Pyrazinoic Acid Research Articles

Study of cytotoxicity and cell death of an active compound from prazinoic acids in chronic myeloid leukemia K562 cell line

Background: Nowadays, more than 100 types of cancer are known worldwide, which leukemia is one of the most common type. Chronic myeloid leukemia (CML) is one of the most recognized forms of leukemia, accounting for between 15% and 20% of total leukemia. Efforts to find new synthetic compounds are one of the basic strategies for finding new drugs for CML treatment. The purpose of this study was to investigate the effects of growth suppression and induction of apoptosis (family of pyrazinoic acid) on the cancer cell line of K562 leukemia. Methods: In this study, the effect of a certain concentration of 2-oxo-1, 2-diphenylethyl-pirazine-2-carboxylate (2 DP.P) was evaluated on the growth, cell cycle and induction of apoptosis of K562 cells using various techniques. MTT test was used to investigate the effects of 2-DP.P on cell viability. For this purpose, 5×104 of K562 cells were cultured at concentrations of 25-100 μM from the 2-DP.P for 24, 48 and 72 hours intervals. Electrophoresis, fluorescence microscopy and flow cytometry were used for analysis of apoptosis. Results: The 2-DP.P inhibited dose and time dependently growth in K562 cells. IC50 of the compound was calculated 25 μM. Also, data from fluorescence microscopy, DNA fragmentation and cell cycle analysis confirmed the reduction of viability and induction of apoptosis by the compound. Conclusion: due to the effects of growth inhibition and induction of apoptosis by the 2-DP.P, this compound can be suggested as a new and effective compound for further studies in treating patients with leukemia.

Structural and free energy landscape of novel mutations in ribosomal protein S1 (rpsA) associated with pyrazinamide resistance

Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type (pncAWT), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncAWT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsACTD) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsACTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsACTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

Exploring the Pyrazinamide Drug Resistance Mechanism of Clinical Mutants T370P and W403G in Ribosomal Protein S1 of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an essential first line antitubercular drug, which plays a crucial role in tuberculosis treatment. The PZA, which is considered as a pro-drug needs an enzyme of mycobacterial pyrazinamidase (PZase) for its conversion into an active form pyrazinoic acid. Further, this active form of PZA inhibits the ribosomal proteins S1, which facilitates the transfer-mRNA complex formation throughout the translation. The spontaneous mutations in RpsA have been found to be associated with PZA drug resistance. However, the drug resistance mechanism is still unclear. Furthermore, there is no such information available about the structural dynamics of RpsA protein because of mutations that confer Pyrazinoic acid resistance. Moreover, a total of 18 clinical PZA-resistant isolates were investigated and found to be pncAWT, which allowed exploration of the resistance mechanism of RpsA in the mutated state. Samples were repeated for the drug susceptibility testing followed by RpsA gene sequencing. A total of 11 clinical isolates harbored a total of 15 mutations. Almost half of the total strains (7/15) were observed to be in the conserved region of RpsA and known as Mycobacterium tuberculosis C-terminal domain. In the current study, (2/7) mutation T370P (mutant 1) and W403G (mutant 2) were explored to ensure the RpsA resistance mechanism through essential dynamics simulation. The essential dynamics study results revealed that the distal loop mutations drastically altered the conformation of RpsA both in the absence (-) and presence (+) of pyrazinoic acid drug for two reasons: (1) dramatic alteration or reduction in the binding pattern of pyrazinoic acid with active site residues observed and (2) a clear image of the opening and closing switching mechanism was seen upon the distal site mutation on nearby 310-helixes beside the pyrazinoic acid binding site. This switch was found to consistently remain closed only in wild type systems, while it was open in the mutant systems. We called such distance impact an "allosteric effect." The overall mechanistic investigations will provide useful information behind drug resistance for better understanding to manage tuberculosis.

Mutations in Efflux Pump Rv1258c (Tap) Cause Resistance to Pyrazinamide, Isoniazid, and Streptomycin in M. tuberculosis.

