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Pyrazolo[1,5- a ]pyridine Inhibitor of the Respiratory Cytochrome bcc Complex for the Treatment of Drug-Resistant Tuberculosis
Abstract
Respiration is a promising target for the development of new antimycobacterial agents, with a growing number of compounds in clinical development entering this target space. However, more candidate inhibitors are needed to expand the therapeutic options available for drug-resistant Mycobacterium tuberculosis infection. Here, we characterize a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5-a]pyridine-3-carboxamide. TB47 is active (MIC between 0.016 - 0.500 µg/mL) against a panel of 56 M. tuberculosis clinical isolates, including 37 multi-drug resistant and 2 extensively-drug resistant strains. Pharmacokinetic and toxicity studies showed promising profiles, including negligible CYP450 interactions, cytotoxicity and hERG channel inhibition. Consistent with other reported QcrB inhibitors, TB47 inhibits oxygen consumption only when the alternative oxidase, cytochrome bd, is deleted. A point mutation in the qcrB cd2-loop (H190Y, M. smegmatis numbering) rescues the inhibitory effects of TB47. Metabolomic profiling of TB47-treated M. tuberculosis H37Rv cultures revealed accumulation of steps in TCA cycle and pentose phosphate pathway that are linked to reducing-equivalents, suggesting that TB47 causes metabolic redox stress. In mouse infection models, a TB47 monotherapy was not bactericidal. However, TB47 was strongly synergistic with pyrazinamide and rifampicin, suggesting a promising role in combination therapies. We propose that TB47 is an effective lead compound for the development of novel Tuberculosis chemotherapies.
... An analogous pyrazolo [1,5-a]pyridine-3-carboxamide derivative TB47 ( Figure 6) was also identified as a preclinical anti-TB candidate that inhibits QcrB [48,49,60]. TB47 exhibited potent anti-TB activities (MIC = 0.016-0.500 ...
... TB47 exhibited potent anti-TB activities (MIC = 0.016-0.500 µg/mL) against a panel of M. tb clinical isolates, including various MDR and XDR strains [60]. TB47 also showed promising PK and toxicity profiles, whereby it displayed negligible cytotoxicity (IC 50 > 100 µM against both Vero and HepG2 cell lines), CYP450 interactions (IC 50 > 20 µM) and hERG channel inhibition (IC 50 > 30 µM) [60]. ...
... µg/mL) against a panel of M. tb clinical isolates, including various MDR and XDR strains [60]. TB47 also showed promising PK and toxicity profiles, whereby it displayed negligible cytotoxicity (IC 50 > 100 µM against both Vero and HepG2 cell lines), CYP450 interactions (IC 50 > 20 µM) and hERG channel inhibition (IC 50 > 30 µM) [60]. In mouse infection models, although TB47 was not bactericidal as a monotherapy, it displayed a strong synergism with PZA and RIF, indicating its potential when combined with other anti-TB drugs [60]. ...
Mycobacterium tuberculosis (M. tb), the causative agent of TB, is a recalcitrant pathogen that is rife around the world, latently infecting approximately a quarter of the worldwide population. The asymptomatic status of the dormant bacteria escalates to the transmissible, active form when the host’s immune system becomes debilitated. The current front-line treatment regimen for drug-sensitive (DS) M. tb strains is a 6-month protocol involving four different drugs that requires stringent adherence to avoid relapse and resistance. Poverty, difficulty to access proper treatment, and lack of patient compliance contributed to the emergence of more sinister drug-resistant (DR) strains, which demand a longer duration of treatment with more toxic and more expensive drugs compared to the first-line regimen. Only three new drugs, bedaquiline (BDQ) and the two nitroimidazole derivatives delamanid (DLM) and pretomanid (PMD) were approved in the last decade for treatment of TB—the first anti-TB drugs with novel mode of actions to be introduced to the market in more than 50 years—reflecting the attrition rates in the development and approval of new anti-TB drugs. Herein, we will discuss the M. tb pathogenesis, current treatment protocols and challenges to the TB control efforts. This review also aims to highlight several small molecules that have recently been identified as promising preclinical and clinical anti-TB drug candidates that inhibit new protein targets in M. tb.
... To identify alternative targets of HM2-16F, we challenged M. tuberculosis H37Rv with varying concentrations of HM2-16F (1-10× MIC) and identified changes in intracellular metabolites by LC/MS-MS ( Supplementary Fig. 6). We considered two different scenarios for biological significance: (1) sub-saturated changes in metabolites, where changes are in a dose-dependent manner 30 and (2) saturated changes, where the three measurements showed a consistent fold-change in metabolites regardless of HM2-16F concentration. ...
... However, addition of HM2-16F to M. smegmatis ΔcydAB-MtbCydABDC + caused significant inhibition of the OCR suggesting this inhibition was dependent on the presence of cytochrome bd. When IMVs were preincubated with the potent cytochrome bcc-aa 3 oxidase inhibitor TB47 30 , the OCR in the M. smegmatis ΔcydAB-pYUB28b mutant was completely inhibited demonstrating that oxygen consumption in this genetic background was entirely mediated by cytochrome bcc-aa 3 oxidase. Oxygen consumption was restored in M. smegmatis ΔcydAB-MtbCydABDC + that was TB47-insensitive as this was mediated by cytochrome bd, and HM2-16F addition inhibited this OCR (Fig. 6b, top panel). ...
... Liquid chromatography-mass spectrometry (LC-MS) metabolomics. Filtercultured M. tuberculosis strain H37Rv was first grown for 5 days in 7H10 agar media to expand biomass, and then moved to fresh 7H10 medium containing compounds or a vehicle control (DMSO) for a 24 h exposure 30 . M. tuberculosis metabolism was quenched by plunging M. tuberculosis-laden filters into extraction buffer (acetonitrile: methanol: H 2 O = 40:40:20), which was precooled to −40°C on dry ice 49 . ...
Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis. Derivatives of the FDA-approved drug, amiloride, can eliminate drug-resistant Mycobacterium tuberculosis in vitro by interfering with bacterial energy conservation.
... The reliance on the PMF and ATP homeostasis thus highlights the importance of the mycobacterial proton-pumping cytochrome bcc-aa 3 supercomplex, which consists of a bcc menaquinol reductase (complex III, CIII) and an aa 3 oxidase (complex IV, CIV) that are tightly associated (Gong et al., 2018;Kim et al., 2015;Megehee et al., 2006). Several studies support the attractiveness of cytochrome bccaa 3 for mycobacterial drug development (de Jager et al., 2020;Liu et al., 2019;Lu et al., 2019;Pethe et al., 2013;Scherr et al., 2018). Given the strict sequence conservation of this complex (Figure 1), broad-spectrum activity of a therapeutic within the pathogenic mycobacteria is likely (Lee et al., 2020b). ...
... Interestingly, all the cytochrome bcc-aa 3 inhibitors published to date appear to target the QcrB subunit ( Figure 1) of the cytochrome bcc complex and are likely bound to the menaquinol-binding (Qp) site of the QcrB subunit (Lee et al., 2020b). The most advanced of these are Q203 and TB47, which have been shown to clear infections due to M. tuberculosis (de Jager et al., 2020;Lu et al., 2019;Pethe et al., 2013) and Mycobacterium ulcerans Scherr et al., 2018). Q203 has recently completed phase II clinical trials for TB treatment ( ClinicalTrials. ...
... The highly conserved residues that are involved in the binding of these two molecules in this region ( Figure 6-figure supplement 1) suggest a consistent overall fold and binding site exists in mycobacteria. This is also in agreement with the fact that Q203 and TB47 show antimycobacterial activity across many species (de Jager et al., 2020;Liu et al., 2019;Lu et al., 2019;Pethe et al., 2013;Scherr et al., 2018). It is worth noting that the structures of cytochrome bcc from M. tuberculosis and M. smegmatis have high similarity ( Figure 2-figure supplement 5), and no steric hindrance is observed between the Q203 and M. smegmatis cytochrome bcc ( Figure 6). ...
Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrB Thr ³¹³ and QcrB Glu ³¹⁴ , residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.
... More seriously, TB resistant to isoniazid and rifampin plus other drug(s) needs to be treated longer and results in a higher death rate (3). Considering side effects of injectable drugs and accompanying extensive resistance to fluoroquinolones (FQs) (4)(5)(6) and pyrazinamide (Z) (7-10) by drug-resistant TB (DR-TB), we sought to identify a fully oral pan-TB regimen by evaluating the effect of replacing FQ and/or Z with the clinically effective off-patent linezolid (N) (11) in regimens containing a new antimycobacterial candidate, TB47 (T) (12)(13)(14)(15)(16), and clofazimine (C), the effect of which further stresses the benefit of dual or multiple targeting of the electron transport chain in enhanced killing of mycobacteria (15,(17)(18)(19)(20). ...
... Such steps have been taken with the recent FDA's approval of the combination of bedaquiline, pretomanid, and linezolid (BPaL) (11,31). TB47, a novel antimycobacterial candidate (12,14) that entered good laboratory practice and safety assessment in 2021, has demonstrated good synergy with other anti-TB agents (14)(15)(16)21). A regimen from our previous study (16) was used as a positive control, which is a second-line regimen containing C (22) and T. The use of this second-line regimen is limited by prevalence of resistance to FQs and Z in many DR-TB patients (4,7), especially in countries with a high TB burden (5,6). ...
