Towards a new tuberculosis drug: pyridomycin – nature's isoniazid
2012; Springer Nature; Volume: 4; Issue: 10 Linguagem: Inglês
10.1002/emmm.201201689
ISSN1757-4684
AutoresRuben C. Hartkoorn, Claudia Sala, João Neres, Florence Pojer, Sophie Magnet, Raju Mukherjee, Swapna Uplekar, Stefanie Boy‐Röttger, Karl‐Heinz Altmann, Stewart T. Cole,
Tópico(s)Pneumocystis jirovecii pneumonia detection and treatment
ResumoResearch Article17 September 2012Open Access Towards a new tuberculosis drug: pyridomycin – nature's isoniazid Ruben C. Hartkoorn Ruben C. Hartkoorn Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Claudia Sala Claudia Sala Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author João Neres João Neres Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Florence Pojer Florence Pojer Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Sophie Magnet Sophie Magnet Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Raju Mukherjee Raju Mukherjee Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Swapna Uplekar Swapna Uplekar Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Stefanie Boy-Röttger Stefanie Boy-Röttger Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Karl-Heinz Altmann Karl-Heinz Altmann Eidgenössische Technische Hochschule Zürich, Institut für Pharmazeutische Wissenschaften, HCI H 405, Zürich, Switzerland Search for more papers by this author Stewart T. Cole Corresponding Author Stewart T. Cole [email protected] Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Ruben C. Hartkoorn Ruben C. Hartkoorn Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Claudia Sala Claudia Sala Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author João Neres João Neres Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Florence Pojer Florence Pojer Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Sophie Magnet Sophie Magnet Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Raju Mukherjee Raju Mukherjee Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Swapna Uplekar Swapna Uplekar Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Stefanie Boy-Röttger Stefanie Boy-Röttger Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Karl-Heinz Altmann Karl-Heinz Altmann Eidgenössische Technische Hochschule Zürich, Institut für Pharmazeutische Wissenschaften, HCI H 405, Zürich, Switzerland Search for more papers by this author Stewart T. Cole Corresponding Author Stewart T. Cole [email protected] Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland Search for more papers by this author Author Information Ruben C. Hartkoorn1, Claudia Sala1, João Neres1, Florence Pojer1, Sophie Magnet1, Raju Mukherjee1, Swapna Uplekar1, Stefanie Boy-Röttger1, Karl-Heinz Altmann2 and Stewart T. Cole *,1 1Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland 2Eidgenössische Technische Hochschule Zürich, Institut für Pharmazeutische Wissenschaften, HCI H 405, Zürich, Switzerland *Tel: +41 21 693 1851; Fax +41 21 693 1790 EMBO Mol Med (2012)4:1032-1042https://doi.org/10.1002/emmm.201201689 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA-inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti-tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin-resistant mutants of Mycobacterium tuberculosis, whole-genome sequencing and genetic validation, we identified the NADH-dependent enoyl- (Acyl-Carrier-Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH-binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid-resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development. →See accompanying article http://dx.doi.org/10.1002/emmm.201201811 The paper explained PROBLEM: Even today, infection with Mycobacterium tuberculosis accounts for up to two million deaths annually. The effectiveness of current anti-tuberculosis drugs to combat these infections is increasingly compromised by the escalating prevalence of multi- and extensively drug-resistant tuberculosis. For these cases, the most effective anti-tubercular compounds such as isoniazid and rifampicin are no longer effective and this can result in mortality rates approaching 100% for patients with extensively drug-resistant tuberculosis. For these reasons, it is imperative to ensure that the pipeline of drug candidates to treat tuberculosis is well filled. RESULTS: We show here that the natural product pyridomycin is a very selective bactericidal compound against mycobacteria including Mycobacterium tuberculosis, the causative bacterium of tuberculosis in humans. By selecting and isolating M. tuberculosis mutants resistant to pyridomycin and sequencing their genome, we