Although drug resistance in Mycobacterium tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in the possible role of efflux in causing drug resistance. Previously, efflux genes have been shown to be upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance in M. tuberculosis. We constructed point mutations V219A and S292L in Rv1258c in the chromosome of M. tuberculosis and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed M. tuberculosis Rv1258c mutants to different tuberculosis drugs was assessed using conventional drug susceptibility testing in 7H11 agar in the presence and absence of efflux pump inhibitor piperine. A C14-labeled PZA uptake experiment was performed to demonstrate higher efflux activity in the M. tuberculosis Rv1258c mutants. Interestingly, the V219A and S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in M. tuberculosis. While V219A point mutation conferred low-level drug resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. The S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance, including PZA resistance, and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance in M. tuberculosis and address their role in affecting treatment outcome in vivo.

Pyrazinamide resistance and mutations in pncA among isolates of Mycobacterium tuberculosis from Khyber Pakhtunkhwa, Pakistan

BackgroundPyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan.MethodsWe performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations.ResultsA total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n = 4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C > T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%).ConclusionMutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.

Population Pharmacokinetic Modelling of Pyrazinamide and Pyrazinoic Acid in Patients with Multi-Drug Resistant Tuberculosis.

Pyrazinamide, a drug used in the regimen for the treatment of drug-sensitive tuberculosis, is also used for the treatment of multidrug-resistant tuberculosis (MDR-TB). We aimed to describe the population pharmacokinetics of pyrazinamide and its major metabolite, pyrazinoic acid, in patients with MDR-TB and characterise the effects of demographic variables. This was a non-randomised clinical study involving 51 adult patients admitted for the intensive phase of MDR-TB treatment. Blood samples were collected at pre-dose and at 0.5, 1, 1.5, 2, 3, 4, 8, 16 and 24h after drug administration. Plasma concentrations of pyrazinamide and pyrazinoic acid were analysed using a validated LC-MS/MS method. Nonlinear mixed-effects modelling using Monolix 2018R1 software was employed to estimate population pharmacokinetic parameters. A one-compartment pharmacokinetic model with transit compartment absorption process and first-order elimination best described the pyrazinamide and pyrazinoic acid concentration-time data. The estimated population pharmacokinetic parameters were 0.7h, 3.38h-1, 57.1l, 4.37L/h and 10.5L/h for mean transit time, absorption rate constant, apparent distribution volume for pyrazinamide, and apparent clearance for pyrazinamide and pyrazinoic acid (CLm/F), respectively. These parameters were not affected by patient age, HIV status or sex. The parameter variability in CLm/F was the highest (83.5%), while the rest of the parameters ranged from 16.2 to 58%. The developed population pharmacokinetic model adequately described the disposition of pyrazinamide and pyrazinoic acid and can be useful for dose determination of pyrazinamide in patients with MDR-TB.

Synthesis, computational studies, antimycobacterial and antibacterial properties of pyrazinoic acid-isoniazid hybrid conjugates.

Benzotriazole and microwave mediated syntheses led to a new set of hybrid conjugates of pyrazinoic acid with isoniazid via amino acid linkers in excellent yields with retention of chirality. Microbiological screening of the synthesized conjugates revealed an exceptionally high activity against some of the pathogenic bacterial strains at low concentrations. Promising antimycobacterial properties were observed against tuberculous and non-tuberculous mycobacteria. Robust molecular models (2D-QSAR and 3D-pharmacophore) support the observed biological properties. Safety profile of the synthesized conjugates against human normal cell (RPE1) was evaluated by MTT technique.

PYRAZINAMIDE/PYRAZINOIC ACID RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS: RECENT FINDINGS AND IMPLICATIONS FOR IMPROVING THE TREATMENT OF TUBERCULOSIS

Abstract. Pyrazinamide (PZA) is unique in that it is a component of the first line therapy for drug sensitive tuberculosis and in most current and experimental treatments also for multi drug resistant tuberculosis. Furthermore, PZA has been shown to help to ensure lasting cure and prevent relapse in shorter multi drug regimens. PZA is a prodrug. Mycobacterial tuberculosis (MTB) PncA enzyme activates the anti-mycobacterial prodrug PZA by transforming it into pyrazinoic acid (POA). The majority of clinical PZA resistant isolates contain mutations within the pncA gene and therefore remain sensitive to POA as they no longer activate PZA. Resistance to the active compound POA requires an alternative resistance mechanism and in vitro selected spontaneous MTB POA resistant mutants typically acquire a range of mutations in panD or mutations in one of a series of genes most of which are associated with the regulation of the bacterial stringent response. Clinically isolated PZA resistant MTB strains resistant to PZA and POA with mutations in any of these genes are unusual. Thus, it is likely the stringent response is critical for MTB in vivo and a damaged stringent response results in at least a reduction in fitness. Various lead compounds that disrupt the MTB stringent response have been identified that might form the basis for drugs with activity against latent mycobacteria with the potential to shorten tuberculosis treatment. Here we discuss the role of latency in the lifecycle of MTB and possible links to the activity PZA with a focus on potential new targets and drugs.