... Such steps have been taken with the recent FDA's approval of the combination of bedaquiline, pretomanid, and linezolid (BPaL) (11,31). TB47, a novel antimycobacterial candidate (12,14) that entered good laboratory practice and safety assessment in 2021, has demonstrated good synergy with other anti-TB agents (14)(15)(16)21). A regimen from our previous study (16) was used as a positive control, which is a second-line regimen containing C (22) and T. The use of this second-line regimen is limited by prevalence of resistance to FQs and Z in many DR-TB patients (4,7), especially in countries with a high TB burden (5,6). ...
TB47, a new drug candidate targeting QcrB in the electron transport chain, has shown a unique synergistic activity with clofazimine and formed a highly sterilizing combination. Here, we investigated the sterilizing effects of several all-oral regimens containing TB47 + clofazimine + linezolid as a block and the roles of fluoroquinolones and pyrazinamide in them. All these regimens cured tuberculosis within 4 to 6 months in a well-established mouse model and adding pyrazinamide showed significant difference in bactericidal effects.
... Like Q203, TB47 acts by targeting the QcrB subunit; therefore, the pyrazolopyridine carboxyamides class is identified as a novel scaffold for QcrB inhibitors [172]. TB47 blocks the cytochrome bc1 complex, thereby causing a reduction in intracellular ATP and eventually inhibiting the growth of Mtb [172,173]. In order to understand its mode of action, TB47 was docked into the quinol oxidation site (Qp) of the QcrB, whereby potential hydrogen In vitro studies on interactions of SQ-109 with other drugs showed that it does not interact positively with either ethambutol or pyrazinamide; however, SQ-109 was synergistic with isoniazid when both drugs were used at 0.5 MIC [140]. ...
... This eventually led to the discovery of TB47, a pyrazolo[1,5-a]pyridine analog of Q203 discovered through scaffold hopping [171]. Like Q203, TB47 acts by targeting the QcrB subunit; therefore, the pyrazolopyridine carboxyamides class is identified as a novel scaffold for QcrB inhibitors [172]. TB47 blocks the cytochrome bc1 complex, thereby causing a reduction in intracellular ATP and eventually inhibiting the growth of Mtb [172,173]. ...
... Like Q203, TB47 acts by targeting the QcrB subunit; therefore, the pyrazolopyridine carboxyamides class is identified as a novel scaffold for QcrB inhibitors [172]. TB47 blocks the cytochrome bc1 complex, thereby causing a reduction in intracellular ATP and eventually inhibiting the growth of Mtb [172,173]. In order to understand its mode of action, TB47 was docked into the quinol oxidation site (Qp) of the QcrB, whereby potential hydrogen bonding interactions between the amide of TB47 and the glutamate residue (Glu314) of the Qp binding site were observed [174]. ...
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a curable airborne disease currently treated using a drug regimen consisting of four drugs. Global TB control has been a persistent challenge for many decades due to the emergence of drug-resistant Mtb strains. The duration and complexity of TB treatment are the main issues leading to treatment failures. Other challenges faced by currently deployed TB regimens include drug-drug interactions, miss-matched pharmacokinetics parameters of drugs in a regimen, and lack of activity against slow replicating sub-population. These challenges underpin the continuous search for novel TB drugs and treatment regimens. This review
summarizes new TB drugs/drug candidates under development with emphasis on their chemical classes, biological targets, mode of resistance generation, and pharmacokinetic properties. As effective TB treatment requires a combination of drugs, the issue of drug-drug interaction is, therefore, of great concern; herein, we have compiled drug-drug interaction reports, as well as efficacy reports for drug combinations studies involving antitubercular agents in clinical development.
... TB47 is an imidazopyridine amide class of antibiotics [5,6] that targets the QcrB subunit in cytochrome bc1 oxidase complex [6,7]. TB47 can block the cytochrome bc1 complex, thereby causing a reduction of intracellular ATP and eventually inhibiting the growth of M. tuberculosis. ...
... The target of TB47 is similar to Q203 [8,9], however, little is known about the regimen containing Q203 in combination with drugs having expired patents (DEP). Our recently published studies revealed that TB47 had a bacteriostatic activity against M. tuberculosis and highly bactericidal activity against M. ulcerans [6,7]. It is worth noting that the complementary pathway, which consists of cytochrome bd oxidase (Cyt-bds) in M. tuberculosis, can also protect M. tuberculosis against the killing effect of TB47 [6]. ...
... An acute TB mouse model was used to test the activity of TB47 alone, as depicted in Table 1a. Briefly, BALB/c mice infected with UAlRv were continuously treated by oral gavage for twelve days, starting from day 1 post-infection [7,13]. To test the efficacy of regimens, studies 2 and 3 were designed as shown in Table 1b and 1c, respectively. ...
Multidrug-resistant tuberculosis (MDR-TB) remains a serious public health threat worldwide. To date, the anti-TB activity of TB47 (T), an imidazopyridine amide class of antibiotics targeting QcrB in the electron transport chain, has not been systematically evaluated, especially in a new regimen against MDR-TB. This study employed both macrophage infection and a mouse model to test the activity of T alone or in combination with other antimicrobial agents. Different regimens containing amikacin (A), levofloxacin (L), ethambutol (E), and pyr-azinamide (Z) + clofazimine (C)/T were evaluated in the mouse model. The bacterial burdens of mice from different groups were monitored at different time points while relapse was assessed 6 months after treatment cessation. Colonies obtained at relapse underwent drug susceptibility testing. We found that T exhibited highly synergistic bactericidal activity with C in all models. Adding T to ALEZC might shorten the MDR-TB treatment duration from ≥ 9 months to ≤ 5months, as five months of treatment with ALEZCT achieved zero relapse rates in 2 animal experiments. These findings indicate that T exhibits a highly synergistic sterilizing activity when combined with C. All isolates from relapsing mice remained sensitive to each drug, suggesting that the relapse was not due to drug resistance but rather associated with the type of regimen.
... The menaquinol oxidation site of the mycobacterial QcrB was determined by Lu et al. [36] to be the target of the lead compound 48. This led to the discovery of pyrazolopyridine carboxyamides as a fresh framework for QcrB inhibitors. ...
... Additionally, the in vivo activity of 46 was examined. Depending on the cell line used (THP-1, VERO, or HepG2), the selectivity index (bacterial MIC/human cell line IC 50 ) of compound 48 ranges from 1200 to >3330 and exhibits no action against a panel of seven cytochromes P450 enzymes up to a concentration of 20 M. Similarly, in hERG channel activity no cardiotoxicity was observed above the IC 50 value of 30 M. Compound 48 exhibited high oral bioavailability in rats and the absence of toxicity in both human cell lines and rat models[36]. The structures of Pyrazolo[1,5-a] pyridine derivatives displaying potent anti-tubercular activity are shown inFig. ...
Background
Fused nitrogen-containing heterocyclic compounds have been identified to display a prominent role in medicinal chemistry, biochemistry, and other streams of science. Countless derivatives of pyrazolo[1,5-a] pyridine have been investigated by researchers for their distinct pharmacological characterization. In this article, we have revealed and arranged the various routes of synthesis and therapeutic activities such as dopamine binding affinity, kinase inhibitory activity, and PDE inhibitors of pyrazolo[1,5-a]pyridine-containing compounds which have been explored till now. The remarkable outcomes obtained via in vitro as well as in vivo profile screening of this moiety and its derivatives lead this scaffold to be recognized to a greater extent and examined further for better results.
Conclusion
This review will give medicinal chemists a flying-bird eye catch view of pyrazolo[1,5-a] pyridine derivatives which will help them to design and synthesize potential compounds bearing this moiety.
... TB47 has a close structural similarity with Telecebec (Fig. 3A). The mechanism of action, binding site in QcrB, interaction with the residues of QcrB, and SAR of TB47 is similar to Telecebec (Fig. 3A and Fig. 3B) [21,43,44]. TB47 has demonstrated MIC values in the 0.16-0.50 ...
... TB47 also showed negligible cytotoxicity, CYP450 interactions, and hERG channel inhibition in animal models. TB47 also displayed a strong synergistic anti-TB effect in combination with rifampicin, pyrazinamide, and clofazimine [44,45]. TB47 is unveiled in Example 24 as compound TJ830047 in US10155756B2, which claims many pyrazolo[1,5-a]pyridine derivatives as ant-TB agents [46]. ...
The unmet medical need for drug-resistant tuberculosis (DRTB) is a significant concern. Accordingly, identifying new drug targets for tuberculosis (TB) treatment and developing new therapies based on these drug targets is one of the strategies to tackle DRTB. QcrB is an innovative drug target to create treatments for DRTB. This article highlights QcrB inhibitors and their therapeutic compositions for treating TB. The literature for this article was gathered from PubMed and free patent databases utilizing different keywords related to QcrB inhibitor-based inventions. The data was collected from the conceptualization of telacebec (2010) QcrB to December 2022. A little interesting and encouraging research has been performed on QcrB inhibitors. Telacebec and TB47 are established QcrB inhibitors in the clinical trial. The inventive QcrB inhibitor-based drug combinations can potentially handle DRTB and reduce the TB therapy duration. The authors anticipate great opportunities in fostering QcrB inhibitor-based patentable pharmaceutical inventions against TB. Drug repurposing can be a promising strategy to get safe and effective QcrB inhibitors. However, developing drug resistance, drug tolerance, and selectivity of QcrB inhibitors for Mtb will be the main challenges in developing effective QcrB inhibitors. In conclusion, QcrB is a promising drug target for developing effective treatments for active, latent, and drug-resistant TB. Many inventive and patentable combinations and compositions of QcrB inhibitors with other anti-TB drugs are anticipated as future treatments for TB.