found that a single mutation in a gene named inhA is responsible for the resistance. InhA is already the target of the current frontline anti-tuberculosis agent isoniazid. However, most interestingly, no cross resistance was observed between pyridomycin and isoniazid, both in laboratory strains containing mutations in InhA or in the most frequently encountered isoniazid-resistant clinical isolates that contain mutations in katG (a gene required to activate isoniazid). We then present detailed genetic and biochemical studies to confirm that pyridomycin itself inhibits InhA and that in live bacteria, this inhibition leads to the depletion of mycolic acids, an essential cell wall component. Finally, studies of the crystal structure of the InhA protein and the pyridomycin-resistant form give valuable insight into the binding pocket of pyridomycin. IMPACT: Inhibition of InhA is one of the most effective means of killing Mycobacterium tuberculosis, and this is the mechanism behind one of the most potent anti-tubercular agents currently used: isoniazid. The increasing emergence of multi- and extensively drug-resistant tuberculosis (both of which are resistant to isoniazid) means that for these cases, this target can no longer be effectively inhibited by current therapy. Our finding that pyridomycin kills M. tuberculosis by inhibiting InhA (even in isoniazid-resistant clinical isolates) provides a promising basis for the development of pyridomycin or a related agent for the treatment of isoniazid-resistant tuberculosis. INTRODUCTION Today, infection with Mycobacterium tuberculosis accounts for up to two million deaths annually (Glaziou et al, 2009). Major confounding factors such as poverty, homelessness and the prevalence of HIV/AIDS (Harrington, 2010) mean that tuberculosis will indefinitely remain an important cause of morbidity and mortality throughout the world. Furthermore, despite the small, but growing number of drugs that are effective at killing M. tuberculosis, the current treatment is still burdened by its duration (typically 6 months for drug-sensitive strains) and the ever increasing number of multidrug (MDR) and extensively drug resistant (XDR) clinical isolates of M. tuberculosis (Cegielski, 2010). Together, this underlines the need for alternative therapeutic entities that can be used both to shorten the duration of therapy and to combat the growing problem of clinical drug resistance. Natural products have long provided a rich source of effective anti-tuberculosis agents. The most active of these in current use, the rifamycins (rifampicin, rifabutin and rifapentine), inhibit RNA polymerase and are crucial for front-line treatment of the disease. Furthermore, several other natural products such as the aminoglycosides (streptomycin, amikacin and kanamycin) and the peptide antibiotic (capreomycin) are part of the current portfolio of anti-tuberculosis drugs. The rich diversity of natural products represents a powerful tool for drug discovery, firstly, in the form of leads for potential anti-microbial agents and secondly, as a means of identifying those targets that are most vulnerable in the bacterium. In 1953, pyridomycin was first described as an antibiotic that exhibited specific activity against different mycobacteria including M. tuberculosis and M. smegmatis (Maeda et al, 1953). Pyridomycin (Fig 1A) is produced by Streptomyces pyridomyceticus (Maeda et al, 1953; Yagishita, 1954, 1955, 1957a, b) or Dactylosporangium fulvum (Shomura et al, 1986). Its biosynthesis was first studied in 1968 (Ogawara et al, 1968) and more recently in 2011 (Huang et al, 2011) when the involvement of both non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) was proposed. Despite this body of work, the mechanism of action of pyridomycin against M. tuberculosis is unknown, and its potential as an anti-tuberculosis compound has not been assessed. Figure 1. Chemical structure and intracellular activity of pyridomycin. A.. Chemical structure of pyridomycin. B.. The activity of pyridomycin on intracellular M. tuberculosis was tested in activated THP-1-derived macrophages. Cells were infected at an MOI of 1:1 with M. tuberculosis Erdman and treated with isoniazid (INH) at 1 µg/ml, rifampicin (RIF) at 1 µg/ml, streptomycin (STR) at 10 µg/ml or pyridomycin (PYR) at 10 µg/ml. Colony forming units (CFU) were determined after 7 days exposure to drugs. NT refers to the untreated sample and NT0 to untreated sample at time 0. The experiment was performed in duplicate and results are shown as mean values and standard errors. Download