Pharmacological and Molecular Mechanisms Behind the Sterilizing Activity of Pyrazinamide
Authors: Pooja Gopal, Gerhard Grüber, Véronique Dartois, Thomas Dick
Journal: Trends in Pharmacological Sciences (2019)

PZA is able to penetrate all TB lung lesion types, including necrotic caseous granulomas, where it kills nongrowing drug-tolerant Mtb, explaining the in vivo sterilizing activity of the drug.Pyrazinoic acid (POA), the bioactive component of the prodrug PZA, inhibits bacterial synthesis of Coenzyme A by binding to aspartate decarboxylase, PanD, and blocking this enzymatic step.Rather than inhibiting the catalytic activity of PanD, we propose that binding of POA triggers the degradation of its target by the bacterial caseinolytic protease.The hypothesis that the bioactive component of PZA acts as a target degrader supports selective target degradation as a novel antibacterial drug discovery approach.Identification of the pharmacological and molecular mechanisms of the activity of PZA provides the basis for the rational discovery of the next-generation PZA with improved in vitro potency while maintaining the attractive lesion-penetration properties of PZA. Inclusion of pyrazinamide (PZA) in the tuberculosis (TB) drug regimen during the 1970s enabled a reduction in treatment duration from 12 to 6 months. PZA has this remarkable effect in patients despite displaying poor potency against Mycobacterium tuberculosis (Mtb) in vitro. The pharmacological basis for the in vivo sterilizing activity of the drug has remained obscure and its bacterial target controversial. Recently it was shown that PZA penetrates necrotic caseous TB lung lesions and kills nongrowing, drug-tolerant bacilli. Furthermore, it was uncovered that PZA inhibits bacterial Coenzyme A biosynthesis. It may block this pathway by triggering degradation of its target, aspartate decarboxylase. The elucidation of the pharmacological and molecular mechanisms of PZA provides the basis for the rational discovery of the next-generation PZA with improved in vitro potency while maintaining attractive pharmacological properties. Inclusion of pyrazinamide (PZA) in the tuberculosis (TB) drug regimen during the 1970s enabled a reduction in treatment duration from 12 to 6 months. PZA has this remarkable effect in patients despite displaying poor potency against Mycobacterium tuberculosis (Mtb) in vitro. The pharmacological basis for the in vivo sterilizing activity of the drug has remained obscure and its bacterial target controversial. Recently it was shown that PZA penetrates necrotic caseous TB lung lesions and kills nongrowing, drug-tolerant bacilli. Furthermore, it was uncovered that PZA inhibits bacterial Coenzyme A biosynthesis. It may block this pathway by triggering degradation of its target, aspartate decarboxylase. The elucidation of the pharmacological and molecular mechanisms of PZA provides the basis for the rational discovery of the next-generation PZA with improved in vitro potency while maintaining attractive pharmacological properties. Mtb, the causative agent of TB, is now the largest infectious disease killer worldwide and a major cause of death of patients living with AIDS. In immune-competent populations, drug therapy and immunity join forces to win the lengthy war against Mtb. By contrast, in patients who are HIV positive, chemotherapy must fully sterilize all infection sites. With the rampant spread of drug resistance, and to shorten the 6–24 month-long treatment regimens for drug-susceptible and multidrug-resistant TB, there is an urgent medical need to develop more potent sterilizing drugs. In 1952, the anti-TB activity of PZA was discovered in animal models [1Solotorovsky M. et al.Pyrazinoic acid amide; an agent active against experimental murine tuberculosis.Proc. Soc. Exp. Biol. Med. 1952; 79: 563-565Crossref PubMed Scopus (29) Google Scholar,2Malone L. et al.The effect of pyrazinamide (aldinamide) on experimental tuberculosis in mice.Am. Rev. Tuberc. 