functionalized-MWCNT can strengthen the nanocarrier's stability in the biological matrix, make more cavities, and control the internalization to the cells for drug/gene delivery. Egg white-based nanocarrier has revealed 29.1% ± 1.5% GFP positive transfection efficiency. Also, MTT assay (HEK-293, MCF-7 and PC-12) and antibacterial activity and in vivo testing on the mice (H&E stain and AST:ALT analysis) have been screened.
The cytidine triphosphate synthases (CTPS) are a highly conserved family of enzymes responsible for the de novo synthesis of CTP using UTP, ATP and glutamine as their substrate. The CTP synthases form inactive dimers, and active tetramers in presence of their substrates ATP and UTP as well as of their product CTP. These tetramers can further assemble as massive intracellular structures called filaments whose role in their regulation is still debated. There are two CTP synthases in human: CTPS1 and CTPS2. Previous works published in this lab demonstrated the crucial role of CTPS1 in the immune system, especially during lymphocyte expansion after TCR (T-cell receptor) stimulation in response to pathogen infections. Moreover, numerous anterior studies link increased CTP synthase activity to cancer development and progression. The available data suggest that specifically targeting the activity of CTPS1 could be a relevant therapeutic approach for the treatment of pathologies that include cancers and some immune diseases. We have generated and characterized CTPS1- and CTPS2-deficient cellular models using the HEK 293T and Jurkat cell lines. Jurkat cells do not express CTPS2, and the inactivation of CTPS1 abrogates their CTPS activity and ability to proliferate, leading to apoptosis. CTPS1 and CTPS2 were found to be partially redundant in HEK cells. In CTPS2 knock-out (KO) HEK, no detectable changes in CTPS activity and proliferation were observed. CTPS1-KO HEK were found to have a strongly decreased CTPS activity associated with a moderate decrease in proliferation. CTPS1 and CTPS2-KO HEK were unable to proliferate, yet able to survive a prolonged CTP deprivation despite a null CTPS activity. Exogenous complementation approaches confirmed that both CTPS1 and CTPS2 were able to restore CTPS-deficient cell viability and proliferation. The enzymatic activity of CTPS2 seems lower than that of CTPS1, and CTPS2 appears to have a reduced capacity to complement cellular models deficient in CTP synthases. Finally, our data suggest that CTPS1 and CTPS2 might interact at the tetramer level. The CTPS1-KO Jurkat model was used to study two CTPS1 mutants expressed in patients with immunodeficiency. CTPS1Δ18 results from the mutation of a splice site, leading to exon skipping and to the expression of an alternative C-terminal domain. Our data confirms that CTPS1Δ18 is an hypomorphic, yet active mutant. The second mutant, CTPS1Δ19, for which we had neither functional data nor patient samples, lacks the last 19 residues of the wild-type protein. Although our preliminary data suggests that his mutant is active and more stable than CTPS1Δ18, further studies are required to fully characterize the impact of this mutation. Our cellular models have allowed us to study the impact of mutations previously used in the literature but tested on other species as well as on non-CTPS-deficient models. In particular, we have been interested in their impact on the capacity of CTPS1 to sustain cell proliferation, form filaments and interact with CTPS2. We have also been interested in testing the impact of targeted mutations of CTPS1 expressed in CTP synthase-deficient cellular models on its capacity to sustain cell proliferation, form filaments and interact with CTPS2. This project has been established in collaboration with a company, Step Pharma, who develops CTPS1-specific inhibitors. We have observed that small molecule inhibitors developed by Step Pharma that were shown to be highly selective for human CTPS1 in cellular and enzyme models unexpectedly lost their selectivity on murine enzymes. Through sequence analysis, mutagenesis and generation of cellular models, we have validated the key role of a residue the selectivity for CTPS1 of Step Pharma compounds.