... 117,249 In 2019, Lu et al. published additional assessments of the QcrB inhibitor TB47 (99) (Figure 12), a Q203 mimic designed by scaffold hopping that was disclosed by these investigators in 2015. 253,254 Pyrazolo[1,5-a]pyridine 99 was noncytotoxic (CC 50 > 100 μM on Vero and HepG2 cells) and displayed high potency, equal to that of Q203 (23), against Mtb H37Rv (MIC 90 0.011 μM). Remarkably, the Caco-2 permeability of 99 was negligible, and it was 100% bound to human plasma proteins, yet it still demonstrated an excellent rat PK profile, with a half-life of 19 h and an oral bioavailability of 94%. ...
... Remarkably, the Caco-2 permeability of 99 was negligible, and it was 100% bound to human plasma proteins, yet it still demonstrated an excellent rat PK profile, with a half-life of 19 h and an oral bioavailability of 94%. 254 This lead showed no early toxicity liabilities (hERG IC 50 > 30 μM and IC 50 s > 20 μM for a panel of 7 CYPs) and provided an MTD of >2 g/kg in rats. Furthermore, 99 was efficacious in a BALB/c mouse model of acute Mtb infection; oral administration for 4 weeks (starting 1 day after infection) gave lung bacterial burden reductions ranging from 2.2 log 10 CFU at 3.1 mg/kg up to 3.9 log 10 CFU at 200 mg/kg (relative to the vehicle control group). ...
Over the past 2000 years, tuberculosis (TB) has claimed more lives than any other infectious disease. In 2020 alone, TB was responsible for 1.5 million deaths worldwide, comparable to the 1.8 million deaths caused by COVID-19. The World Health Organization has stated that new TB drugs must be developed to end this pandemic. After decades of neglect in this field, a renaissance era of TB drug discovery has arrived, in which many novel candidates have entered clinical trials. However, while hundreds of molecules are reported annually as promising anti-TB agents, very few successfully progress to clinical development. In this Perspective, we critically review those anti-TB compounds published in the last 6 years that demonstrate good in vivo efficacy against Mycobacterium tuberculosis. Additionally, we highlight the main challenges and strategies for developing new TB drugs and the current global pipeline of drug candidates in clinical studies to foment fresh research perspectives.
... During the characterization of TB47, a novel pyrazolopyridine inhibitor of the cytochrome bcc complex, reduced killing of M. tuberculosis by Inh was demonstrated when administered in combination with TB47 in vivo. 23 This data provides evidence that the inhibition of respiration leads to the attenuation of Inh killing, and the authors attributed this to perturbations in NADH/NAD + ratios as a result of inhibition of the ETC. 23 Supporting this, Lee et al. showed that both Bdq and Q203 inhibit the early bactericidal activity of Inh. ...
... 23 This data provides evidence that the inhibition of respiration leads to the attenuation of Inh killing, and the authors attributed this to perturbations in NADH/NAD + ratios as a result of inhibition of the ETC. 23 Supporting this, Lee et al. showed that both Bdq and Q203 inhibit the early bactericidal activity of Inh. 57 In addition, it has been shown that chemical inhibition of different respiratory complexes, including ATP synthase, NADH dehydrogenase, succinate dehydrogenase, and cytochrome bcc, all impaired the killing activity of Inh. ...
... Targeting simultaneously multiple respiratory enzyme complexes in Mycobacterium tuberculosis is regarded as one of the most effective options to treat TB, shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance 5,6 . In this light, new lines of evidence point to a crucial role of the cytochrome bd oxidase for mycobacterial pathophysiology [7][8][9][10][11] . This quinol-oxidizing terminal oxygen reductase is encoded by the cydAB gene cluster and is a central component of the branched respiratory chain of M. tuberculosis. ...
... The cytochrome bd oxidase from M. tuberculosis (M. tb) was produced in Mycobacterium smegmatis mc 2 155 ΔcydAB cells transformed with the pLHcyd plasmid carrying genes of the cytochrome bd oxidase from M. tb with a C-terminal FLAG-tag modification at cydB, the CydDC assembly factor of the cytochrome bd oxidase from M. tb, and a hygromycin resistance cassette 10 . Mycobacteria were grown in LB Lennox media (10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl) with 0.05% w/v Tween 80 and 50 µg/ml hygromycin B added before use. ...
New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.
... The mechanism of action was elucidated and it was shown that nanomolar concentrations significantly lower the levels of ATP, which led to the suggestion that the compound inhibits cytochrome bc1 activity [78]. Based on the structure of 39a, bioisosteric pyrazolo[1,5-a]pyridine-3-carboxamide analogs were prepared and identified as potential new antitubercular agents with high activity against Mtb H37Rv (MIC 90 ¼ 0.125 mg/mL) [79]. More importantly, lead compound 39b (Fig. 5) had remarkable activity against 56 Mtb isolates, including 37 MDR and 2 XDR strains. ...
... Docking studies of 39b in the Q p binding site revealed p-p interactions with the aromatic side chains of TYR321 and PHE156, similar to the interactions observed with Q203. Surprisingly, 39b exhibited a strong synergy with subtherapeutic concentrations of PZA and RMP, causing a 4-and 5-fold reduction in lung CFU [79]. ...
Causing approximately 10 million incident cases and 1.3 - 1.5 million deaths every year, Mycobacterium tuberculosis remains a global health problem. The risk is further exacerbated with latent tuberculosis (TB) infection, the HIV pandemic, and increasing anti-TB drug resistance. Therefore, unexplored chemical scaffolds directed towards new molecular targets are increasingly desired. In this context, mycobacterial energy metabolism, particularly the oxidative phosphorylation (OP) pathway, is gaining importance. Mycobacteria possess primary dehydrogenases to fuel electron transport; aa3-type cytochrome c oxidase and bd-type menaquinol oxidase to generate a protonmotive force; and ATP synthase, which is essential for both growing mycobacteria as well as dormant mycobacteria because ATP is produced under both aerobic and hypoxic conditions. Small organic molecules targeting OP are active against latent TB as well as resistant TB strains. FDA approval of the ATP synthase inhibitor bedaquiline and the discovery of clinical candidate Q203, which both interfere with the cytochrome bc1 complex, have already confirmed mycobacterial energy metabolism to be a valuable anti-TB drug target. This review highlights both preferable molecular targets within mycobacterial OP and promising small organic molecules targeting OP. Progressive research in the area of mycobacterial OP revealed several highly potent anti-TB compounds with nanomolar-range MICs as low as 0.004 μM against Mtb H37Rv. Therefore, we are convinced that targeting the OP pathway can combat resistant TB and latent TB, leading to more efficient anti-TB chemotherapy.
... Moreover, various chemical classes of derivatives, including imidazopyridine amides [7], phenoxyalkylbenzimidazoles [8], 2-(quinoline-4-yloxy)acetamides [9], arylvinylpiperazine amides [10] , morpholino thiophenes [11], pyrrolo [3,4-c]pyridine-1,3(2H)-diones [12], pyrazolo [1,5-a]pyridine-3-carboxamides [13], 4-amino-thieno [2,3-d]pyrimidines [14] and 2ethylthio-4-methylaminoquinazolines [15] were synthesized to target QcrB for the treatment of tuberculosis. Many recent research work on Mtb are mainly focused on the identification of novel inhibitors of QcrB [16], [17]. ...
QcrB is an essential subunit of cytochrome-bc1 complex and is anticipated as a therapeutic target for tuberculosis. In recent years, significant attempts have been made to develop different chemical classes of QcrB inhibitors. Among them, a series of 31 phenoxyalkylimidazoles (PABs) showed anti-tuberculosis activity with MIC 90 values ranging from 0.10µM to 20µM were used to develop a pharmacophore and 3D-QSAR model. Five featured pharmacophore model, HHRRR, which consists of two hydrophobic regions (H) and three aromatic ring features (R), was chosen as the best-fitted model based on the highest survival score and molecular occupancy. The statistically significant 3D-QSAR model constructed using the HHRRR hypothesis possessed good predictive power with an excellent correlation coefficient (R ² = 0.9859) and cross-validation coefficient (Q ² = 0.8593). The contour map analysis provided crucial structural insights into the activity of active compounds. Furthermore, comparative binding mode analysis of a known clinical candidate, Q203, and the most active compound through induced fit docking approach revealed that these two compounds share a similar binding mode with the Q P site of QcrB and form hydrogen bonds with the critical residue T313. Substitution of different chemical scaffolds at the R position of PABs would lead to developing potential QcrB inhibitors.
... Over the recent years, ibudilast has been paid much attention as an agent for therapy of multiple sclerosis and neurodegenerative disorders [3,4]. Recently, pyrazolo [1,5-a]pyridine scaffolds have been utilized in the design of DDX3X helicase [5], Pan-JAK kinase [6], C-terminal Src kinase [7], human dihydroorotate dehydrogenase [8], p110α-selective PI3 kinase [9], p38 kinase [10], ERK inhibitors [11], 5-HT4 [12], EP1 [13], D3 dopamine receptors antagonists, antitubercular [14] and antimalarial [15] agents, etc. Numerous general approaches have been reported for the construction of this heterocyclic core [16,17]. Among them, the 1,3-dipolar cycloaddition of electron-deficient alkynes and alkenes, such as unsaturated carbonyl compound [18] or nitroalkenes [19,20], with pyridinium-N-imines followed by oxidation is one of the most common methods of pyrazolo [1,5-a]pyridine synthesis. ...
