Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes.
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ABSTRACT:
SUBMITTER: Levine SR
PROVIDER: S-EPMC8096124 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMycobacterium tuberculosis continues to be a major pathogen in the third world, killing almost 2 million people a year by the most recent estimates. Even in industrialized countries, the emergence of multi-drug resistant (MDR) strains of tuberculosis hails the need to develop additional medications for treatment. Many of the drugs used for treatment of tuberculosis target metabolic enzymes. Genome-scale models can be used for analysis, discovery, and as hypothesis generating tools, which will hopefully assist the rational drug development process. These models need to be able to assimilate data from large datasets and analyze them.ResultsWe completed a bottom up reconstruction of the metabolic network of Mycobacterium tuberculosis H37Rv. This functional in silico bacterium, iNJ661, contains 661 genes and 939 reactions and can produce many of the complex compounds characteristic to tuberculosis, such as mycolic acids and mycocerosates. We grew this bacterium in silico on various media, analyzed the model in the context of multiple high-throughput data sets, and finally we analyzed the network in an 'unbiased' manner by calculating the Hard Coupled Reaction (HCR) sets, groups of reactions that are forced to operate in unison due to mass conservation and connectivity constraints.ConclusionAlthough we observed growth rates comparable to experimental observations (doubling times ranging from about 12 to 24 hours) in different media, comparisons of gene essentiality with experimental data were less encouraging (generally about 55%). The reasons for the often conflicting results were multi-fold, including gene expression variability under different conditions and lack of complete biological knowledge. Some of the inconsistencies between in vitro and in silico or in vivo and in silico results highlight specific loci that are worth further experimental investigations. Finally, by considering the HCR sets in the context of known drug targets for tuberculosis treatment we proposed new alternative, but equivalent drug targets.
| S-EPMC1925256 | biostudies-literature
Project description:The value of the β-lactam antibiotics for the control of bacterial infection has eroded with time. Three Gram-positive human pathogens that were once routinely susceptible to β-lactam chemotherapy-Streptococcus pneumoniae, Enterococcus faecium, and Staphylococcus aureus-now are not. Although a fourth bacterium, the acid-fast (but not Gram-positive-staining) Mycobacterium tuberculosis, has intrinsic resistance to earlier β-lactams, the emergence of strains of this bacterium resistant to virtually all other antibiotics has compelled the evaluation of newer β-lactam combinations as possible contributors to the multidrug chemotherapy required to control tubercular infection. The emerging molecular-level understanding of these resistance mechanisms used by these four bacteria provides the conceptual framework for bringing forward new β-lactams, and new β-lactam strategies, for the future control of their infections.
| S-EPMC4852796 | biostudies-literature
Project description:Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including highthroughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNAsynthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl- AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases.
| S-EPMC3808994 | biostudies-literature
Project description:Recent clinical studies indicate that meropenem, a β-lactam antibiotic, is a promising candidate for therapy of drug-resistant tuberculosis. However, meropenem is chemically unstable, requires frequent intravenous injection, and must be combined with a β-lactamase inhibitor (clavulanate) for optimal activity. Here, we report that faropenem, a stable and orally bioavailable β-lactam, efficiently kills Mycobacterium tuberculosis even in the absence of clavulanate. The target enzymes, L,D-transpeptidases, were inactivated 6- to 22-fold more efficiently by faropenem than by meropenem. Using a real-time assay based on quantitative time-lapse microscopy and microfluidics, we demonstrate the superiority of faropenem to the frontline antituberculosis drug isoniazid in its ability to induce the rapid cytolysis of single cells. Faropenem also showed superior activity against a cryptic subpopulation of nongrowing but metabolically active cells, which may correspond to the viable but nonculturable forms believed to be responsible for relapses following prolonged chemotherapy. These results identify faropenem to be a potential candidate for alternative therapy of drug-resistant tuberculosis.
| S-EPMC4335862 | biostudies-literature
Project description:We have developed a software program that weights and integrates specific properties on the genes in a pathogen so that they may be ranked as drug targets. We applied this software to produce three prioritized drug target lists for Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease for which a new drug is desperately needed. Each list is based on an individual criterion. The first list prioritizes metabolic drug targets by the uniqueness of their roles in the M. tuberculosis metabolome ("metabolic chokepoints") and their similarity to known "druggable" protein classes (i.e., classes whose activity has previously been shown to be modulated by binding a small molecule). The second list prioritizes targets that would specifically impair M. tuberculosis, by weighting heavily those that are closely conserved within the Actinobacteria class but lack close homology to the host and gut flora. M. tuberculosis can survive asymptomatically in its host for many years by adapting to a dormant state referred to as "persistence." The final list aims to prioritize potential targets involved in maintaining persistence in M. tuberculosis. The rankings of current, candidate, and proposed drug targets are highlighted with respect to these lists. Some features were found to be more accurate than others in prioritizing studied targets. It can also be shown that targets can be prioritized by using evolutionary programming to optimize the weights of each desired property. We demonstrate this approach in prioritizing persistence targets.