Drug-resistant tuberculosis (TB) infections are on the rise and anti-tuberculosis drugs that inhibit Mycobacterium tuberculosis (M. tuberculosis) through a new novel mechanism could be an important component of evolving TB therapy. Pantothenate kinase (PanK) and CTP synthetase (PyrG) are both essential for de novo pyrimidine biosynthesis. Given the extensive knowledge base on de novo pyrimidine biosynthesis inhibition of M. tuberculosis growth and survival, these enzymes present an interesting opportunity for anti-mycobacterial drug discovery. A recent experimental study shows that CDD-823953 and GSK-735826A act as dual PanK and PyrG inhibitors, respectively. However, the molecular mechanisms of their selective inhibition remain elusive. Herein, density functional theory (DFT) calculation was applied to unveil the molecular and reactivity properties of two lead compounds targeting these enzymes in a shot. Molecular dynamics simulations were then employed to investigate the inhibitory mechanism as well as selectivity impact of these potential inhibitors for their enzymes. Computational modeling of the ligands and the enzyme–ligand systems reveal that CDD-823953 and GSK-735826A lead compounds can potentially inhibit both PanK and PyrG thereby creating a pathway via the use of double target approach in tuberculosis treatment.
Molecular dynamics (MD) simulations of wild-type and V91W mutant Mycobacterium tuberculosis-LprG (Mtb-LprG) were performed with the goal to provide a comprehensive understanding of the Mtb-LprG as a potential antimycobacterial target. A long-range MD simulations and post-MD analyzes led us to various results that plainly explained the impact of V91W mutation on Mtb-LprG. Herein, the results revealed that the wild-type is less stable compared to V91W mutant. This was further supported by root mean square fluctuation, where the V91W mutant showed a higher degree of flexibility compared to the wild-type. Dynamic cross-correlation analysis revealed that induced mutation leads to higher residual flexibility in the mutant structure as compared to the wild-type structure thus resulting in the existence of negatively correlated motions. The difference in principal component analysis scatter plot across the first two normal modes suggests a greater mobility of the V91W mutant conformation compared to the wild-type. Thermodynamic calculations revealed that the van der Waals (Evdw) forces contribute the most towards binding free energy in a case of the V91W mutant as compared to the wild-type LprG complex. In addition, the residue interaction networks revealed more of Evdw interaction existence among residues in case of the V91W mutant. This study supports the Mtb-LprG as a potential antimycobacterial target and also serves as a cornerstone to identifying new potential targets that have no inhibitors.
Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.
The emergence and dissemination of drug-resistant Mycobacterium tuberculosis is a global threat to health. In this report, surveillance of drug-resistant tuberculosis during the past 20 years is described.
To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.
Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Despite the almost 50 yrs since the introduction of curative antituberculosis drugs, Mycobacterium tuberculosis continues to exert an enormous toll on world health, and tuberculosis remains the world's leading cause of death due to a single infectious agent. This has stimulated research efforts into finding new tools to tackle the continuing tuberculosis pandemic. One of the few successes to date has been the development of a new discipline, molecular epidemiology. This has added a further dimension to the classical epidemiology of tuberculosis and enhanced understanding of how M. tuberculosis continues to be successfully transmitted within populations. In the process, inadequacies in tuberculosis control programmes have been identified, helping accumulate resources for their improvement. Other technologies, based on knowledge of the complete genome sequence of M. tuberculosis, which will provide newer tools for probing the epidemiology of tuberculosis, are now emerging. In spite of these advances, tuberculosis continues to remain a devastating infectious disease, disproportionately impacting on the world's poorest countries. The future challenge for molecular epidemiology is to provide better understanding of the transmission dynamics of tuberculosis in these settings and to stimulate the implementation of control measures on a more global scale.
Introduction:
The molecular mechanics energies combined with the Poisson-Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA and MM/GBSA) methods are popular approaches to estimate the free energy of the binding of small ligands to biological macromolecules. They are typically based on molecular dynamics simulations of the receptor-ligand complex and are therefore intermediate in both accuracy and computational effort between empirical scoring and strict alchemical perturbation methods. They have been applied to a large number of systems with varying success.
Areas covered:
The authors review the use of MM/PBSA and MM/GBSA methods to calculate ligand-binding affinities, with an emphasis on calibration, testing and validation, as well as attempts to improve the methods, rather than on specific applications.
Expert opinion:
MM/PBSA and MM/GBSA are attractive approaches owing to their modular nature and that they do not require calculations on a training set. They have been used successfully to reproduce and rationalize experimental findings and to improve the results of virtual screening and docking. However, they contain several crude and questionable approximations, for example, the lack of conformational entropy and information about the number and free energy of water molecules in the binding site. Moreover, there are many variants of the method and their performance varies strongly with the tested system. Likewise, most attempts to ameliorate the methods with more accurate approaches, for example, quantum-mechanical calculations, polarizable force fields or improved solvation have deteriorated the results.