Covalent coupling of tuberculostatic agents and graphene oxide: A promising approach for enhancing and extending their antimicrobial applications

In this work, we present a simple, two-stage method for the preparation of new and efficient antimicrobial hybrid systems, based on isoniazid and pyrazine-2-carbohydrazide tuberculostatic agents covalently linked to graphene oxide. In the first step, the carboxyl groups of graphene oxide are activated by transforming them into the corresponding acid chloride groups, which, in the second step, will react with the amino groups of the two drugs. Pyrazine-2-carbohydrazide was obtained from pyrazinoic acid and hydrazine hydrate and was confirmed by nuclear magnetic resonance spectroscopy. The materials have been characterized by elemental analysis, infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy.Their antimicrobial activity was tested on mycobacterial (Mycobacterium terrae), as well as on Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853), Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923) and fungal (Candida albicans ATCC 10231), in planktonic and biofilm growth state. The biocompatibility of the tested compounds was assessed in vitro by live-dead fluorescence staining and flow cytometry. The results of this study have shown that the functionalization of graphene oxide with isoniazid and pyrazine-2-carbohydrazide both enhanced the anti-mycobacterial activity of the respective drugs and extended their antimicrobial spectrum towards other microbial strains, in planktonic and biofilm growth state, showing a promising potential for mitigating the impact of antimicrobial resistance and the deleterious effects of microbial biofilms. At the active tuberculostatic concentrations, the tested compounds exhibit very low cytotoxicity and do not interfere with the cellular cycle of Hep-2 cells. The flow cytometry and live/dead fluorescence staining proved that the tested compounds exhibit a microbicidal effect through induction of cellular lesions, consecutive to membrane depolarization.

Crystal structure of a 1:1 salt of 4-amino-benzoic acid (vitamin B10) with pyrazinoic acid.

The title 1:1 salt, C7H8NO2 +·C5H3N2O2 - (systematic name: 4-carb-oxy-anilinium pyrazine-2-carboxyl-ate), was synthesized successfully by slow evaporation of a saturated solution from water-ethanol (1:1 v/v) mixture and characterized by X-ray diffraction (SCXRD, PXRD) and calorimetry (DSC). The crystal structure of the salt was solved and refined at 150 and 293 K. The salt crystallizes with one mol-ecule of 4-amino-benzoic acid (PABA) and one mol-ecule of pyrazinoic acid (POA) in the asymmetric unit. In the crystal, the PABA and POA mol-ecules are associated via COOH⋯Narom heterosynthons, which are connected by N-H⋯O hydrogen bonds, creating zigzag chains. The chains are further linked by N-H⋯O hydrogen bonds and π-π stacking inter-actions along the b axis [centroid-to-centroid distances = 3.7377 (13) and 3.8034 (13) Å at 150 and 293 K, respectively] to form a layered three-dimensional structure.

Pyrazinamide resistance and mutations L19R, R140H, and E144K in Pyrazinamidase of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important component of first-line antituberculosis drugs activated by Mycobacterium tuberculosis pyrazinamidase (PZase) into its active form pyrazinoic acid. Mutations in the pncA gene have been recognized as the major cause of PZA resistance. We detected some novel mutations, Leucine19Arginine (L19R), Arginine140Histidine (R140H), andGlutamic acid144 Lysine (E144K), in the pncA gene of PZA-resistant isolates in our wet lab PZA drug susceptibility testing and sequencing. As the molecular mechanism of resistance of these variants has not been reported earlier, we have performed multiple analyses to unveil different mechanisms of resistance because of PZase mutations L19R, R140H, and E144K. The mutants and native PZase structures were subjected to comprehensive computational molecular dynamics (MD) simulations at 100 nanoseconds in apo and drug-bound form. Mutants and native PZase binding pocket were compared to observe the consequence of mutations on the binding pocket size. Hydrogen bonding, Gibbs free energy, and natural ligand Fe +2 effect were also analyzed between native and mutants. A significant variation between native and mutant PZase structure activity was observed. The native PZase protein docking score was found to be the maximum, showing strong binding affinity in comparison with mutants. MD simulations explored the effect of the variants on the biological function of PZase. Hydrogen bonding, metal ion Fe +2 deviation, and fluctuation also seemed to be affected because of the mutations L19R, R140H, and E144K. The variants L19R, R140H, and E144K play a significant role in PZA resistance, altering the overall activity of native PZase, including metal ion Fe +2 displacement and free energy. This study offers valuable evidence for better management of drug-resistant tuberculosis.