figure Download PowerPoint The aim of this study was to determine how pyridomycin kills M. tuberculosis and to identify its target. To achieve this, a combination of approaches involving resistance mapping, genetic validation, biochemistry, enzyme inhibition and X-ray crystallographic analysis of the target are described. The combined results unambiguously indicate that pyridomycin is a competitive inhibitor of the NADH-binding site of InhA, NADH-dependent enoyl-[Acyl-Carrier-Protein] reductase, the target of the two anti-tuberculosis pro-drugs isoniazid and ethionamide (Banerjee et al, 1994; Vilcheze et al, 2006). RESULTS Purification of pyridomycin Several strains of Streptomyces pyridomyceticus (NRRL B-2517, ISP-5024 and DSM40024) were initially tested for pyridomycin production with limited success, likely due to the presence of producing and non-producing populations in the same culture. Pyridomycin (Fig 1A) was, however, readily produced by and purified from Dactylosporangium fulvum (NRRL B-16292) with a yield of 20–40 mg/L at a purity >99% and with an NMR spectrum as previously reported (Kinoshita et al, 1989). Anti-bacterial properties of pyridomycin Pyridomycin has been described to act specifically against mycobacteria, with little or no activity against other Gram-positive and Gram-negative species (Maeda et al, 1953). In order to verify its spectrum of activity, the resazurin reduction microplate assay (REMA) was used to determine the minimum inhibitory concentration (MIC) for various bacteria. From Table 1, it can be clearly seen that pyridomycin is effective against all members of the Mycobacterium genus tested including M. tuberculosis (strain H37Rv, MIC = 0.31–0.63 µg/ml) and M. smegmatis (strain mc2 155, MIC = 0.62–1.25 µg/ml). Pyridomycin, however, showed no detectable activity against other bacteria, including the close relative C. glutamicum (all MIC > 100 µg/ml). These data therefore agree with earlier observations (Maeda et al, 1953; Maeda, 1957) and suggest that pyridomycin targets a mycobacterial component that is either sufficiently divergent or absent in other genera. Table 1. Bacterial susceptibility to pyridomycin as measured by resazurin reduction microtitre assay Bacterium Pyridomycin MIC (µg/ml) Mycobacterium tuberculosis 0.39 Mycobacterium bovis BCG 0.39 Mycobacterium smegmatis 0.78 Mycobacterium marinum 3.13 Mycobacterium abscessus 6.25 Mycobacterium bolletii 6.25 Mycobacterium massiliense 6.25 Mycobacterium avium 12.5 Corynebacterium glutamicum >100 Corynebacterium diphtheriae >100 Micrococcus luteus >100 Listeria monocytogenes >100 Staphylococcus aureus >100 Bacillus subtilis >100 Enterococcus faecalis >100 Escherichia coli >100 Pseudomonas putida >100 Pseudomonas aeruginosa >100 Salmonella typhimurium >100 Candida albicans >100 To further understand the properties of pyridomycin against M. tuberculosis, its minimum bactericidal concentration (MBC) was determined and its activity against non-replicating and intracellular M. tuberculosis measured. MBC data demonstrated that pyridomycin is bactericidal against M. tuberculosis H37Rv at concentrations of 0.62–1.25 µg/ml. Evaluation of pyridomycin activity against non-replicating M. tuberculosis using the streptomycin-starved 18b (ss18b) model (Sala et al, 2010) revealed that pyridomycin is not effective, thereby implying that it may target a function involved in active growth. Finally, the intracellular killing activity of pyridomycin was assessed ex vivo after infection of activated THP1-derived macrophages. The results indicated that, when left untreated for a 7-day period, intracellular M. tuberculosis grew by at least 3 logs, whilst exposure to both pyridomycin (10 µg/ml) and rifampicin (1 µg/ml) prevented any multiplication within the macrophages (Fig 1B). Further controls showed that streptomycin (10 µg/ml) had no impact on the growth of intracellular bacteria while isoniazid (1 µg/ml) was able to reduce the intracellular M. tuberculosis load by 1 log (Fig 1B). Pyridomycin is therefore clearly able to enter macrophages and arrest bacterial replication. Cytotoxicity of pyridomycin on human cell lines To determine whether pyridomycin is cytotoxic to human cells, the concentration-dependent cytotoxicity of the compound was assessed on two human cell lines. Data indicated that the amount of pyridomycin needed to kill 50% of HepG2 cells (human hepatic cell line) or A549 cells (human lung epithelium cell line) was 100 and 50 µg/ml, respectively. Pyridomycin therefore shows higher selectivity for M. tuberculosis compared to the human