The aggregation of Aβ-peptide (Aβ) is widely considered to be the critical step in the pathology of Alzheimer's disease. Small, soluble Aβ oligomers have been shown to be more neurotoxic than large, insoluble aggregates and fibrils. Recent studies suggest that biometal ions, including Zn(II), may play an important role in the aggregation process. Experimentally determining the details of the binding process is complicated by the kinetic lability of zinc. To study the dynamic nature of the zinc-bound Aβ complexes and the potential mechanisms by which Zn(II) affects Aβ oligomerization we have performed atomistic molecular dynamics (MD) simulations of Zn(Aβ) and Zn(Aβ)2. The models were based on NMR data and predicted coordination environments from previous density functional theory calculations. When modeled as 4-coordinate covalently bound Zn(Aβ) n complexes (where n = 1 or 2), zinc imposes conformational changes in the surrounding Aβ residues. Moreover, zinc reduces the helix content and increases the random coil content of the full peptide. Although zinc binds at the N-terminus of Aβ, β-sheet formation is observed exclusively at the C-terminus in the Zn(Aβ) and most of the Zn(Aβ)2 complexes. Furthermore, initial binding to zinc promotes the formation of intra-chain salt-bridges, while subsequent dissociation promotes the formation of inter-chain salt-bridges. These results suggest that Zn-binding to Aβ accelerates the aggregation of Aβ by unfolding the helical structure in Aβ peptide and stabilizing the formation of vital salt-bridges within and between Aβ peptides.
Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.
Background
Pantothenate kinase (PanK), an essential enzyme for Mycobacterium tuberculosis, catalyzes the rate-limiting step in the CoA pathway.
Results
Structures of M. tuberculosis PanK, complexed with new inhibitors, were determined, and their inhibitions were evaluated biochemically.
Conclusion
Inhibitor binding overlaps with the substrate/product sites; also, an alternative mode of ATP binding is proposed.
Significance
These are the first structures of a type I PanK complexed with inhibitors.
The degree of similarity of two protein three-dimensional structures is usually measured with the root-mean-square distance between equivalent atom pairs. Such a similarity measure depends on the dimension of the proteins, that is, on the number of equivalent atom pairs. The present communication presents a simple procedure to make the root-mean-square distances between pairs of three-dimensional structures independent of their dimensions. This normalization may be useful in evolutionary and fold classification studies as well as in simple comparisons between different structural models.
In molecular dynamics (MD) simulations the need often arises to maintain such parameters as temperature or pressure rather than energy and volume, or to impose gradients for studying transport properties in nonequilibrium MD. A method is described to realize coupling to an external bath with constant temperature or pressure with adjustable time constants for the coupling. The method is easily extendable to other variables and to gradients, and can be applied also to polyatomic molecules involving internal constraints. The influence of coupling time constants on dynamical variables is evaluated. A leap‐frog algorithm is presented for the general case involving constraints with coupling to both a constant temperature and a constant pressure bath.
The main thrust of the World Health Organization's global tuberculosis (TB) control strategy is to ensure effective and equitable delivery of quality assured diagnosis and treatment of TB. Options for including preventive efforts have not yet been fully considered. This paper presents a narrative review of the historical and recent progress in TB control and the role of TB risk factors and social determinants. The review was conducted with a view to assess the prospects of effectively controlling TB under the current strategy, and the potential to increase epidemiological impact through additional preventive interventions. The review suggests that, while the current strategy is effective in curing patients and saving lives, the epidemiological impact has so far been less than predicted. In order to reach long-term epidemiological targets for global TB control, additional interventions to reduce peoples' vulnerability for TB may therefore be required. Risk factors that seem to be of importance at the population level include poor living and working conditions associated with high risk of TB transmission, and factors that impair the host's defence against TB infection and disease, such as HIV infection, malnutrition, smoking, diabetes, alcohol abuse, and indoor air pollution. Preventive interventions may target these factors directly or via their underlying social determinants. The identification of risk groups also helps to target strategies for early detection of people in need of TB treatment. More research is needed on the suitability, feasibility and cost-effectiveness of these intervention options.
We present a new method for isothermal rigid body simulations using the quaternion representation and Langevin dynamics. It can be combined with the traditional Langevin or gradient (Brownian) dynamics for the translational degrees of freedom to correctly sample the canonical distribution in a simulation of rigid molecules. We propose simple, quasisymplectic second-order numerical integrators and test their performance on the TIP4P model of water. We also investigate the optimal choice of thermostat parameters.