1952; 65: 511-518PubMed Google Scholar], and was shortly thereafter confirmed in humans [3Yeager R.L. et al.Pyrazinamide (aldinamide*) in the treatment of pulmonary tuberculosis.Trans. Annu. Meet. Natl. Tuberc. Assoc. 1952; 48: 178-201PubMed Google Scholar]. The drug displayed remarkable sterilizing activity in mouse tissues [4McCune R.M. et al.THE fate of mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique : II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug.J. Exp. Med. 1956; 104: 763-802Crossref PubMed Scopus (216) Google Scholar] and its inclusion in the regimen of drug-susceptible patients during the 1970s resulted in a significant shortening of therapy duration from 12 to 6 months. The present-day 'short-course' regimen was finalized during the 1980s, and comprises an initial 2-month intensive phase of isoniazid, rifampicin, PZA, and ethambutol, followed by a 4-month treatment continuation phase of only isoniazid and rifampicin [5Mitchison D.A. The action of antituberculosis drugs in short-course chemotherapy.Tubercle. 1985; 66: 219-225Abstract Full Text PDF PubMed Scopus (423) Google Scholar,6Fox W. et al.Studies on the treatment of tuberculosis undertaken by the British Medical Research Council Tuberculosis Units, 1946-1986, with relevant subsequent publications.Int. J. Tuberc. Lung Dis. 1999; 3: S231-S279PubMed Google Scholar]. Owing to its relapse-preventing properties, PZA is also included in most new drug combinations that are in clinical and preclinical development for both drug-sensitive and multidrug-resistant TB [7Diacon A.H. et al.14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial.Lancet. 2012; 380: 986-993Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar,8Tasneen R. et al.Sterilizing activity of novel TMC207- and PA-824-containing regimens in a murine model of tuberculosis.Antimicrob. Agents Chemother. 2011; 55: 5485-5492Crossref PubMed Scopus (162) Google Scholar]. The basis of the remarkable sterilizing activity of PZA in patients remains puzzling since this fragment-size drug (MW = 123 g/mol) exhibits poor in vitro potency [minimum inhibitory concentration (MIC) = 30–100 μg/ml], concentrations that are barely exceeded in plasma and lesions [9Gopal P. Dick T. Reactive dirty fragments: implications for tuberculosis drug discovery.Curr. Opin. Microbiol. 2014; 21: 7-12Crossref PubMed Scopus (28) Google Scholar,10Blanc L. et al.Impact of immunopathology on the antituberculous activity of pyrazinamide.J. Exp. Med. 2018; 215: 1975-1986Crossref PubMed Scopus (22) Google Scholar]. As a structural analog of niacin, a vitamin with anti-inflammatory properties [11Walocko F.M. et al.The role of nicotinamide in acne treatment.Dermatol. Ther. 2017; 30: e12481Crossref Scopus (27) Google Scholar], it was suggested that PZA exerts host-directed effects [12Mendez S. et al.The antituberculosis drug pyrazinamide affects the course of cutaneous leishmaniasis in vivo and increases activation of macrophages and dendritic cells.Antimicrob. Agents Chemother. 2009; 53: 5114-5121Crossref PubMed Scopus (42) Google Scholar]. This was explored by Almeida et al., who hypothesized that, if PZA has host-directed activity, this should be detectable in mice infected with PZA-resistant Mtb. However, no such activity could be detected, pointing to a lack of significant host-directed activity [13Almeida D.V. et al.Revisiting anti-tuberculosis activity of pyrazinamide in mice.Mycobact. Dis. 2014; 4: 145Crossref PubMed Google Scholar]. In addition to the mysterious in vivo–in vitro disconnect of PZA activity, the antibacterial mechanism of action (MoA) of the drug remained elusive for decades, despite intense research. PZA is a prodrug that is converted to its bioactive form, pyrazinoic acid (POA), by the bacterial pyrazinamidase PncA, as well as by host enzymes [14Scorpio A. Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus.Nat. 