A series of pyrazolo[1,5-a]pyridine-3-ylphosphonates were prepared with moderate to good yields by the oxidative [3+2]cycloaddition of 2-subtituted ethynylphosphonates with in situ generated pyridinium-N-imines and their annulated analogs. 2-Aliphatic and 2-Ph acetylenes demonstrate low activity, and the corresponding pyrazolopyridines were achieved with a moderate yield in the presence of 10 mol% Fe(NO3)3·9H2O. At the same time, tetraethyl ethynylbisphosphonate, diethyl 2-TMS- and 2-OPh-ethynylphosphonates possess much greater reactivity and the corresponding pyrazolo[1,5-a]pyridines, and their annulated derivatives were obtained with good to excellent yields without any catalyst. 2-Halogenated ethynylphosphonates also readily reacted with pyridinium-N-imines, forming complex mixtures containing poor amounts of 2-halogenated pyrazolopyridines.
... Oxygen consumption rate measurements. Oxygen consumption rate measurements of exponentially growing M. tuberculosis cells were performed as previously described using an Oroboros O2k FluoRespirometer (61,67). In brief, M. tuberculosis strains expressing sgRNA targeting sdhA1, sdhA2, and frdA in single or multiplexed constructs were inoculated at a starting OD 600 of 0.005 in 10 mL 7H9 medium supplemented with either OADC, glycerol, or succinate and 0 or 100 ng/mL ATc. ...
New drugs are urgently required to combat the tuberculosis epidemic that claims 1.5 million lives annually. Inhibitors of mycobacterial energy metabolism have shown significant promise clinically; however, further advancing this nascent target space requires a more fundamental understanding of the respiratory enzymes and pathways used by Mycobacterium tuberculosis .
... As a continuous flow of PMF and ATP is essential even for the viability of nonreplicating Mtb, inhibition of this complex can eliminate nonreplicating subpopulations. 7 The approval of BDQ for the treatment of DR-TB, and Q203 being in phase II clinical development, demonstrates that mycobacterial respiration could be valuable for therapeutic interventions. ...
It has always been a challenge to develop interventional therapies for Mycobacterium tuberculosis. Over the years, several attempts at developing such therapies have hit a dead-end owing to rapid mutation rates of the tubercular bacilli and their ability to lay dormant for years. Recently, cytochrome bcc complex (QcrB) has shown some promise as a novel target against the tubercular bacilli, with Q203 being the first molecule acting on this target. In this paper, we report the deployment of several ML-based approaches to design molecules against QcrB. Machine learning (ML) models were developed based on a data set of 350 molecules using three different sets of molecular features, i.e., MACCS keys, ECFP6 fingerprints, and Mordred descriptors. Each feature set was trained on eight ML classifier algorithms and optimized to classify molecules accurately. The support vector machine-based classifier using the ECFP6 feature set was found to be the best classifier in this study. Further, screening of the known imidazopyridine amide inhibitors demonstrated that the model correctly classified the most potent molecules as actives, hence validating the model for future applications.
... We demonstrated that mutations in either the target QcrB or the putative phosphodiesterase Rv1339 lead to resistance. This information adds another series of interest to the list of known or proposed QcrB inhibitors, which include the imidazopyridine amides (5), morpholino thiophenes (6), quinolinyl acetamides (19), pyrazolopyridines (20), and arylvinylpiperazine amides (21). Since QcrB is a clinically validated target (22), this series is an attractive one to develop further. ...
We previously identified a series of triazolopyrimidines with antitubercular activity. We determined that Mycobacterium tuberculosis strains with mutations in QcrB, a subunit of the cytochrome bcc-aa3 supercomplex, were resistant. A cytochrome bd oxidase deletion strain was more sensitive to this series. We isolated resistant mutants with mutations in Rv1339. Compounds led to the depletion of intracellular ATP levels and were active against intracellular bacteria, but they did not inhibit human mitochondrial respiration. These data are consistent with triazolopyrimidines acting via inhibition of QcrB.
... The energy of PMF is used to yield ATP through ATP synthase. As continuous flow of PMF and ATP are essential even for the viability of non-replicating Mtb, inhibition of this complex can eliminate non-replicating subpopulations 6 . The approval of BDQ for the treatment of DR-TB, and Q203 being in phase II clinical development confirm that mycobacterial respiration is a promising target for Mtb. ...
Mycobacterium tuberculosis has been a challenging target with respect to developing interventional therapies. Over the years, several attempts at developing anti-tubercular agents have hit a dead-end due to the propensity of the tubercular bacilli to mutate rapidly. Recently, cytochrome bcc complex (QcrB) has shown some promise as a novel target of the tubercular bacilli; with Q203 being the first molecule acting on this target. In this paper, we report the deployment of several ML-based approaches to design molecules against QcrB. Machine Learning (ML) models were developed based on a large dataset of 350 molecules using three different sets of molecular features, i.e. MACCS keys, ECFP6 fingerprints and Mordred descriptors. Each feature set was trained on eight ML classifier algorithms and optimised to classify molecules accurately. Of the 24 models generated, the best performing model was one built with support vector machine and based on the ECFP6 feature set. A further screening of the known imidazopyridine amide inhibitors demonstrated that the model correctly classified the most potent molecules as actives. Thus validating the model for future applications.
... Various experiments carried out by Harrison et al. indicated that these 4-aminothieno [2,3-d]pyrimidines could target QcrB, a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase [64]. A recent study suggests that combination of QcrB inhibitors and current treatments tends to amplify the antimycobacterial activity of the treatments and presents a possible alternative to improve current antitubercular drugs [65]. However, the safety of such approach needs to be confirmed. ...
Thienopyrimidines are widely represented in the literature, mainly due to their structural relationship with purine base such as adenine and guanine. This current review presents three iso-mers-thieno[2,3-d]pyrimidines, thieno[3,2-d]pyrimidines and thieno[3,4-d]pyrimidines-and their anti-infective properties. Broad-spectrum thienopyrimidines with biological properties such as an-tibacterial, antifungal, antiparasitic and antiviral inspired us to analyze and compile their structure-activity relationship (SAR) and classify their synthetic pathways. This review explains the main access route to synthesize thienopyrimidines from thiophene derivatives or from pyrimidine analogs. In addition, SAR study and promising anti-infective activity of these scaffolds are summarized in figures and explanatory diagrams. Ligand-receptor interactions were modeled when the biological target was identified and the crystal structure was solved.
... The success of bedaquiline, a mycobacterial ATPase inhibitor, has made respiration an attractive target for new therapeutics. Multiple small molecule inhibitor screens have identified drugs that target the QcrB component of the proton-pumping cytochrome bc 1 /aa 3 [3,5,39,40], most notably is Q203 (Telacebec) which is currently in clinical trials [5,6]. However, the flexibility of the mycobacterial respiratory chain has raised concerns about the potential efficacy of this drug [2,4,29,41]. ...
In order to sustain a persistent infection, Mycobacterium tuberculosis ( Mtb ) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc 1 /aa 3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFNγ), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that ΔcydA mutants were hypersusceptible to the low pH encountered in IFNγ-activated macrophages. Unlike wild type Mtb , cytochrome bd -deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc 1 /aa 3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc 1 /aa 3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs.
... These insights into the binding mode of MKH 2 may be useful to probe the potential binding modes of Q203 and other known QcrB inhibitors e.g. imidazo[1,2-a]pyridines [54,55], pyrazolo[1,5-a]pyridine [56], pyrrolo[3,4-c]pyridine-1,3(2H)-diones [57] and arylvinylpiperazine amides [58] that may facilitate the structure-guided drug design of the next-generation cytochrome bcc complex inhibitors. ...
... The four green triangles indicate the mutation sites from the second step of screening and amino acid residues at these positions in M. tuberculosis may interact with Q203 when Q203 binds to QcrB from a report 31 . The mutation site in the QcrB of TB47-resistant M. smegmatis is indicated in the black background and spot 30 . ...
Buruli ulcer (BU), caused by Mycobacterium ulcerans, is currently treated with rifampin-streptomycin or rifampin-clarithromycin daily for 8 weeks recommended by World Health Organization (WHO). These options are lengthy with severe side effects. A new anti-tuberculosis drug, TB47, targeting QcrB in cytochrome bc1:aa3 complex is being developed in China. TB47-containing regimens were evaluated in a well-established murine model using an autoluminescent M. ulcerans strain. High-level TB47-resistant spontaneous M. ulcerans mutants were selected and their qcrB genes were sequenced. The in vivo activities of TB47 against both low-level and high-level TB47-resistant mutants were tested in BU murine model. Here, we show that TB47-containing oral 3-drug regimens can completely cure BU in ≤2 weeks for daily use or in ≤3 weeks given twice per week (6 doses in total). All high-level TB47-resistant mutants could only be selected using the low-level mutants which were still sensitive to TB47 in mice. This is the first report of double mutations in QcrB in mycobacteria. In summary, TB47-containing regimens have promise to cure BU highly effectively and prevent the emergence of drug resistance. Novel QcrB mutations found here may guide the potential clinical molecular diagnosis of resistance and the discovery of new drugs against the high-level resistant mutants.
... These insights into the binding mode of MKH 2 may be useful to probe the potential binding modes of Q203 and other known QcrB inhibitors e.g. imidazo[1,2-a]pyridines [54,55], pyrazolo[1,5-a]pyridine [56], pyrrolo[3,4-c]pyridine-1,3(2H)-diones [57] and arylvinylpiperazine amides [58] that may facilitate the structure-guided drug design of the next-generation cytochrome bcc complex inhibitors. ...