The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits.
Control of coenzyme A synthesis was studied in isolated, perfused rat hearts. Pantothenic acid (PA), coenzyme A, and intermediates in the the pathway were separated by high pressure liquid chromatography. The amount of 14C label in each of the metabolites was determined in tissue extracts when [14C]PA was supplied in the perfusate. The rate-controlling steps in the pathway were determined by measuring the net rate of [14C]PA flux through each of the reactions. The data indicated that the primary site of control in the pathway was the pantothenate kinase-catalyzed reaction, the first intracellular step in the conversion of PA to CoA. The rate of this reaction was inhibited by including glucose, pyruvate, fatty acids, or beta-hydroxybutyrate in the perfusate of isolated hearts. Pyruvate and beta-hydroxybutyrate caused a much greater inhibition than did glucose. Insulin was a strong inhibitor, but only in the presence of glucose. Insulin had no effect in hearts receiving either no substrate or palmitate as substrate. Collectively, these data indicated that an unknown tissue metabolite whose level changed with each of these substrates and insulin is a strong regulator of pantothenate kinase. Synthesis of CoA occurred in both the cytosolic and mitochondrial compartments. Accelerated mitochondrial CoA synthesis appeared to be dependent upon the production and accumulation of 4'-phosphopantotheine, which occurred only when pantothenate kinase was stimulated.
The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific
drugs have been discovered in 40 years. We identified a diarylquinoline, R207910, that potently inhibits both drug-sensitive
and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 μg/ml). In mice, R207910 exceeded the bactericidal activities of isoniazid
and rifampin by at least 1 log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis
treatment regimen (rifampin, isoniazid, and pyrazinamide) with R207910 accelerated bactericidal activity, leading to complete
culture conversion after 2 months of treatment in some combinations. A single dose of R207910 inhibited mycobacterial growth
for 1 week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected
in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.
Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine. Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium. Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E. coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content. Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis. Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis. Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E. coli.
Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized at atomic resolution using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. In the following chapter, we present an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of a similar protocol has resulted in models of useful accuracy for domains in more than half of all known protein sequences.
Mycobacterium tuberculosis relies on its own ability to biosynthesise coenzyme A in order to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterised a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro whereas silencing of panB, panC or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection, or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice, and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.
Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
We present an implementation of explicit solvent all atom classical molecular dynamics (MD) within the AMBER program package that runs entirely on CUDA-enabled GPUs. First released publicly in April 2010 as part of version 11 of the AMBER MD package and further improved and optimized over the last two years, this implementation supports the three most widely used statistical mechanical ensembles (NVE, NVT, and NPT), uses particle mesh Ewald (PME) for the long-range electrostatics, and runs entirely on CUDA-enabled NVIDIA graphics processing units (GPUs), providing results that are statistically indistinguishable from the traditional CPU version of the software and with performance that exceeds that achievable by the CPU version of AMBER software running on all conventional CPU-based clusters and supercomputers. We briefly discuss three different precision models developed specifically for this work (SPDP, SPFP, and DPDP) and highlight the technical details of the approach as it extends beyond previously reported work [Götz et al., J. Chem. Theory Comput. 2012, DOI: 10.1021/ct200909j; Le Grand et al., Comp. Phys. Comm. 2013, DOI: 10.1016/j.cpc.2012.09.022].We highlight the substantial improvements in performance that are seen over traditional CPU-only machines and provide validation of our implementation and precision models. We also provide evidence supporting our decision to deprecate the previously described fully single precision (SPSP) model from the latest release of the AMBER software package.
Current tuberculosis (TB) therapies take too long and the regimens are complex and subject to adverse effects and drug-drug interactions with concomitant medications. The emergence of drug-resistant TB strains exacerbates the situation. Drug discovery for TB has resurged in recent years, generating compounds (hits) with varying potential for progression into developable leads. In parallel, advances have been made in understanding TB pathogenesis. It is now possible to apply the lessons learned from recent TB hit generation efforts and newly validated TB drug targets to generate the next wave of TB drug leads. Use of currently underexploited sources of chemical matter and lead-optimization strategies may also improve the efficiency of future TB drug discovery. Novel TB drug regimens with shorter treatment durations must target all subpopulations of Mycobacterium tuberculosis existing in an infection, including those responsible for the protracted TB treatment duration. This review proposes strategies for generating improved hits and leads that could help achieve this goal.