cells tested (selectivity index >100-fold), in agreement with a previous finding that pyridomycin shows low toxicity in an acute murine model following 800 mg/kg intraperitoneal injection (Maeda et al, 1953). Identification of the pyridomycin target The strategy to identify the target and mechanism of action of pyridomycin was to raise pyridomycin-resistant mutants and to pinpoint the genetic mutations responsible for this phenotype, anticipating that these mutations would be in the gene for the drug target. Resistant mutants of strain H37Rv were selected on solid medium containing pyridomycin at 10× MIC (3 µg/ml) and arose at a frequency of around 10−6. Of the 10 colonies selected for further analysis (PYR1 to 10), nine showed no change in the MIC to pyridomycin when re-tested by REMA, whereas mutant PYR7 retained a near 10-fold increase in its resistance level compared to the parent H37Rv (Fig 2). This phenotype was stably maintained and mutant PYR7 remained fully susceptible to isoniazid, moxifloxacin and rifampicin like wild-type H37Rv (Fig 2). Figure 2. Genetic validation of InhA as the target of pyridomycin. A-C.. The compound susceptibility of wild-type H37Rv transformed with the control vector pMV261 (filled squares), pMVinhA (open squares), pMVinhA (S94A) (open triangle) or pMVinhA (D148G) (open circle) to: (A) moxifloxacin, (B) isoniazid or (C) pyridomycin. D-F.. The compound susceptibility of pyridomycin-resistant mutant PYR7 transformed with the control vector pMV261 (filled squares), pMVinhA (open squares), pMVinhA (S94A) (open triangle) or pMVinhA (D148G) (open circle) to: (D) moxifloxacin, (E) isoniazid or (F) pyridomycin. Download figure Download PowerPoint To identify the single nucleotide polymorphisms (SNPs) or insertion/deletions (INDELs) responsible for the pyridomycin resistance, the genomes of both PYR7 and the parental strain were sequenced to near completion by the Illumina protocol. Ninety-eight percent of the reads were successfully mapped to the H37Rv reference genome (Cole et al, 1998) resulting in an average 300-fold coverage. Comparison of the PYR7 and H37Rv assemblies revealed 63 SNPs of which 53 mapped to the repetitive PE and PPE gene families and were therefore discarded. Of the remaining 10 SNPs, nine were synonymous. The only non-synonymous mutation found was an a443g transition in inhA resulting in replacement of the aspartic acid at position 148 by a glycine (D148G). This missense mutation was subsequently confirmed by conventional Sanger sequencing. With reference to previously published structures of the NADH-dependent enoyl-ACP reductase InhA, Asp148 was found to be located near the NADH binding pocket (Dessen et al, 1995; Dias et al, 2007; Molle et al, 2010; Oliveira et al, 2006; Rozwarski et al, 1999; Vilcheze et al, 2006). Genetic validation of InhA as the target of pyridomycin To genetically validate InhA as the target of pyridomycin, we evaluated whether over expression of inhA caused an increase in resistance to the antibiotic in wild-type M. tuberculosis. For this purpose, we transformed strain H37Rv with a plasmid carrying the inhA gene under the control of the hsp60 promoter (pMVinhA; Larsen et al, 2002). In Fig 2, it can be seen that H37Rv::pMVinhA displayed a 15-fold higher MIC for pyridomycin compared to the control strain H37Rv::pMV261 (from 0.31 to 5 µg/ml). When the same experiment was performed in the PYR7 background, no complementation of the resistant phenotype was observed, indicating that the associated mutation was dominant. Similar to the wild-type strain, we noticed a four-fold increase in resistance in PYR7::pMVinhA compared to the empty vector (PYR7::pMV261; from 2.5 to 10 µg/ml; Fig 2). In control experiments, overexpression of inhA also led to increased isoniazid resistance in both strains whilst not impacting the MIC of moxifloxacin (Fig 2). Together, these genetic data strongly suggest that InhA is the target of pyridomycin. To further corroborate that the D148G mutation in inhA was indeed responsible for pyridomycin resistance, we overexpressed this allele in H37Rv and compared its effect with that of a well-characterized mutation associated with isoniazid resistance, InhA (S94A) (Vilcheze et al, 2006). Results presented in Fig 2 clearly show that, compared to overexpression of wild-type InhA (pMVinhA), overexpression of InhA (D148G) causes four-fold greater resistance to pyridomycin while overexpression of InhA (S94A) conferred only two-fold resistance. Furthermore, overexpression of InhA (D148G) had no impact on the MIC for isoniazid compared to