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Several MoA of POA were proposed, only to be later questioned or disproven (see later). Understanding the pharmacological and molecular mechanisms of the drug is critical to rationally discover the next-generation PZA with improved sterilizing activity. In this opinion article, we discuss major recent advances in this field. Pharmacological explanations for the unique sterilizing property of PZA are proposed based on lesion-centric analyses in animal models, and a target is identified based on genetic and biochemical evidence. At the same time, the modest on-target potency of PZA constitutes a clear limitation and points to an obvious area for improvement. Together, these findings provide the basis for a rational mechanism-based chemical optimization to discover more efficacious next-generation PZA analogs. A spectrum of lesion types exists in human pulmonary TB: cellular lesions comprising primarily immune cells, caseous (cheese-like) granulomas containing a necrotic core of bacterial and host cell debris, and cavities where lesions erode into major airways. In these lesions, Mtb can reside either intracellularly within various immune cells or extracellularly. Within these different compartments, the bacilli can either be growing or quiescent and nongrowing. Caseous lesions are believed to harbor mostly extracellular nongrowing bacteria. The response of these physiologically different bacterial populations to antibiotic treatment is proposed to be heterogeneous, with nongrowing bacilli assumed to display phenotypic drug tolerance (i.e., they are not as readily killed by antibiotics) [19Dartois V. The path of anti-tuberculosis drugs: from blood to lesions to mycobacterial cells.Nat. Rev. Microbiol. 2014; 12: 159Crossref PubMed Scopus (265) Google Scholar]. We hypothesized that the two key elements in the clinical efficacy of PZA may be that the drug: (i) reaches the bacilli residing in all these different lesions; and (ii) is able to kill nongrowing drug-tolerant forms of the pathogen. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based imaging of drug distribution in TB-infected human lung tissue, we found that the distribution of drugs into the various lesion types is both drug and lesion type dependent. Most drugs do not penetrate all compartments in all lesions. By contrast, PZA distributes rapidly and homogenously into all lesions types, both cellular and caseous lesions, where larger and more hydrophobic drugs diffuse less effectively [20Prideaux B. et al.The association between sterilizing activity and drug distribution into tuberculosis lesions.Nat. Med. 2015; 21: 1223-1227Crossref PubMed Scopus (314) Google Scholar] (Figure 1A). Thus, PZA can reach pathogens residing within the various sites of infection within the lungs of patients with TB. To determine whether Mtb in caseum is indeed nongrowing and drug tolerant, we developed an ex vivo assay with caseum isolated from Mtb-infected rabbits. Indeed, under these conditions, Mtb does not grow (Figure 1B) and displays extreme drug tolerance to most drugs [21Sarathy J.P. et al.Extreme drug tolerance of Mycobacterium tuberculosis in caseum.Antimicrob. Agents Chemother. 2018; 62 (e02266–17)Crossref PubMed Scopus (107) Google Scholar]. PZA was found to display bactericidal activity in caseum, albeit at rather high concentrations, suggesting that it not only reaches drug-tolerant bacilli, but also kills them [21Sarathy J.P. et al.Extreme drug tolerance of Mycobacterium tuberculosis in caseum.Antimicrob. Agents Chemother. 2018; 62 (e02266–17)Crossref PubMed Scopus (107) Google Scholar] (Figure 1C). Despite these attractive pharmacokinetic and pharmacodynamic (PK/PD) properties, efficacy studies in different mouse and guinea pig models have yielded contradicting results [22Lanoix J.-P. et al.Selective inactivity of pyrazinamide against tuberculosis in C3HeB/FeJ mice Is best explained by neutral pH of caseum.Antimicrob. Agents Chemother. 2016; 60: 735-743Crossref PubMed Scopus (50) Google Scholar, 23Ahmad Z. et al.Dose-dependent activity of pyrazinamide in animal models of intracellular and extracellular tuberculosis infections.Antimicrob. Agents Chemother. 