OriginPro 9.1. OriginLab Corporation, One Roundhouse Plaza, Suite 303, Northampton, MA 01060, United States. 1800-969-7720. www.OriginLab.com. See Web site for pricing information.
We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the analysis and processing of time series of three-dimensional atomic positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange molecular dynamics), image with periodic boundary conditions, create average structures, strip subsets of the system, and perform calculations such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized at atomic resolution using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. In this chapter, we present an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of a similar protocol has resulted in models of useful accuracy for domains in more than half of all known protein sequences.
AutoDock Vina, a new program for molecular docking and virtual screening, is presented. AutoDock Vina achieves an approximately two orders of magnitude speed-up compared with the molecular docking software previously developed in our lab (AutoDock 4), while also significantly improving the accuracy of the binding mode predictions, judging by our tests on the training set used in AutoDock 4 development. Further speed-up is achieved from parallelism, by using multithreading on multicore machines. AutoDock Vina automatically calculates the grid maps and clusters the results in a way transparent to the user.
Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed.
A method and parametrization scheme which allow fast and accurate calculations of hydration free energies are described. The solute is treated as a polarizable cavity of a shape defined by the molecular surface, containing point charges at the location of atomic nuclei. Electrostatic contributions to solvation are derived from:finite difference solutions of the Poisson equation (FDPB method). Nonpolar (cavity/van der Waals) energies are added as a surface area dependent term, with a single surface tension coefficient (gamma) derived from hydrocarbon solubility in water. Atomic charges and radii are obtained by modifying existing force-field or quantum-mechanically-derived values, by fitting to experimental solvation energies of small organic molecules. A new, simple parameter set (parameters for solvation energy, PARSE) is developed specifically for the FDPB/gamma method, by choosing atomic charges and radii which reproduce the estimated contributions to solvation of simple functional groups. The PARSE parameters reproduce hydration free energies for a test set of 67 molecules with an average error of 0.4 kcal/mol. For amino acid side chain and peptide backbone analogs the average error is only 0.1 kcal/mol.
Despite the introduction 40 years ago of the inexpensive and effective four-drug (isoniazid, rifampicin, pyrazinamide and ethambutol) treatment regimen, tuberculosis (TB) continues to cause considerable morbidity and mortality worldwide. For the first time since the 1960s, new and novel drugs and regimens for all forms of TB are emerging. Such regimens are likely to utilize both repurposed drugs and new chemical entities, and several of these regimens are now progressing through clinical trials. This article covers current concepts and recent advances in TB drug discovery and development, including an update of ongoing TB treatment trials, newer clinical trial designs, TB biomarkers and adjunct host-directed therapies.
Ab initio molecular orbital calculations were used to study hydrogen bonding interactions and interatomic distances of a number of hydrogen bonded complexes that are germane to biomolecular structure and function. The calculations were carried out at the STO-3G, 3-21G, 6-31G*, and MP2/6-31G* levels (geometries were fully optimized at each level). For anionic species, 6-31 + G* and MP2/6-31 + G* were also used. In some cases, more sophisticated calculations were also carried out. Whenever possible, the corresponding enthalpy, entropy, and free energy of complexation were calculated. The agreement with the limited quantity of experimental data is good. For comparison, we also carried out semiempirical molecular orbital calculations. In general, AM1 and PM3 give lower interaction enthalpies than the best ab initio results. With regard to structural results, AM1 tends to favor bifurcated structures for OH-O and NHO types of hydrogen bonds, but not for hydrogen bonds involving O-HS and S-HO, where the usual hydrogen bond patterns are observed. Overall, AM1 geometries are in general in poor agreement with ab initio structural results. On the other hand, PM3 gives geometries similar to the ab initio ones. Hence, from the structural point of view PM3 does show some improvement over AM1. Finally, insights into the formation of cyclic or open formate–water hydrogen bonded complexes are presented. © 1992 by John Wiley & Sons, Inc.