overexpression of wild type InhA, while, as expected, overexpressing InhA (S94A) increased resistance around four-fold (Fig 2). None of the mutations affected the MIC for moxifloxacin (Fig 2). Collectively, the data prove that the D148G mutation in InhA is responsible for resistance to pyridomycin, whilst not noticeably affecting isoniazid activity. In addition to isoniazid, InhA is also the target of ethionamide and triclosan. Susceptibility studies using these compounds on H37Rv and PYR7 indicate that both strains are equally sensitive with MICs of 2.0 and 12.5 µg/ml, respectively. This lack of cross-resistance indicates that D148G in InhA is likely to have no impact on the binding to InhA of either triclosan or the active metabolite of ethionamide, the ethionamide-NAD adduct. Susceptibility of isoniazid-resistant clinical isolates to pyridomycin Since our findings indicated that pyridomycin has the same target as isoniazid, we investigated whether isoniazid-resistant clinical isolates of M. tuberculosis retained susceptibility to pyridomycin. As isoniazid is a pro-drug, clinically relevant mutations that confer resistance are frequently found in the katG gene encoding the catalase–peroxidase required for isoniazid bio-activation or, less commonly, in the promoter region of inhA, which increases expression of the protein. Of the eight independent isoniazid-resistant clinical isolates analysed, four had mutations in katG (S315T), three in the promoter region of inhA [c (−15)t] and one isolate carried both mutations (Table 2). Analysis of the drug susceptibility of these isolates confirmed that all strains carrying the katG mutation displayed a high level of resistance to isoniazid (MIC >10 µg/ml) and those isolates carrying only the inhA promoter mutation showed intermediate isoniazid resistance (MIC = 1.25 µg/ml) compared to H37Rv (0.16 µg/ml; Table 2). On the contrary, isolates carrying the katG mutations showed no resistance to pyridomycin (MIC = 0.3–0.6 µg/ml), while a mutation in the inhA promoter resulted in increased pyridomycin resistance (MIC = 2.5–5 µg/ml; Table 2). For all clinical isolates tested, the susceptibility to moxifloxacin was similar to wild-type (MIC = 0.03–0.10 µg/ml). Thus, isoniazid-resistant clinical isolates carrying the inhA [c (−15)t] promoter mutation displayed cross-resistance with pyridomycin, whereas the more common katG (S315T) isoniazid-resistant mutants retained full sensitivity to the antibiotic. Table 2. Pyridomycin activity against isoniazid-resistant clinical isolates of M. tuberculosis Isolate KatG genotypea inhA promoter genotypeb MIC (µg/ml) Isoniazid Pyridomycin Moxifloxacin H37Rv wt wt 0.16 0.31 0.03 1 S315T wt >10 0.63 0.03 2 S315T wt >10 0.50 0.05 3 S315T wt >10 0.50 0.05 4 S315T wt >10 0.31 0.03 5 S315T c (−15)t >10 2.5 0.02 6 wt c (−15)t 1.25 3.75 0.10 7 wt c (−15)t 1.25 3.75 0.06 8 wt c (−15)t 1.25 5 0.06 wt, wild-type. a Numbering refers to the KatG protein sequence. b Numbering refers to the inhA coding sequence, with +1 corresponding to the first base of the ATG start codon. Inhibition of mycolic acid synthesis by pyridomycin It has been elegantly demonstrated that inhibition of InhA by isoniazid in M. tuberculosis leads to the specific depletion of mycolic acids from the bacterial cell wall without affecting fatty acid synthesis (Vilcheze et al, 2006). To show that pyridomycin inhibition of InhA also results in inhibition of mycolic acid synthesis, the mycolic and fatty acid content of M. tuberculosis was determined in the presence and absence of pyridomycin by radiometric thin layer chromatography (TLC). We found that pyridomycin caused a concentration-dependent reduction of mycolic acid synthesis (alpha-, methoxy- and keto-mycolic acids) whilst not affecting the fatty acid content (Fig 3). Furthermore, when performing the same experiment on PYR7, over five-fold higher pyridomycin concentrations were needed to inhibit mycolic acid biosynthesis consistent with the resistance level observed. Both H37Rv and PYR7 behaved similarly when the assay was repeated in the presence of isoniazid (Fig 3). Indeed, the latter caused a concentration-dependent decrease in the amount of mycolic acids in H37Rv and was equally effective at inhibiting mycolic acid synthesis in PYR7. Taken together, these data confirm that pyridomycin targets mycolic acid synthesis and demonstrate that the InhA (D148G) enzyme in PYR7 is much less susceptible to pyridomycin inhibition. Figure 3. Inhibition of mycolic acid synthesis by pyridomycin. The fatty acid methyl ester (FAMEs) and mycolic acid