Insights into the Mechanisms of the Pyrazinamide Resistance of Three Pyrazinamidase Mutants N11K, P69T, and D126N.

In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncA gene contributes 72-99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe2+ ion. Among the mutations, D126N allosterically disturbed the position of the Fe2+ ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.

Correction: Efficacy of pyrazinoic acid dry powder aerosols in resolving necrotic and non-necrotic granulomas in a guinea pig model of tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0204495.].

Pyrazinamide drug resistance in RpsA mutant (∆438A) of Mycobacterium tuberculosis: Dynamics of essential motions and free-energy landscape analysis.

Pyrazinamide is an essential first-line antitubercular drug which plays pivotal role in tuberculosis treatment. It is a prodrug that requires amide hydrolysis by mycobacterial pyrazinamidase enzyme for conversion into pyrazinoic acid (POA). POA is known to target ribosomal protein S1 (RpsA), aspartate decarboxylase (PanD), and some other mycobacterial proteins. Spontaneous chromosomal mutations in RpsA have been reported for phenotypic resistance against pyrazinamide. We have constructed and validated 3D models of the native and Δ438A mutant form of RpsA protein. RpsA protein variants were then docked to POA and long range molecular dynamics simulations were carried out. Per residue binding free-energy calculations, free-energy landscape analysis, and essential dynamics analysis were performed to outline the mechanism underlying the high-level PZA resistance conferred by the most frequently occurring deletion mutant of RpsA. Our study revealed the conformational modulation of POA binding site due to the disruptive collective modes of motions and increased conformational flexibility in the mutant than the native form. Residue wise MMPBSA decomposition and protein-drug interaction pattern revealed the difference of energetically favorable binding site in the wild-type (WT) protein in comparison with the mutant. Analysis of size and shape of minimal energy landscape area delineated higher stability of the WT complex than the mutant form. Our study provides mechanistic insights into pyrazinamide resistance in Δ438A RpsA mutant, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis.

A simplified pyrazinamidase test for pyrazinamide drug susceptibility in Mycobacterium tuberculosis

We modified Wayne's pyrazinamidase test against Mycobacterium tuberculosis to indirectly measure pyrazinamidase activity via pyrazinoic acid in liquid medium. The modified pyrazinamidase test was easy to perform and its results were in complete agreement with those of the conventional Wayne's method, highlighting its potential application in phenotypic pyrazinamide susceptibility testing.

Pyrazinoates as antiparasitic agents against Trypanosoma cruzi.

This work reports a repurposing study of pyrazinoic acid (1) and methyl (2), ethyl (3) and 2-chloroethyl (4) ester derivatives with antimycobacterial activity, in assays against Trypanosoma cruzi. The compounds and benznidazole, the standard antitrypanosoma drug, were evaluated in concentrations ranging from 100 to 6.25 μg/mL. The results showed that compounds 2 and 3 (EC50 = 182 and 447 μM) significantly reduced the infection rate of the parasite into the mammalian cells at 100 μg/mL (p < 0.05) in a similar way to benznidazole. In addition, all the compounds also significantly reduced the number of intracellular parasites (compound 1 at 50 μg/mL, and compounds 2-4 at 100 μg/mL, p < 0.05) in comparison to the control. Compounds 1 and 2 were more effective than benznidazole at 50 μg/mL (p < 0.001). Moreover, compounds 1-4 did not show significant cytotoxicity against THP-1, J774, and HeLa cells (>1000 μM), indicating that they possess considerable selectivity against the parasites. This report represents the first study of such compounds against T. cruzi, indicating the potential of pyrazinoates as antiparasitic agents.

Efficacy of pyrazinoic acid dry powder aerosols in resolving necrotic and non-necrotic granulomas in a guinea pig model of tuberculosis.