Introduction: Tuberculosis (TB) is a leading infectious disease worldwide whose chemotherapy is challenged by the continued rise of drug resistance. This epidemic urges the need to discover anti-TB drugs with novel modes of action.
Areas covered: The mycobacterial electron transport chain (ETC) pathway represents a hub of anti-TB drug targets. Herein, the authors highlight the various targets within the mycobacterial ETC and highlight some of the promising ETC-targeted drugs and clinical candidates that have been discovered or repurposed. Furthermore, recent breakthroughs in the availability of X-ray and/or cryo-EM structures of some targets are discussed, and various opportunities of exploiting these structures for the discovery of new anti-TB drugs are emphasized.
Expert opinion: The drug discovery efforts targeting the ETC pathway have led to the FDA approval of bedaquiline, a FOF1-ATP synthase inhibitor, and the discovery of Q203, a clinical candidate drug targeting the mycobacterial cytochrome bcc-aa3 supercomplex. Moreover, clofazimine, a proposed prodrug competing with menaquinone for its reduction by mycobacterial NADH dehydrogenase 2, has been repurposed for TB treatment. Recently available structures of the mycobacterial ATP synthase C9 rotary ring and the cytochrome bcc-aa3 supercomplex represent further opportunities for the structure-based drug design (SBDD) of the next-generation of inhibitors against Mycobacterium tuberculosis.
... Kalia et al. subsequently showed that genetic inactivation of Cyt-bd combined with Q203 caused a de novo lethality, demonstrating a synthetic lethal interaction (Kalia et al., 2017). Another QcrB inhibitor, TB47, was also shown to synergize with both pyrazinamide and RIF in a mouse model of infection (Lu et al., 2019). Importantly though, it has been shown by Berube et al., that additive and synergistic effects of drug combinations varied significantly between different Mtb strains (Berube and Parish, 2017). ...
Development of novel anti-tuberculosis combination regimens that increase efficacy and reduce treatment timelines will improve patient compliance, limit side-effects, reduce costs, and enhance cure rates. Such advancements would significantly improve the global TB burden and reduce drug resistance acquisition. Bioenergetics has received considerable attention in recent years as a fertile area for anti-tuberculosis drug discovery. Targeting the electron transport chain (ETC) and oxidative phosphorylation machinery promises not only to kill growing cells but also metabolically dormant bacilli that are inherently more drug tolerant. Over the last two decades, a broad array of drugs targeting various ETC components have been developed. Here, we provide a focused review of the current state of art of bioenergetic inhibitors of Mtb with an in-depth analysis of the metabolic and bioenergetic disruptions caused by specific target inhibition as well as their synergistic and antagonistic interactions with other drugs. This foundation is then used to explore the reigning theories on the mechanisms of antibiotic-induced cell death and we discuss how bioenergetic inhibitors in particular fail to be adequately described by these models. These discussions lead us to develop a clear roadmap for new lines of investigation to better understand the mechanisms of action of these drugs with complex mechanisms as well as how to leverage that knowledge for the development of novel, rationally-designed combination therapies to cure TB.
Drug-resistant tuberculosis is a global health care threat calling for novel effective treatment options. Here, we report on two novel cytochrome bc1 inhibitors (MJ-22 and B6) targeting the Mycobacterium tuberculosis respiratory chain with excellent intracellular activities in human macrophages. Both hit compounds revealed very low mutation frequencies and distinct cross-resistance patterns with other advanced cytochrome bc1 inhibitors.
Advancement in the area of anti-tubercular drug development has been full-fledged, yet, a very less number of drug molecules have reached phase II clinical trials, and therefore "End-TB" is still a global challenge. Inhibitors to specific metabolic pathways of Mycobacterium tuberculosis (Mtb) gain importance in strategizing anti-tuberculosis drug discovery. The lead compounds that target DNA replication, protein synthesis, cell wall biosynthesis, bacterial virulence and energy metabolism are emerging as potential chemotherapeutic options against Mtb growth and survival within the host. In recent times, the in silico approaches have become most promising tools in the identification of suitable inhibitors for specific protein targets of Mtb. An update in the fundamental understanding of these inhibitors and the mechanism of interaction may bring hope to future perspectives in novel drug development and delivery approaches. This review provides a collective impression of the small molecules with potential antimycobacterial activities and their target pathways in Mtb such as cell wall biosynthesis, DNA replication, transcription and translation, efflux pumps, antivirulence pathways and general metabolism. The mechanism of interaction of specific inhibitor with their respective protein targets has been discussed. The comprehensive knowledge of such an impactful area of research would essentially reflect in the discovery of novel drug molecules and effective delivery approaches. This narrative review encompasses the knowledge of emerging targets and promising n that could potentially translate in to the anti-TB-drug discovery.
Over the past 2000 years, tuberculosis (TB) has killed more people than any other infectious disease. In 2021, TB claimed 1.6 million lives worldwide, making it the second leading cause of death from an infectious disease after COVID-19. Unfortunately, TB drug discovery research was neglected in the last few decades of the twentieth century. Recently, the World Health Organization has taken the initiative to develop new TB drugs. Imidazopyridine, an important fused bicyclic 5,6 heterocycle has been recognized as a “drug prejudice” scaffold for its wide range of applications in medicinal chemistry. A few examples of imidazo[1,2-a]pyridine exhibit significant activity against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). Here, we critically review anti-TB compounds of the imidazo[1,2-a]pyridine class by discussing their development based on the structure–activity relationship, mode-of-action, and various scaffold hopping strategies over the last decade, which is identified as a renaissance era of TB drug discovery research.
Drug metabolism is generally associated with liver enzymes. However, in the case of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), Mtb-mediated drug metabolism plays a significant role in treatment outcomes. Mtb is equipped with enzymes that catalyse biotransformation reactions on xenobiotics with consequences either in its favour or as a hindrance by deactivating or activating chemical entities, respectively. Considering the range of chemical reactions involved in the biosynthetic pathways of Mtb, information related to the biotransformation of antitubercular compounds would provide opportunities for the development of new chemical tools to study successful TB infections while also highlighting potential areas for drug discovery, host-directed therapy, dose optimization and elucidation of mechanisms of action. In this Review, we discuss Mtb-mediated biotransformations and propose a holistic approach to address drug metabolism in TB drug discovery and related areas.
The increasing incidence of drug resistance in Mycobacterium tuberculosis has diminished the efficacy of almost all available antibiotics, complicating efforts to combat the spread of this global health burden. Alongside the development of new drugs, optimised drug combinations are needed to improve treatment success and prevent the further spread of antibiotic resistance. Typically, antibiotic resistance leads to reduced sensitivity, yet in some cases the evolution of drug resistance can lead to enhanced sensitivity to unrelated drugs. This phenomenon of collateral sensitivity is largely unexplored in M. tuberculosis but has the potential to identify alternative therapeutic strategies to combat drug-resistant strains that are unresponsive to current treatments. Here, by using drug susceptibility profiling, genomics and evolutionary studies we provide evidence for the existence of collateral drug sensitivities in an isogenic collection M. tuberculosis drug-resistant strains. Furthermore, in proof-of-concept studies, we demonstrate how collateral drug phenotypes can be exploited to select against and prevent the emergence of drug-resistant strains. This study highlights that the evolution of drug resistance in M. tuberculosis leads to collateral drug responses that can be exploited to design improved drug regimens.
A [3 + 2] cycloaddition reaction of N-aminopyridines, N-aminoquinolines, and N-aminoisoquinolines with 1-bromoethene-1-sulfonyl fluoride (BESF) was performed to obtain optimum yields of various useful pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]quinolinyl, and pyrazolo[5,1-a]isoquinolinyl sulfonyl fluorides (43-90% yield). The transformation process showed broad substrate specificity, mild reaction conditions, and operational simplicity. Therefore, the reaction has great applicable value in the field of medicinal chemistry and other disciplines.
The increasing incidence of drug resistance in Mycobacterium tuberculosis has diminished the efficacy of almost all available antibiotics, complicating efforts to combat the spread of this global health burden. Alongside the development of new drugs, optimised drug combinations are needed to improve treatment success and prevent the further spread of antibiotic resistance. Typically, antibiotic resistance leads to reduced sensitivity, yet in some cases the evolution of drug resistance can lead to enhanced sensitivity to unrelated drugs. This phenomenon of collateral sensitivity is largely unexplored in M. tuberculosis but has the potential to identify alternative therapeutic strategies to combat drug-resistant strains that are unresponsive to current treatments. To investigate the collateral impacts of drug resistance in M. tuberculosis, we generated an isogenic collection of mono-resistant strains in a PC2-approved avirulent background of M. tuberculosis against 23 structurally and functionally diverse antibiotics. Through drug susceptibility profiling, genomics, and evolutionary studies we provide evidence for the existence of collateral drug sensitivity in M. tuberculosis. In proof-of-concept studies, we demonstrate how collateral drug phenotypes can be targeted to select against and prevent the emergence of drug-resistant strains of M. tuberculosis. This study highlights that the evolution of drug resistance in M. tuberculosis leads to collateral drug responses that can be exploited to design improved drug regimens.
A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 μM).
Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Mycobacterial bioenergetics has emerged as a promising space for the development of novel therapeutics. Further to this, unique combinations of respiratory inhibitors have been shown to have synergistic or synthetic lethal interactions, suggesting that combinations of bioenergetic inhibitors could drastically shorten treatment times. Realizing the full potential of this unique target space requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest interactions and potential in a clinical setting. In this review, we discuss (i) chemical-interaction, (ii) genetic-interaction and (iii) chemical-genetic interaction studies to explore the consequences of inhibiting multiple mycobacterial respiratory components. We provide potential mechanisms to describe the basis for the strongest interactions. Finally, whilst we place an emphasis on interactions that occur with existing bioenergetic inhibitors, by highlighting interactions that occur with alternative respiratory components we envision that this information will provide a rational to further explore alternative proteins as potential drug targets and as part of unique drug combinations.