methyl ester (MAMEs) profiles of wild-type H37Rv and pyridomycin-resistant mutant PYR7 were evaluated by thin-layer chromatography. Both strains were treated with different concentrations of isoniazid and pyridomycin for 3 h and labeled with [1,2-14C]-acetate. A.. 14C-labeled FAMEs and MAMEs were separated by thin-layer chromatography and detected by autoradiography. B,C.. Quantification of the MAME band intensity relative to the density of the FAMEs illustrates the inhibition of MAME synthesis by pyridomycin (B) and isoniazid (C) in H37Rv (black squares) and PYR7 (open circles). Download figure Download PowerPoint In vitro inhibition of InhA by pyridomycin Inhibition of purified InhA by pyridomycin was studied to investigate if pyridomycin alone can inhibit the enzyme or whether in vivo bio-activation by an intracellular process is needed. InhA, InhA (S94A) and InhA (D148G) were successfully expressed and purified. All three enzymes were catalytically active and oxidized NADH in the presence of the substrate 2-trans-octenoyl-CoA (OcCoA). Initial experiments determined the NADH-binding constant (Km) and confirmed that for InhA (S94A) it was around 6.5 times higher than for wild-type InhA (Table 3) as reported previously (Quemard et al, 1995; Rawat et al, 2003; Vilcheze et al, 2006). Surprisingly, we found that the Km of InhA (D148G) for NADH was 14-fold greater than for wild-type InhA (Table 3) suggesting a lower affinity for NADH in the D148G mutant. All the enzymes had a similar Vmax (around 0.52 µmol/min/mg) (Table 3). Enzyme inhibition studies showed that pyridomycin was able to inhibit both wild-type InhA and InhA (S94A) at a similar Ki (6.5 and 4.55 µM, respectively) (Table 3). InhA (D148G) could not be inhibited at all by pyridomycin at concentrations below 18.6 µM. Statistical analysis of inhibition of both wild-type InhA and InhA (S94A) by pyridomycin favours a model of competitive inhibition with NADH as indicated by similar y-axis intercepts on Lineweaver–Burk plots (Fig 4). These data prove that pyridomycin is the pharmacophore that inhibits InhA, and this activity is achieved by competitive inhibition of the NADH-binding site. Additionally, these biochemical and enzymological results confirm that InhA (D148G) is more resistant to pyridomycin while InhA (S94A) is as susceptible as the wild-type enzyme. Table 3. In vitro kinetic parameters of M. tuberculosis InhA and its inhibition by pyridomycin Wild-type InhA InhA (S94A) InhA (D148G) NADH Km (µM) 13.5 ± 2.3 83.5 ± 9.5 190 ± 16 NADH Vmax (µmol/min/mg) 0.52 ± 0.03 0.50 ± 0.02 0.54 ± 0.3 Pyridomycin Ki (µM) 6.5 ± 1.2 5.0 ± 0.4 No inhibition at 18.6 µM Figure 4. Inhibition of purified InhA by pyridomycin. A-C.. Lineweaver–Burk plot showing the competitive inhibition of wild-type InhA (A), InhA (S94A) (B) and InhA (D148G) (C) by pyridomycin in the presence of NADH. Download figure Download PowerPoint Crystal structures of InhA (D148G), wild-type InhA and InhA (S94A) To further investigate the mode of binding of pyridomycin to InhA at the atomic level, we crystallized and solved the structures of wild-type InhA and InhA (S94A) mutant in complex with NADH as previously published (Dessen et al, 1995; Dias et al, 2007; Molle et al, 2010; Oliveira et al, 2006; Rozwarski et al, 1999; Vilcheze et al, 2006). In an attempt to obtain crystal structures of InhA in complex with pyridomycin, pre-crystallized InhA:NADH or InhA (S94A):NADH crystals were soaked in a pyridomycin solution. On penetration of pyridomicin, the crystals turned yellow but lost their ability to diffract. This suggests that pyridomycin may induce major conformational changes upon binding to the NADH co-factor pocket of InhA. For control purposes, InhA:NADH crystals were also soaked with triclosan, an inhibitor of the enoyl-ACP substrate binding site of InhA, and the structure successfully solved, thereby ruling out technical issues with soaking (data not shown). As an alternative strategy to define the pyridomycin binding site in InhA attempts were made to co-crystallize InhA or InhA (S94A) in the presence of pyridomycin alone or with the octenoyl CoA substrate; however, despite testing over 1000 conditions, no diffracting crystals have been obtained to date. The D148G mutation leads to pyridomycin resistance as well as to a decrease in NADH affinity (Table 3). To determine the molecular basis for this resistance, we crystallized InhA (D148G) in the presence or absence of NADH. As with InhA and InhA (S94A), we obtained crystals only in presence o
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