New therapeutic strategies are needed to treat drug resistant tuberculosis (TB) and to improve treatment for drug sensitive TB. Pyrazinamide (PZA) is a critical component of current first-line TB therapy. However, the rise in PZA-resistant TB cases jeopardizes the future utility of PZA. To address this problem, we used the guinea pig model of TB and tested the efficacy of an inhaled dry powder combination, referred to as Pyrazinoic acid/ester Dry Powder (PDP), which is comprised of pyrazinoic acid (POA), the active moiety of PZA, and pyrazinoic acid ester (PAE), which is a PZA analog. Both POA and PAE have the advantage of being able to act on PZA-resistant Mycobacterium tuberculosis. When used in combination with oral rifampicin (R), inhaled PDP had striking effects on tissue pathology. Effects were observed in lungs, the site of delivery, but also in the spleen and liver indicating both local and systemic effects of inhaled PDP. Tissue granulomas that harbor M. tuberculosis in a persistent state are a hallmark of TB and they pose a challenge for therapy. Compared to other treatments, which preferentially cleared non-necrotic granulomas, R+PDP reduced necrotic granulomas more effectively. The increased ability of R+PDP to act on more recalcitrant necrotic granulomas suggests a novel mechanism of action. The results presented in this report reveal the potential for developing therapies involving POA that are optimized to target necrotic as well as non-necrotic granulomas as a means of achieving more complete sterilization of M. tuberculosis bacilli and preventing disease relapse when therapy ends.

Lead compounds and key residues of ribosomal protein S1 in drug-resistant Mycobacterium tuberculosis

Ribosomal protein S1 (RpsA) has been identified as a novel target of pyrazinoic acid (POA), which is the active form of pyrazinamide (PZA), in vivo. RpsA plays a crucial role in trans-translation, which is widespread in microbes. In our investigation, we first described the discovery of promising RpsA antagonists for drug-resistant mycobacterium (MtRpsAd438A) and M. smegmatis, as well as wild-type M. tuberculosis. These antagonists were discovered via structure/ligand-based virtual screening approaches. A total of 21 targeted compounds were selected by virtual screening, combined scores, affinity, similarities and rules for potential as drugs. Next, the affinities of these compounds for three targeted proteins were tested in vitro by applying various technologies, including fluorescence quenching titration (FQT), saturation transfer difference (STD), and chemical shift perturbation (CSP) assays. The results showed that seven compounds had a high affinity for the targeted proteins. Our discovery set the stage for discovering new chemical entities (NCEs) for PZA-resistant tuberculosis and providing key residues for rational drug design to target RpsA.

Identification of Novel Mutations in LprG (rv1411c), rv0521, rv3630, rv0010c, ppsC, and cyp128 Associated with Pyrazinoic Acid/Pyrazinamide Resistance in Mycobacterium tuberculosis.

Despite progress, the mechanisms of action and resistance of pyrazinamide (PZA) are not well understood. We characterized 109 mutants resistant to pyrazinoic acid (POA), the active form of PZA, and found that while most (n = 101 [93%]) mutants had panD mutations and 4 (4%) had clpC1 mutations (S91G), new mutations in lprG (rv1411c) and rv0521 (n = 4 [4%]), rv3630, rv0010c, ppsC, and cyp128 (cytochrome P450 128) were identified, shedding new light on the mechanisms of action and resistance of PZA in M. tuberculosis.

'Happy the man, who, studying nature's laws, Thro' known effects can trace the secret cause.' Do we have enough pieces to solve the pyrazinamide puzzle?

A low pH was assumed to be required for the activity of pyrazinoic acid (the active form of pyrazinamide) against Mycobacterium tuberculosis, but recently activity has been demonstrated at neutral pH. Renewed interest in pyrazinamide has led to an increasing number of potential targets and the suspicion that pyrazinamide is a 'dirty drug'. However, it is our opinion that the recent demonstration that pyrazinoic acid is active against PanD provides an alternative explanation for the secret of pyrazinamide's unusual activity. In this article we propose that PanD is the primary target of pyrazinoic acid but expression of pyrazinoic acid susceptibility requires an intact stress response. As the mycobacterial stress response requires the interaction of a number of genes, disruption of any could result in an inability to enter the susceptible phenotype. We believe this model can explain most of the recent observations of the seemingly diverse spectrum of activity of pyrazinamide.