The synthesis of a range of 3,3'-bipyrazolo[1,5-a]pyridine derivatives via direct cross-dehydrogenative coupling of pyrazolo[1,5-a]pyridine precursors is herein presented. This simple and efficient methodology involving palladium(II)-catalyzed C-H bond activation showed good functional group tolerance and product yield (up to 94%). Through the mechanistic insights gained from both kinetic isotope effect experimental studies and density functional theory calculations, a plausible reaction mechanism was outlined. Furthermore, subsequent derivatizations of the resulting 7,7'-diaryl-3,3'-bipyrazolo[1,5-a]pyridines, executed by performing palladium-mediated ortho C-H bond activation followed by hypervalent iodine-induced chlorination, rendered this series of compounds more extended π-conjugation and twisted conformations. Our study on these bipyrazolo[1,5-a]pyridine-based luminogens provides new opportunities for tailor-made organic luminescent materials.
Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions with sterilizing activity, suggesting that regimens with multiple bioenergetic inhibitors could shorten treatment times. However, realising this potential requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest consequences on bacterial growth and viability. Here we have used multiplex CRISPR interference (CRISPRi) and Mycobacterium smegmatis as a physiological and molecular model for mycobacterial respiration to identify interactions between respiratory complexes. In this study, we identified synthetic lethal and synergistic interactions between respiratory complexes and demonstrate how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.
The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than fifty years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII 2 CIV 2 ). In mycobacterial electron transport chains, CIII 2 CIV 2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII 2 CIV 2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII 2 CIV 2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII 2 CIV 2 activity.
With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and extensive drug-resistant strains (XDR-TB), there is an urgent need to develop novel drugs for the treatment of tuberculosis. Here, we designed and synthesized a series of 5-methylpyrimidopyridone analogues as potential antitubercular agents. The most potent compound 6q exhibited a MIC value of 4 μM in vitro against Mycobacterium tuberculosis. The antitubercular activities of the synthesized compounds were impacted by the amantadine and 2-chlorophenyl groups, and were enhanced by the presence of 3-methyl(4-dimethylamino)piperidinylphenyl. Molecular modeling and binding studies suggest that PknB is the potential molecular target of 5-methylpyrimidopyridone compounds. This study provides insights for the future development of new antimycobacterial agents with novel mechanisms of action.
The anti-tuberculosis drug telacebec is ineffective against Mycobacterium abscessus . A recent study suggested that TB47, a telacebec analogue, potentiated the efficacy of clofazimine against M. abscessus . Here, we report that TB47 is not only ineffective against M. abscessus in vitro , it also does not potentiate the activity of clofazimine.
The bicyclic 5–6 system with one bridgehead nitrogen atom has gained much popularity in the present decade. The best example could be denoted as pyrazolo[1,5-a]pyridine, which is devoted a marketed drug ‘Selpercatinib’ employed against cancer. However, the N-iminopyridinium salts, α,β-unsaturated esters and enynylpyrazoles are positively investigated as best raw materials for construction of pyrazolo[1,5-a]pyridine core. Whereas, isoxazolo[2,3-a]pyridines are much more focused toward the synthesis of partially saturated aromatic analogues. The synthesis and reactivity of imidazo[1,5-a]pyridines are involved with different metal catalysts, most of the synthetic reports are shown substituted aminomethyl pyridines, 1,2-dipyrdyls/2-benzoyl pyridines as emerging starting materials. The oxazolo[3,2-a]-pyridines are obtained from different heterocycles such as pyridines and 1,3 oxazoles, partially saturated oxazolo[3,2-a]-pyridines are accessed by the use of alkynes, alkylidines, malonates and alcohols. The thiazolo[3,2-a]pyridines are synthesized by various approaches involved with numerous heterocyclic derivatives. The synthesis of imidazo[1,2-a]pyridines are mainly associated with aminopyridine analogues, the reactivity has been investigated with multiple catalysts. The imidazo[1,2-a]pyridines are the most reactive substances reported with huge reactivity profile and emerging as potential medicinal scaffold in the current time period. The imidazo[1,2-a]pyridine provided a significant clinical agent GLPG1690 developed based autotaxin enzyme to treat idiopathic pulmonary fibrosis (IPF).
Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis (Mtb) and one of the deadliest infectious diseases in the world. Mtb has the ability to become dormant within the host and to develop resistance. Hence, new antitubercular agents are required to overcome problems in the treatment of multidrug resistant‐Tb (MDR‐Tb) and extensively drug resistant‐Tb (XDR‐Tb) along with shortening the treatment time. Several efforts are being made to develop very effective new drugs for Tb, within the pharmaceutical industry, the academia, and through public private partnerships. This review will address the anti‐tubercular activities, biological target, mode of action, synthetic approaches and thoughtful concept for the development of several new drugs currently in the clinical trial pipeline (up to October 2019) for tuberculosis. The aim of this review may be very useful in scheming new chemical entities (NCEs) for Mtb. This review will address the anti‐tubercular activities, biological target, mode of action, synthetic approaches and thoughtful concept for the development of several new drugs currently in the clinical trial pipeline (up to October 2019) for tuberculosis. The aim of this review may be very useful in scheming new chemical entities (NCEs) for Mtb.
Novel inhibitors are needed to tackle tuberculosis. Herein, we report the 3‐aryl‐substituted imidazo[1,2‐a]pyridines as potent antituberculosis agents. A small library of 3‐aryl‐substituted imidazo[1,2‐a]pyridines was synthesized using direct arylation, followed by nitro reduction and finally Pd‐catalyzed C−N coupling reactions. The compounds thus obtained were evaluated against Mycobacterium tuberculosis H37Rv. Compound 26 was identified as an antituberculosis lead with a minimum inhibitory concentration of 2.3 μg/ml against M. tuberculosis H37Rv. This compound showed a selectivity index of 35. The docking of 26 in the active site of the M. tuberculosis cytochrome bc1 complex cytochrome b subunit (Mtb QcrB) revealed key π–π interactions of compound 26 with the Tyr389 and Trp312 residues of Mtb QcrB. A small library of 3‐aryl‐substituted imidazo[1,2‐a]pyridines was synthesized using direct arylation, followed by nitro reduction and Pd‐catalyzed C–N coupling reactions. The obtained compounds were evaluated against Mycobacterium tuberculosis H37Rv, identifying compound 26 as an antituberculosis lead with a minimum inhibitory concentration of 2.3 μg/ml against M. tuberculosis H37Rv and a selectivity index of 35.
Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the TCA cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bi-directional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1 , sdhA2 , and frdA in M. tuberculosis . We show that the simultaneous knockdown of sdhA1 + sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis , with the combined knockdown of sdhA1 + sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of several antitubercular drugs against M. tuberculosis , including potentiating the activity of bioenergetic inhibitors and attenuating the activity of cell wall inhibitors. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility.
Importance
New drugs are urgently required to combat the tuberculosis epidemic that claims 1.5 million lives annually. Inhibitors of mycobacterial energy metabolism have shown significant promise clinically; however, further advancing this nascent target space requires a more fundamental understanding of the respiratory enzymes and pathways used by Mycobacterium tuberculosis . Succinate is a major focal point in mycobacterial metabolism and respiration; yet the essentiality of succinate oxidation, and the consequences of inhibiting this process, are poorly defined. In this study, we demonstrate that impaired succinate oxidation prevents the optimal growth of M. tuberculosis on a range of carbon sources and significantly reduces the activity of the electron transport chain. Moreover, we show that impaired succinate oxidation both positively and negatively influences the activity of a variety of anti-tuberculosis drugs. Combined, these findings provide fundamental insights into mycobacterial physiology and drug susceptibility that will be useful in the continued development of bioenergetic inhibitors.
Mycobacterium tuberculosis ( Mtb ), the causative agent of human tuberculosis, harbors a branched electron transport chain preventing the bactericidal action of cytochrome bc 1 inhibitors (e.g. TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc 1 inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited Mtb oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc 1 inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g. the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc 1 inhibitors with PMD inhibited Mtb respiration and killed the bacilli. The activity of cytochrome bc 1 inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.
The rapid rise of antibiotic resistance causes an urgent need for new antimicrobial agents with unique and different mechanisms of action. The respiratory chain is one such target involved in the redox balance and energy metabolism. As a natural quinone compound isolated from the root of Salvia miltiorrhiza Bunge, cryptotanshinone (CT) has been previously demonstrated against a wide range of Gram-positive bacteria including multidrug-resistant pathogens. Although superoxide radicals induced by CT are proposed to play an important role in the antibacterial effect of this agent, its mechanism of action is still unclear. In this study, we have shown that CT is a bacteriostatic agent rather than a bactericidal agent. Metabolome analysis suggested that CT might act as an antibacterial agent targeting the cell membrane. CT did not cause severe damage to the bacterial membrane but rapidly dissipated membrane potential, implying that this compound could be a respiratory chain inhibitor. Oxygen consumption analysis in staphylococcal membrane vesicles implied that CT acted as respiratory chain inhibitor probably by targeting type II NADH:quinone dehydrogenase (NDH-2). Molecular docking study suggested that the compound would competitively inhibit the binding of quinone to NDH-2. Consistent with the hypothesis, the antimicrobial activity of CT was blocked by menaquinone, and the combination of CT with thioridazine but not 2-n-heptyl-4-hydroxyquinoline-N-oxide exerted synergistic activity against Staphylococcus aureus. Additionally, combinations of CT with other inhibitors targeting different components of the bacterial respiratory chain exhibit potent synergistic activities against S. aureus, suggesting a promising role in combination therapies.