Tuberculosis is still one of the most important causes of death worldwide. The 2010 Lancet tuberculosis series provided a comprehensive overview of global control efforts and challenges. In this update we review recent progress. With improved control efforts, the world and most regions are on track to achieve the Millennium Development Goal of decreasing tuberculosis incidence by 2015, and the Stop TB Partnership target of halving 1990 mortality rates by 2015; the exception is Africa. Despite these advances, full scale-up of tuberculosis and HIV collaborative activities remains challenging and emerging drug-resistant tuberculosis is a major threat. Recognition of the effect that non-communicable diseases--such as smoking-related lung disease, diet-related diabetes mellitus, and alcohol and drug misuse--have on individual vulnerability, as well as the contribution of poor living conditions to community vulnerability, shows the need for multidisciplinary approaches. Several new diagnostic tests are being introduced in endemic countries and for the first time in 40 years a coordinated portfolio of promising new tuberculosis drugs exists. However, none of these advances offer easy solutions. Achievement of international tuberculosis control targets and maintenance of these gains needs optimum national health policies and services, with ongoing investment into new approaches and strategies. Despite growing funding in recent years, a serious shortfall persists. International and national financial uncertainty places gains at serious risk. Perseverance and renewed commitment are needed to achieve global control of tuberculosis, and ultimately, its elimination.
Mycobacterium tuberculosis, which belongs to the genus Mycobacterium, is the pathogenic agent for most tuberculosis (TB). As TB remains one of the most rampant infectious diseases, causing morbidity and death with emergence of multi-drug-resistant and extensively-drug-resistant forms, it is urgent to identify new drugs with novel targets to ensure future therapeutic success. In this regards, the structural genomics of M. tuberculosis provides important information to identify potential targets, perform biochemical assays, determine crystal structures in complex with potential inhibitor(s), reveal the key sites/residues for biological activity, and thus validate drug targets and discover novel drugs. In this review, we will discuss the recent progress on novel targets for structure-based anti-M. tuberculosis drug discovery.
The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) and the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) methods calculate binding free energies for macromolecules by combining molecular mechanics calculations and continuum solvation models. To systematically evaluate the performance of these methods, we report here an extensive study of 59 ligands interacting with six different proteins. First, we explored the effects of the length of the molecular dynamics (MD) simulation, ranging from 400 to 4800 ps, and the solute dielectric constant (1, 2, or 4) on the binding free energies predicted by MM/PBSA. The following three important conclusions could be observed: (1) MD simulation length has an obvious impact on the predictions, and longer MD simulation is not always necessary to achieve better predictions. (2) The predictions are quite sensitive to the solute dielectric constant, and this parameter should be carefully determined according to the characteristics of the protein/ligand binding interface. (3) Conformational entropy often show large fluctuations in MD trajectories, and a large number of snapshots are necessary to achieve stable predictions. Next, we evaluated the accuracy of the binding free energies calculated by three Generalized Born (GB) models. We found that the GB model developed by Onufriev and Case was the most successful model in ranking the binding affinities of the studied inhibitors. Finally, we evaluated the performance of MM/GBSA and MM/PBSA in predicting binding free energies. Our results showed that MM/PBSA performed better in calculating absolute, but not necessarily relative, binding free energies than MM/GBSA. Considering its computational efficiency, MM/GBSA can serve as a powerful tool in drug design, where correct ranking of inhibitors is often emphasized.
The 3D-QSAR method comparative molecular field analysis (CoMFA) involves the estimation of atomic partial charges as part of the process of calculating molecular electrostatic fields. Using 30 data sets from the literature the effect of using different common partial charge calculation methods on the predictivity (cross-validated R2) of CoMFA was studied. The partial charge methods ranged from the popular Gasteiger and the newer MMFF94 electronegativity equalization methods, to the more complex and computationally expensive semiempirical charges AM1, MNDO, and PM3. The MMFF94 and semiempirical MNDO, AM1, and PM3 methods for computing charges were found to result in statistically significantly more predictive CoMFA models than the Gasteiger charges. Although there was a trend toward the semiempirical charges performing better than the MMFF94 charges, the difference was not statistically significant. Thus, semiempirical partial charge calculation methods are suggested for the most predictive CoMFA models, but the MMFF94 charge calculation method is a very good alternative if semiempirical methods are not available or faster calculation speed is important.