2011; 55: 1527-1532Crossref PubMed Scopus (54) Google Scholar, 24Irwin S.M. et al.Bedaquiline and pyrazinamide treatment responses are affected by pulmonary lesion heterogeneity in Mycobacterium tuberculosis infected C3HeB/FeJ mice.ACS Infect. Dis. 2016; 2: 251-267Crossref PubMed Scopus (83) Google Scholar]. To resolve these conflicting data, we used the rabbit model of active TB, a disease model that recapitulates the major lung lesions observed in human TB [25Subbian S. et al.Chronic pulmonary cavitary tuberculosis in rabbits: a failed host immune response.Open Biol. 2011; 1: 110016Crossref PubMed Scopus (83) Google Scholar]. With this model, we confirmed that PZA not only penetrates all lesion types, including caseous necrotic compartments, but also reduces bacterial burden and sterilizes both cellular and necrotic lesions [10Blanc L. et al.Impact of immunopathology on the antituberculous activity of pyrazinamide.J. Exp. Med. 2018; 215: 1975-1986Crossref PubMed Scopus (22) Google Scholar]. In-depth analyses of bacterial burden and cumulative burden at the lesion level revealed that the onset of bacterial killing by PZA is slow, suggesting that the rather weak in vitro and ex vivo activities of PZA limit its efficacy in vivo at clinically approved dosages [10Blanc L. et al.Impact of immunopathology on the antituberculous activity of pyrazinamide.J. Exp. Med. 2018; 215: 1975-1986Crossref PubMed Scopus (22) Google Scholar]. PK/PD modeling of PZA penetration in human lesions [20Prideaux B. et al.The association between sterilizing activity and drug distribution into tuberculosis lesions.Nat. Med. 2015; 21: 1223-1227Crossref PubMed Scopus (314) Google Scholar] was carried out to identify lesion types and lesion compartments where PZA achieves target concentrations and at what fraction of the dosing interval. The model revealed that PZA performed suboptimally in all lesion types due to its high MIC, with only a small fraction of the dosing interval above adequate concentrations, and resulting in a significant proportion of 'at-risk' patients (i.e., those achieving relevant concentrations for <4 h) [26Strydom N. et al.Tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: a mechanistic model and tool for regimen and dose optimization.PLoS Med. 2019; 16: e1002773Crossref PubMed Scopus (97) Google Scholar]. Given the common adverse effects of PZA due to hepatotoxicity, higher dosage is not a viable option. Taken together, these recent pharmacological findings provide an explanation for the clinically observed sterilizing and treatment-shortening effects of PZA: the drug penetrates all TB lung lesion compartments, including difficult-to-penetrate lesions, and kills nongrowing persisters that reside in caseum. However, consistent with its poor in vitro potency, concentrations required to kill Mtb in caseum are high and achieved for a small fraction of the dosing interval, and onset of lesion sterilization in vivo is slow. The finding that efficacy is potency limited suggests a way forward for the discovery of next-generation PZA: improving potency, while maintaining existing lesion penetration properties. However, structure-driven optimization of PZA will require elucidation of its molecular mechanism. Following early studies in which PZA demonstrated improved potency in media adjusted to acidic pH [27McDermott W. Tompsett R. Activation of pyrazinamide and nicotinamide in acidic environments in vitro.Am. Rev. Tuberc. 1954; 70: 748-754PubMed Google Scholar], it became common belief that PZA is active only at acidic pH and, therefore, that TB lung lesions are acidic [28Zhang Y. Mitchison D. The curious characteristics of pyrazinamide: a review.Int. J. Tuberc. Lung Dis. 2003; 7: 6-21PubMed Google Scholar]. In this context, and since POA is a weak acid (pKa = 2.9), an ionophore model was proposed and became the most widely accepted hypothesis of the MoA of PZA. POA, a carboxylic acid, shuttles protons from the extracellular acidic environment into the bacilli, causing collapse of membrane energetics and cytoplasmic acidification [29Zhang Y. et al.Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid.J. Antimicrob. Chemother. 2003; 52: 790-795Crossref PubMed Scopus (366) Google Scholar]. However, this model has been opposed by three lines of evidence. PZA and POA can inhibit growth of Mtb at neutral pH [16Via L.E. et al.Host-mediated bioactivation of pyrazinamide: implications for efficacy, resistance, and therapeutic alternatives.ACS Infect. Dis. 2015; 1: 203-214Crossref PubMed Scopus (60) Google Scholar,17Lanoix J.-P. et al.High systemic exposure of pyrazinoic acid has limited antituberculosis activity in murine and rabbit models of tuberculosis.Antimicrob. Agents Chemother. 2016; 60: 4197-4205Crossref PubMed Scopus (17) Google Scholar,30Peterson N.D. et al.Uncoupling environmental pH and intrabacterial acidification from pyrazinamide susceptibility in Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2015; 59: 7320-7326Crossref PubMed Scopus (55) Google Scholar,31den Hertog A.L. et al.PZA is active against Mycobacterium tuberculosis cultures at neutral pH with reduced temperature.Antimicrob. Agents Chemother. 2016; 60: 4956-4960Crossref PubMed Scopus (19) Google Scholar], implying that acidic pH is not essential for antibacterial activity of the drug. TB lesions are not necessarily acidic; rather, they present with a spectrum of pH from acidic to alkaline [10Blanc L. et al.Impact of immunopathology on the antituberculous activity of pyrazinamide.J. Exp. Med. 2018; 215: 1975-1986Crossref PubMed Scopus (22) Google Scholar,21Sarathy J.P. et al.Extreme drug tolerance of Mycobacterium tuberculosis in caseum.Antimicrob. Agents Chemother. 2018; 62 (e02266–17)Crossref PubMed Scopus (107) Google Scholar,22Lanoix J.-P. et al.Selective inactivity of pyrazinamide against tuberculosis in C3HeB/FeJ mice Is best explained by neutral pH of caseum.Antimicrob. Agents Chemother. 2016; 60: 735-743Crossref PubMed Scopus (50) Google Scholar,24Irwin S.M. et al.Bedaquiline and pyrazinamide treatment responses are affected by pulmonary lesion heterogeneity in Mycobacterium tuberculosis infected C3HeB/FeJ mice.ACS Infect. Dis. 2016; 2: 251-267Crossref PubMed Scopus (83) Google Scholar,32Kempker R.R. et al.Lung tissue concentrations of pyrazinamide among patients with drug-resistant pulmonary tuberculosis.Antimicrob. Agents Chemother. 2017; 61 (e00226–17)Crossref Scopus (39) Google Scholar,33Koller F. Leuthardt F. Nekrose und Autolyse Beitrag zur Kenntnis der dystrophischen Verkalkung.Klinische Wochenschrift. 1934; 13: 1527-1529Crossref Scopus (11) Google Scholar]. Importantly, unlike bona fide ionophores, such as carbonyl cyanide m-chlorophenyl hydrazine, POA does not cause rapid collapse of membrane potential or cytoplasmic acidification in Mtb [30Peterson N.D. et al.Uncoupling environmental pH and intrabacterial acidification from pyrazinamide susceptibility in Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2015; 59: 7320-7326Crossref PubMed Scopus (55) Google Scholar]. Thus, POA appears not to function as an ionophore in Mtb. Based primarily on biochemical evidence, several discrete proteins were proposed to be targeted by POA (Table 1). POA was suggested to inhibit fatty acid synthesis via FAS-I [34Zimhony O. et al.Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis.Nat. Med. 2000; 6: 1043Crossref PubMed Scopus (213) Google Scholar] and trans-translation, a process that frees ribosomes stalled during translation, by binding to ribosomal protein S1/RpsA [35Shi W. et al.Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis: a potential mechanism for shortening the duration of tuberculosis chemotherapy.Science. 2011; 333: 1630-1632Crossref PubMed Scopus (404) Google Scholar,36Shi W. et al.Introducing RpsA point mutations Δ438A and D123A into the Chromosome of Mycobacterium tuberculosis confirms their role in causing resistance to pyrazinamide.Antimicrob. Agents Chemother. 