This study improves understanding of how mycobacteria, a group of environmentally and medically important bacteria, use trace gases as energy sources. Here I show that the model organism Mycobacterium smegmatis uses the dependable energy sources atmospheric hydrogen and carbon monoxide to survive nutrient starvation. They do so by synthesizing specific enzymes, namely hydrogenases and carbon monoxide dehydrogenases, in response to organic carbon levels. These enzymes are physically and functionally integrated with the aerobic respiratory chain and, when lost, cause survival defects. In turn, these findings provide new cellular and biochemical insights into how bacteria control the composition of the atmosphere.
Drug‐resistance in mycobacterial infections is a major global health problem that leads to high mortality and socioeconomic pressure in developing countries around the world. From finding new targets to discovering novel chemical scaffolds, there is an urgent need for the development of better approaches for the cure of tuberculosis. Recently, energy metabolism in mycobacteria, particularly the oxidative phosphorylation pathway of cellular respiration, has emerged as a novel target pathway in drug discovery. New classes of antibacterials which target oxidative phosphorylation pathway either by interacting with a protein or any step in the pathway of oxidative phosphorylation can combat dormant mycobacterial infections leading to shortening of tuberculosis chemotherapy. Adenosine triphosphate synthase is one such recently discovered target of the newly approved antitubercular drug bedaquiline. Cytochrome bcc is another new target of the antitubercular drug candidate Q203, currently in phase II clinical trial. Research suggests that b subunit of cytochrome bcc, QcrB, is the target of Q203. The review article describes the structure, function, and importance of targeting QcrB throwing light on all chemical classes of QcrB inhibitors discovered to date. An understanding of the structure and function of validated targets and their inhibitors would enable the development of new chemical entities.
Recently, ATP synthase inhibitor Bedaquiline was approved for the treatment of multi-drug resistant tuberculosis emphasizing the importance of oxidative phosphorylation for the survival of mycobacteria. ATP synthesis is primarily dependent on the generation of proton motive force through the electron transport chain in mycobacteria. The mycobacterial electron transport chain utilizes two terminal oxidases for the reduction of oxygen, namely the bc 1-aa 3 supercomplex and the cytochrome bd oxidase. The bc 1-aa 3 supercomplex is an energy-efficient terminal oxidase that pumps out four vectoral protons, besides consuming four scalar protons during the transfer of electrons from menaquinone to molecular oxygen. In the past few years, several inhibitors of bc 1-aa 3 supercomplex have been developed, out of which, Q203 belonging to the class of imidazopyridine, has moved to clinical trials. Recently, the crystal structure of the mycobacterial cytochrome bc 1-aa 3 supercomplex was solved, providing details of the route of transfer of electrons from menaquinone to molecular oxygen. Besides providing insights into the molecular functioning, crystal structure is aiding in the targeted drug development. On the other hand, the second respiratory terminal oxidase of the mycobacterial respiratory chain, cytochrome bd oxidase, does not pump out the vectoral protons and is energetically less efficient. However, it can detoxify the reactive oxygen species and facilitate mycobacterial survival during a multitude of stresses. Quinolone derivatives (CK-2-63) and quinone derivative (Aurachin D) inhibit cytochrome bd oxidase. Notably, ablation of both the two terminal oxidases simultaneously through genetic methods or pharmacological inhibition leads to the rapid death of the mycobacterial cells. Thus, terminal oxidases have emerged as important drug targets. In this review, we have described the current understanding of the functioning of these two oxidases, their physiological relevance to mycobacteria, and their inhibitors. Besides these, we also describe the alternative terminal complexes that are used by mycobacteria to maintain energized membrane during hypoxia and anaerobic conditions.
Wherever thermodynamics allows, microbial life has evolved to transform and harness energy. Microbial life thus abounds in the most unexpected places, enabled by profound metabolic diversity. Within this diversity, energy is transformed primarily through variations on a few core mechanisms. Energy is further managed by the physiological processes of cell growth and maintenance that use energy. Some aspects of microbial physiology are streamlined for energetic efficiency while other aspects seem suboptimal or even wasteful. We propose that the energy that a microbe harnesses and devotes to growth and maintenance is a product of three broad tradeoffs: (i) economic, trading enzyme synthesis or operational cost for functional benefit, (ii) environmental, trading optimization for a single environment for adaptability to multiple environments, and (iii) thermodynamic, trading energetic yield for forward metabolic flux. Consideration of these tradeoffs allows one to reconcile features of microbial physiology that seem to opposingly promote either energetic efficiency or waste.
As an obligate aerobe Mycobacterium tuberculosis uses its electron-transport chain (ETC) to produce energy via oxidative phosphorylation. This pathway has recently garnered a lot of attention and is a target for several new anti-mycobacterials. We tested the respiratory adaptation of M. tuberculosis to phenoxy alkyl benzimidazoles (PABs), compounds proposed to target QcrB, a component of the cytochrome bc1 complex. We show M. tuberculosis is able to reroute its ETC to provide temporary resistance to PABs. However, combination treatment of PAB with agents targeting other components of the electron-transport chain overcomes this respiratory flexibility. PAB in combination with clofazimine resulted in synergistic killing of M. tuberculosis under both replicating and non-replicating conditions. PABs in combination with bedaquiline demonstrated antagonism at early time-points, particularly in non-replicating conditions. However, this antagonistic effect disappeared within three weeks, when PAB/BDQ combinations became highly bactericidal; in some cases, were better than either drug alone. This study highlights the potential for combination treatment targeting the ETC and supports the development of PABs as part of a novel drug regimen against M. tuberculosis .
The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone: cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drugtolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.
The recent discovery of small molecules targeting the cytochrome bc
1
:aa
3
in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc
1
:aa
3
consists of a menaquinone:cytochrome c reductase (bc
1
) and a cytochrome aa
3
-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc
1
:aa
3
complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc
1
:aa
3
, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.
The NADH:NAD + ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD + ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD + ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD + levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD + ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD + ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further studies on mycobacterial physiology and will create new avenues of research for anti-tuberculosis drug discovery.
A previous phenotypic screen by GSK identified 2-(quinolin-4-yloxy)acetamides as potent growth inhibitors of Mycobacterium tuberculosis (Mtb). We report the results of a preliminary structure-activity relationship (SAR) study of the compound class which has yielded more potent inhibitors. An Mtb cytochrome bd oxidase deletion mutant (cydKO) was found to be hypersensitive to most members of the compound library, while strains carrying single-nucleotide polymorphisms of the qcrB gene, which encodes a subunit of the menaquinol cytochrome c oxidoreductase (bc1) complex, were resistant to the library. These results identify that the 2-(quinolin-4-yloxy)acetamide class of Mtb growth inhibitors can be added to the growing number of scaffolds that target the M. tuberculosis bc1 complex.
SETTING AND OBJECTIVES. Drug-resistant tuberculosis (TB) is a major health threat in Myanmar. We conducted an initial study to explore the potential utility of whole-genome sequencing (WGS) for the diagnosis and management of drug-resistant tuberculosis in Myanmar.
METHODS. Fourteen multidrug-resistant Mycobacterium tuberculosis isolates were sequenced. Known resistance genes for a total of nine antibiotics that are commonly used in treatment of drug-susceptible and multidrug-resistant TB (MDR-TB) in Myanmar were interrogated through WGS.
RESULTS. All 14 isolates were MDR-TB consistent with the results of phenotypic drug susceptibility testing (DST) and the Beijing lineage predominated. Based on the results of WGS, nine of 14 isolates were potentially resistant to at least one of the drugs used in the standard MDR regimen, but for which phenotypic DST is not conducted in Myanmar.
CONCLUSION. This study highlights a need for the introduction of second-line DST as part of routine diagnosis in Myanmar and new classes of TB drugs to construct effective regimens.
Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H(+)K(+)-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.
Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring's ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens.
Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multi-drug resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2- a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome b-cc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a "supercomplex" with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis which has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding, and provide structural insight into a third class of the bc-complex superfamily.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Background:
Bedaquiline (Sirturo, TMC207), a diarylquinoline that inhibits mycobacterial ATP synthase, has been associated with accelerated sputum-culture conversion in patients with multidrug-resistant tuberculosis, when added to a preferred background regimen for 8 weeks.
Methods:
In this phase 2b trial, we randomly assigned 160 patients with newly diagnosed, smear-positive, multidrug-resistant tuberculosis to receive either 400 mg of bedaquiline once daily for 2 weeks, followed by 200 mg three times a week for 22 weeks, or placebo, both in combination with a preferred background regimen. The primary efficacy end point was the time to sputum-culture conversion in liquid broth. Patients were followed for 120 weeks from baseline.
Results:
Bedaquiline reduced the median time to culture conversion, as compared with placebo, from 125 days to 83 days (hazard ratio in the bedaquiline group, 2.44; 95% confidence interval, 1.57 to 3.80; P<0.001 by Cox regression analysis) and increased the rate of culture conversion at 24 weeks (79% vs. 58%, P=0.008) and at 120 weeks (62% vs. 44%, P=0.04). On the basis of World Health Organization outcome definitions for multidrug-resistant tuberculosis, cure rates at 120 weeks were 58% in the bedaquiline group and 32% in the placebo group (P=0.003). The overall incidence of adverse events was similar in the two groups. There were 10 deaths in the bedaquiline group and 2 in the placebo group, with no causal pattern evident.