An analytical formula has been derived for the calculation of the solvent accessible surface area of a protein molecule or equivalently the surface area exterior to an arbitrary number of overlapping spheres. The directional derivative of this function with respect to atomic co-ordinates is provided to facilitate minimization procedures used with molecular docking algorithms and energy calculations. An analytical formula for the calculation of the volume enclosed within the accessible surface, the excluded volume, is also derived. Although the area function is not specific to the structures of proteins, the derivation was motivated by the need for a computationally feasible simulation of the hydrophobic effect in proteins. A computer program using the equations for area has been tested and has had limited application to the docking of protein alpha-helices. Possible relationships of the solvent excluded volume to hydrophobic interaction free energy and transfer free energy of solute molecules are derived from the statistical mechanics of solution.
The intrinsic resistance of many mycobacterial species to chemotherapy is largely attributable to their impermeable cell wall. The composition of the cell wall of a particular species appears to be influenced by the environmental niche that the species occupies. The complex regulatory and biosynthetic pathways involved in cell wall biosynthesis and construction offer useful chemotherapeutic targets against mycobacteria.
We describe here a general Amber force field (GAFF) for organic molecules. GAFF is designed to be compatible with existing Amber force fields for proteins and nucleic acids, and has parameters for most organic and pharmaceutical molecules that are composed of H, C, N, O, S, P, and halogens. It uses a simple functional form and a limited number of atom types, but incorporates both empirical and heuristic models to estimate force constants and partial atomic charges. The performance of GAFF in test cases is encouraging. In test I, 74 crystallographic structures were compared to GAFF minimized structures, with a root-mean-square displacement of 0.26 A, which is comparable to that of the Tripos 5.2 force field (0.25 A) and better than those of MMFF 94 and CHARMm (0.47 and 0.44 A, respectively). In test II, gas phase minimizations were performed on 22 nucleic acid base pairs, and the minimized structures and intermolecular energies were compared to MP2/6-31G* results. The RMS of displacements and relative energies were 0.25 A and 1.2 kcal/mol, respectively. These data are comparable to results from Parm99/RESP (0.16 A and 1.18 kcal/mol, respectively), which were parameterized to these base pairs. Test III looked at the relative energies of 71 conformational pairs that were used in development of the Parm99 force field. The RMS error in relative energies (compared to experiment) is about 0.5 kcal/mol. GAFF can be applied to wide range of molecules in an automatic fashion, making it suitable for rational drug design and database searching.
The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
The activity-exposure-toxicity relationship, which can be described as "the rule of three", presents the single most difficult challenge in the design of drug candidates and their subsequent advancement to the development stage. ADME studies are widely used in drug discovery to optimize the balance of properties necessary to convert lead candidates into drugs that are safe and effective for humans. Metabolite characterization has become one of the key drivers of the drug discovery process, helping to optimize ADME properties and increase the success rate for drugs. Various strategies can influence drug design in the decision-making process in the structural modification of drug candidates to reduce metabolic instability.
Coenzyme A (CoA) is a ubiquitous essential cofactor that plays a central role in the metabolism of carboxylic acids, including short- and long-chain fatty acids. In the last few years, all of the genes encoding the CoA biosynthetic enzymes have been identified and the structures of several proteins in the pathway have been determined. CoA is assembled in five steps from pantothenic acid and pathway intermediates are common to both prokaryotes and eukaryotes. In spite of the identical biochemistry, remarkable sequence differences among some of the prokaryotic and eukaryotic enzymes have been revealed by comparative genomics. Renewed interest in CoA has arisen from the realization that the biosynthetic pathway is a target for antibacterial drug discovery and from the unexpected association of a human neurodegenerative disorder with mutations in pantothenate kinase. The purpose of this review is to integrate previous knowledge with the most recent findings in the genetics, enzymology and regulation of CoA biosynthesis in bacteria, plants and mammals.
The last 15 years of effort in understanding bacterial DNA replication and repair has identified that the donut shaped beta2 sliding clamp is harnessed by very functionally different DNA polymerases throughout the lifecycle of the bacterial cell. Remarkably, the sites of binding of these polymerases, in most cases, appear to be the same shallow pocket on the beta dimer. In every case, binding of beta2 by the polymerase enhances their processivity of DNA synthesis. This binding site is also the same point of interaction between beta2 and the clamp loader complex, which binds beta2, opens and places it onto the DNA strand and then vacates the site. Beta2 may also be involved in the initiation of DNA replication again via contact through this same site. While much of the research effort has focused on Escherichia coli and Bacillus subtilis, conservation of this complex system is becoming apparent in diverse bacteria.
). World Health Organization
World Health Organization. (2019). Global tuberculosis report (2019th ed.).
World Health Organization.