2019; 63 (e02681–18)Crossref Scopus (13) Google Scholar]. However, these suggested mechanisms have been called into question or directly contradicted (Table 1) [37Boshoff H.I. et al.Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I.J. Bacteriol. 2002; 184: 2167-2172Crossref PubMed Scopus (124) Google Scholar,38Dillon N.A. et al.Anti-tubercular activity of pyrazinamide is independent of trans-translation and RpsA.Sci. Rep. 2017; 7: 6135Crossref PubMed Scopus (35) Google Scholar]. Thus, FAS-I and RpsA appear not to present significant targets for POA. Recently, GpsI, a probable guanosine pentaphosphate synthase, was shown to bind POA at high (mM) concentrations. A polymorphism in the corresponding gene was identified in two clinical isolates [39Njire M. et al.Pyrazinoic acid inhibits a bifunctional enzyme in Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2017; 61 (e00070-17)Crossref PubMed Scopus (25) Google Scholar]. Whether GpsI is involved in the MoA of PZA remains to be determined. A summary of the currently proposed MoA of PZA/POA with key evidence arguing against these mechanisms is presented in Table 1. We also refer to the recent review by Anthony and colleagues in which the authors discuss evidence for and against the various proposed mechanisms [40Anthony R.M. et al.'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?.J. Antimicrob. Chemother. 2018; 73: 1750-1754Crossref PubMed Scopus (14) Google Scholar].Table 1Proposed MoA of POA against MtbModelProposed mechanismRefs supporting modelKey evidence opposing modelRefs opposing modelPOA acts as an ionophorePOA acts as a protonophore and shuttles protons from an acidic environment to the cytoplasm, resulting in cytoplasm acidification and collapse of membrane potential[29Zhang Y. et al.Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid.J. Antimicrob. Chemother. 2003; 52: 790-795Crossref PubMed Scopus (366) Google Scholar,60Zhang Y. et al.Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide.J. Bacteriol. 1999; 181: 2044-2049PubMed Google Scholar]➢POA does not induce acidification of cytoplasm or collapse of membrane potential➢POA inhibits Mtb growth at a neutral pH[16Via L.E. et al.Host-mediated bioactivation of pyrazinamide: implications for efficacy, resistance, and therapeutic alternatives.ACS Infect. Dis. 2015; 1: 203-214Crossref PubMed Scopus (60) Google Scholar,17Lanoix J.-P. et al.High systemic exposure of pyrazinoic acid has limited antituberculosis activity in murine and rabbit models of tuberculosis.Antimicrob. Agents Chemother. 2016; 60: 4197-4205Crossref PubMed Scopus (17) Google Scholar,30Peterson N.D. et al.Uncoupling environmental pH and intrabacterial acidification from pyrazinamide susceptibility in Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2015; 59: 7320-7326Crossref PubMed Scopus (55) Google Scholar,31den Hertog A.L. et al.PZA is active against Mycobacterium tuberculosis cultures at neutral pH with reduced temperature.Antimicrob. Agents Chemother. 2016; 60: 4956-4960Crossref PubMed Scopus (19) Google Scholar,42Gopal P. et al.Pyrazinamide resistance is caused by two distinct mechanisms: prevention of Coenzyme A depletion and loss of virulence factor synthesis.ACS Infect. Dis. 2016; 2: 616-626Crossref PubMed Scopus (58) Google Scholar]POA targets fatty acid synthase (FAS)-IPOA inhibits synthesis of fatty acids via inhibition of FAS-I[34Zimhony O. et al.Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis.Nat. Med. 2000; 6: 1043Crossref PubMed Scopus (213) Google Scholar]➢POA does not inhibit activity of FAS-I in vitro➢No in vitro or in vivo isolated PZA-resistant mutants contain mutations in FAS-I[37Boshoff H.I. et al.Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I.J. Bacteriol. 