| S-EPMC1475714 | biostudies-literature
Project description:The increasing threat of drug resistance and a stagnated pipeline of novel therapeutics endanger the eradication of tuberculosis. Beta-lactams constitute promising additions to the current therapeutic arsenal and two carbapenems are included in group C of medicines recommended by the WHO for use in longer multidrug-resistant tuberculosis regimens. However, the determinants underlining diverse Mycobacterium tuberculosis phenotypes to beta-lactams remain largely undefined. To decipher these, we present a proof-of-concept study based on a large-scale beta-lactam susceptibility screening for 172 M. tuberculosis clinical isolates from Portugal, including 72 antimycobacterial drug-resistant strains. MICs were determined for multiple beta-lactams and strains were subjected to whole-genome sequencing to identify core-genome single-nucleotide variant-based profiles. Global and cell wall-targeted approaches were then followed to detect putative drivers of beta-lactam response. We found that drug-resistant strains were more susceptible to beta-lactams, but significant differences were not observed between distinct drug-resistance profiles. Sublineage 4.3.4.2 strains were significantly more susceptible to beta-lactams, while the contrary was observed for Beijing and 4.1.2.1 sublineages. While mutations in beta-lactamase or cell wall biosynthesis genes were uncommon, a rise in beta-lactam MICs was detected in parallel with the accumulation of mutations in peptidoglycan cross-linking or cell division genes. Finally, we exposed that putative beta-lactam resistance markers occurred in genes for which relevant roles in cell wall processes have been ascribed, such as rpfC or pknA. Genetic studies to validate the relevance of the identified mutations for beta-lactam susceptibility and further improvement of the phenotype-genotype associations are needed in the future. IMPORTANCE Associations between differential M. tuberculosis beta-lactam phenotypes and preexisting antimycobacterial drug resistance, strain sublineage, or specific mutational patterns were established. Importantly, we reveal that highly drug-resistant isolates of sublineage 4.3.4.2 have an increased susceptibility to beta-lactams compared with other strains. Thus, directing beta-lactams to treat infections by specific M. tuberculosis strains and refraining its use from others emerges as a potentially important strategy to avoid resistance development. Individual mutations in blaC or genes encoding canonical beta-lactam targets, such as peptidoglycan transpeptidases, are infrequent and do not greatly impact the MICs of potent carbapenem plus clavulanic acid combinations. An improved understanding of the global effect of cumulative mutations in relevant gene sets for peptidoglycan and cell division processes on beta-lactam susceptibility is also provided.
| S-EPMC9431576 | biostudies-literature
Project description:Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis, especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.
| S-EPMC3781026 | biostudies-literature
Project description:Tuberculosis (TB) has been the biggest killer in the human history; currently, Mycobacterium tuberculosis (Mtb) kills nearly 2 million people each year worldwide. The high prevalence of TB obligates the identification of new therapeutic targets and the development of anti-TB vaccines that can control multidrug resistance and latent TB infections. Membrane proteins have recently been suggested as key targets for bacterial viability. Current studies have shown that mycobacteria P-type ATPases may play critical roles in ion homeostasis and in the response of mycobacteria to toxic substances in the intraphagosomal environment. In this review, we bring together the genomic, transcriptomic, and structural aspects of the P-type ATPases that are relevant during active and latent Mtb infections, which can be useful in determining the potential of these ATPases as drug targets and in uncovering their possible roles in the development of new anti-TB attenuated vaccines.
| S-EPMC4119724 | biostudies-literature
Project description:BACKGROUND:High attrition rates in drug discovery call for new approaches to improve target validation. Academia is filling gaps, but often lacks the experience and resources of the pharmaceutical industry resulting in poorly characterized tool compounds. DISCUSSION:The SGC has established an open access chemical probe consortium, currently encompassing ten pharmaceutical companies. One of its mandates is to create well-characterized inhibitors (chemical probes) for epigenetic targets to enable new biology and target validation for drug development. CONCLUSION:Epigenetic probe compounds have proven to be very valuable and have not only spurred a plethora of novel biological findings, but also provided starting points for clinical trials. These probes have proven to be critical complementation to traditional genetic targeting strategies and provided sometimes surprising results.
| S-EPMC4673908 | biostudies-literature
Project description:beta-Lactam antibiotics are extremely effective in disrupting the synthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. However, they are ineffective against Mycobacterium tuberculosis, due to the production of a beta-lactamase enzyme encoded on the chromosome of M. tuberculosis that degrades these antibiotics. Indeed, recent studies have demonstrated that deletion of the blaC gene, the only gene encoding a beta-lactamase in M. tuberculosis, or inhibition of the encoded enzyme resulted in significantly increased sensitivity to beta-lactam antibiotics. In this paper we present a biochemical and structural characterization of M. tuberculosis BlaC. Recombinant BlaC shows a broad range of specificity with almost equal penicillinase and cepholothinase activity. While clavulanate is a mechanism-based inhibitor to class A beta-lactamase with high potency (typically K(i) < 0.1 microM), it is a relatively poor inhibitor of the M. tuberculosis BlaC (K(i) = 2.4 microM). The crystal structure of the enzyme, determined at a resolution of 1.7 A, shows that the overall fold of the M. tuberculosis enzyme is similar to other class A beta-lactamases. There are, however, several distinct features of the active site, such as the amino acid substitutions N132G, R164A, R244A, and R276E, that explain the broad specificity of the enzyme, relatively low penicillinase activity, and resistance to clavulanate.
| S-EPMC1538687 | biostudies-literature