2002; 184: 2167-2172Crossref PubMed Scopus (124) Google Scholar,42Gopal P. et al.Pyrazinamide resistance is caused by two distinct mechanisms: prevention of Coenzyme A depletion and loss of virulence factor synthesis.ACS Infect. Dis. 2016; 2: 616-626Crossref PubMed Scopus (58) Google Scholar,44Gopal P. et al.In vivo-selected pyrazinoic acid-resistant Mycobacterium tuberculosis strains harbor missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1.ACS Infect. Dis. 2017; 3: 492-501Crossref PubMed Scopus (26) Google Scholar,50Gopal P. et al.Pyrazinoic acid inhibits mycobacterial Coenzyme A biosynthesis by binding to aspartate decarboxylase PanD.ACS Infect. Dis. 2017; 3: 807-819Crossref PubMed Scopus (42) Google Scholar]POA targets ribosomal protein S1/RpsAPOA inhibits trans-translation via binding to the ribosomal protein S1/RpsA[35Shi W. et al.Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis: a potential mechanism for shortening the duration of tuberculosis chemotherapy.Science. 2011; 333: 1630-1632Crossref PubMed Scopus (404) Google Scholar,36Shi W. et al.Introducing RpsA point mutations Δ438A and D123A into the Chromosome of Mycobacterium tuberculosis confirms their role in causing resistance to pyrazinamide.Antimicrob. Agents Chemother. 2019; 63 (e02681–18)Crossref Scopus (13) Google Scholar]➢POA does not inhibit trans-translation in vitro➢POA does not bind RpsA➢RpsA polymorphisms apparently associated with POA resistance do not cause resistance to POA➢Overexpression of rspA does not cause resistance➢No in vitro or in vivo isolated PZA-resistant mutants contain mutations in RpsA[38Dillon N.A. et al.Anti-tubercular activity of pyrazinamide is independent of trans-translation and RpsA.Sci. Rep. 2017; 7: 6135Crossref PubMed Scopus (35) Google Scholar,42Gopal P. et al.Pyrazinamide resistance is caused by two distinct mechanisms: prevention of Coenzyme A depletion and loss of virulence factor synthesis.ACS Infect. Dis. 2016; 2: 616-626Crossref PubMed Scopus (58) Google Scholar,44Gopal P. et al.In vivo-selected pyrazinoic acid-resistant Mycobacterium tuberculosis strains harbor missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1.ACS Infect. Dis. 2017; 3: 492-501Crossref PubMed Scopus (26) Google Scholar,50Gopal P. et al.Pyrazinoic acid inhibits mycobacterial Coenzyme A biosynthesis by binding to aspartate decarboxylase PanD.ACS Infect. Dis. 2017; 3: 807-819Crossref PubMed Scopus (42) Google Scholar]POA targets guanosine pentaphosphate synthase (GpsI)POA inhibits GpsI involved in nucleic acid and ppGpp metabolism. Note that POA binds to GpsI only at extremely high concentrations (mM)[39Njire M. et al.Pyrazinoic acid inhibits a bifunctional enzyme in Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2017; 61 (e00070-17)Crossref PubMed Scopus (25) Google Scholar]➢No in vitro or in vivo isolated PZA-resistant mutants contain mutations in GpsI[42Gopal P. et al.Pyrazinamide resistance is caused by two distinct mechanisms: prevention of Coenzyme A depletion and loss of virulence factor synthesis.ACS Infect. Dis. 2016; 2: 616-626Crossref PubMed Scopus (58) Google Scholar,44Gopal P. et al.In vivo-selected pyrazinoic acid-resistant Mycobacterium tuberculosis strains harbor missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1.ACS Infect. Dis. 2017; 3: 492-501Crossref PubMed Scopus (26) Google Scholar]POA targets quinolinic acid phosphoribosyl transferase (QAPRTase)POA, a structural analog of quinolinic acid, inhibits the catalytic activity of QAPRTase and, thus, de novo NAD biosynthesis[61Kim H. et al.Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide.PLoS One. 2014; 9: e100062Crossref PubMed Scopus (19) Google Scholar]➢POA treatment does not cause decrease in metabolites downstream of QAPRTase➢No in vitro or in vivo isolated PZA-resistant mutants contain mutations in QAPRTase[42Gopal P. et al.Pyrazinamide resistance is caused by two distinct mechanisms: prevention of Coenzyme A depletion and loss of virulence factor synthesis.ACS Infect. Dis. 2016; 2: 616-626Crossref PubMed Scopus (58) Google Scholar,44Gopal P. et al.In vivo-selected pyrazinoic acid-resistant Mycobacterium tuberculosis strains harbor missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1.ACS Infect. Dis. 2017; 3: 492-501Crossref PubMed Scopus