Conclusions:
The addition of bedaquiline to a preferred background regimen for 24 weeks resulted in faster culture conversion and significantly more culture conversions at 120 weeks, as compared with placebo. There were more deaths in the bedaquiline group than in the placebo group. (Funded by Janssen Pharmaceuticals; TMC207-C208 ClinicalTrials.gov number, NCT00449644.).
The emergence and spread of drug-resistant pathogens and our inability to develop new antimicrobials to overcome resistance has inspired scientists to consider new targets for drug development. Cellular bioenergetics is an area showing promise for the development of new antimicrobials, particularly in the discovery of new anti-tuberculosis drugs where several new compounds have entered clinical trials. In this review, we have examined the bioenergetics of various bacterial pathogens, highlighting the versatility of electron donor and acceptor utilisation and the modularity of electron transport chain components in bacteria. In addition to re-examining classical concepts, we explore new literature that reveals the intricacies of pathogen energetics, for example, how Salmonella enterica and Campylobacter jejuni exploit host and microbiota to derive powerful electron donors and sinks; the strategies Mycobacterium tuberculosis and Pseudomonas aeruginosa use to persist in lung tissues; and the importance of sodium energetics and electron bifurcation in the chemiosmotic anaerobe Fusobacterium nucleatum. A combination of physiological, biochemical, and pharmacological data suggests that, in addition to the clinically-approved target F1Fo-ATP synthase, NADH dehydrogenase type II, succinate dehydrogenase, hydrogenase, cytochrome bd oxidase, and menaquinone biosynthesis pathways are particularly promising next-generation drug targets. The realisation of cellular energetics as a rich target space for the development of new antimicrobials will be dependent upon gaining increased understanding of the energetic processes utilised by pathogens in host environments and the ability to design bacterial-specific inhibitors of these processes.
Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. In vitro resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ in vitro, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance in vivo. Cross-resistance between BDQ and CFZ may have important clinical implications.
Energetics of Respiration and Oxidative Phosphorylation in Mycobacteria, Page 1 of 2
Abstract
Mycobacteria inhabit a wide range of intracellular and extracellular environments. Many of these environments are highly dynamic, and therefore mycobacteria are faced with the constant challenge of redirecting their metabolic activity to be commensurate with either replicative growth or a nonreplicative quiescence. A fundamental feature in this adaptation is the ability of mycobacteria to respire, regenerate reducing equivalents, and generate ATP via oxidative phosphorylation. Mycobacteria harbor multiple primary dehydrogenases to fuel the electron transport chain, and two terminal respiratory oxidases, an aa 3-type cytochrome c oxidase and a cytochrome bd-type menaquinol oxidase, are present for dioxygen reduction coupled to the generation of a proton motive force (PMF). Hypoxia leads to the downregulation of key respiratory complexes, but the molecular mechanisms regulating this expression are unknown. Despite being obligate aerobes, mycobacteria have the ability to metabolize in the absence of oxygen, and a number of reductases are present to facilitate the turnover of reducing equivalents under these conditions (e.g., nitrate reductase, succinate dehydrogenase/fumarate reductase). Hydrogenases and ferredoxins are also present in the genomes of mycobacteria, suggesting the ability of these bacteria to adapt to an anaerobic type of metabolism in the absence of oxygen. ATP synthesis by the membrane-bound F1F0-ATP synthase is essential for growing and nongrowing mycobacteria, and the enzyme is able to function over a wide range of PMF values (aerobic to hypoxic). The discovery of lead compounds that target respiration and oxidative phosphorylation in Mycobacterium tuberculosis highlights the importance of this area for the generation of new frontline drugs to combat tuberculosis.
Extensively drug-resistant tuberculosis is a burgeoning global health crisis mainly affecting economically active young adults, and has high mortality irrespective of HIV status. In some countries such as South Africa, drug-resistant tuberculosis represents less than 3% of all cases but consumes more than a third of the total national budget for tuberculosis, which is unsustainable and threatens to destabilise national tuberculosis programmes. However, concern about drug-resistant tuberculosis has been eclipsed by that of totally and extremely drug-resistant tuberculosis-ie, resistance to all or nearly all conventional first-line and second-line antituberculosis drugs. In this Review, we discuss the epidemiology, pathogenesis, diagnosis, management, implications for health-care workers, and ethical and medicolegal aspects of extensively drug-resistant tuberculosis and other resistant strains. Finally, we discuss the emerging problem of functionally untreatable tuberculosis, and the issues and challenges that it poses to public health and clinical practice. The emergence and growth of highly resistant strains of tuberculosis make the development of new drugs and rapid diagnostics for tuberculosis-and increased funding to strengthen global control efforts, research, and advocacy-even more pressing.
We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogs of the new tuberculosis (TB) drug SQ109 (1) which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multi-target inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.
New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.
Existing drugs have limited efficacy against the rising threat of drug-resistant TB, have significant side effects, and must be given in combinations of four to six drugs for at least 6 months for drug-sensitive TB and up to 24 months for drug-resistant TB. The long treatment duration has led to increased patient noncompliance with therapy. This, in turn, drives the development of additional drug resistance in a spiral that has resulted in some forms of TB being currently untreatable by existing drugs. New antitubercular drugs in development, particularly those with mechanisms of action that are different from existing first- and second-line TB drugs, are anticipated to be effective against both drug-sensitive and drug-resistant TB. SQ109 is a new TB drug candidate with a novel mechanism of action that was safe and well tolerated in Phase I and early Phase II clinical trials. We describe herein the identification, development and characterization of SQ109 as a promising new antitubercular drug.
The 2-year follow-up results for a randomized placebo-controlled study of 47 patients with multidrug-resistant pulmonary tuberculosis treated with either the new diarylquinoline TMC207, recently renamed bedaquiline, or placebo, added to the first 8 weeks of a background regimen, are presented. Bedaquiline significantly reduced the time to culture conversion over 24 weeks (hazard ratio, 2.253; 95% confidence interval, 1.08 to 4.71; P = 0.031). With the exception of nausea reported in 26% of patients receiving bedaquiline and none receiving placebo, adverse events occurred at similar frequencies in both groups of patients: bilateral hearing impairment, extremity pain, acne, and noncardiac chest pain occurred in 13 and 21%, 17 and 13%, 9 and 17%, and 4 and 17% of patients, respectively, receiving bedaquiline or placebo. Excluding resistance to ethambutol and ethionamide, only one patient receiving bedaquiline acquired resistance to companion drugs, but five patients receiving placebo (4.8% versus 21.7%; P = 0.18) acquired resistance to companion drugs, and resistance to ofloxacin was acquired in four patients receiving placebo and none receiving bedaquiline (0% versus 22%; 0 = 0.066). In all, 23 patients (49%), including 13 receiving placebo (54%) and 10 receiving bedaquiline (44%), discontinued the study prior to its completion, 12 during the first 24 weeks of treatment. Eight subjects were withdrawn for noncompliance or default, and seven withdrew consent, citing the rigorous program of investigations for safety and pharmacokinetic monitoring. Bedaquiline may contribute to the management of multidrug-resistant tuberculosis by effecting more rapid sputum culture negativity and by preventing acquired resistance to companion drugs.
The diarylquinoline TMC207 offers a new mechanism of antituberculosis action by inhibiting mycobacterial ATP synthase. TMC207 potently inhibits drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro and shows bactericidal activity in patients who have drug-susceptible pulmonary tuberculosis.
In the first stage of a two-stage, phase 2, randomized, controlled trial, we randomly assigned 47 patients who had newly diagnosed multidrug-resistant pulmonary tuberculosis to receive either TMC207 (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks) (23 patients) or placebo (24 patients) in combination with a standard five-drug, second-line antituberculosis regimen. The primary efficacy end point was the conversion of sputum cultures, in liquid broth, from positive to negative.
The addition of TMC207 to standard therapy for multidrug-resistant tuberculosis reduced the time to conversion to a negative sputum culture, as compared with placebo (hazard ratio, 11.8; 95% confidence interval, 2.3 to 61.3; P=0.003 by Cox regression analysis) and increased the proportion of patients with conversion of sputum culture (48% vs. 9%). The mean log(10) count of colony-forming units in the sputum declined more rapidly in the TMC207 group than in the placebo group. No significant differences in average plasma TMC207 concentrations were noted between patients with and those without culture conversion. Most adverse events were mild to moderate, and only nausea occurred significantly more frequently among patients in the TMC207 group than among patients in the placebo group (26% vs. 4%, P=0.04).
The clinical activity of TMC207 validates ATP synthase as a viable target for the treatment of tuberculosis. (ClinicalTrials.gov number, NCT00449644.)
Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.
The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific
drugs have been discovered in 40 years. We identified a diarylquinoline, R207910, that potently inhibits both drug-sensitive
and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 μg/ml). In mice, R207910 exceeded the bactericidal activities of isoniazid
and rifampin by at least 1 log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis
treatment regimen (rifampin, isoniazid, and pyrazinamide) with R207910 accelerated bactericidal activity, leading to complete
culture conversion after 2 months of treatment in some combinations. A single dose of R207910 inhibited mycobacterial growth
for 1 week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected
in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.
The front-line antituberculosis drug isoniazid (INH) and the related drug ethionamide (ETH) are prodrugs that upon activation
inhibit the synthesis of mycolic acids, leading to bactericidal activity. Coresistance to INH and ETH can be mediated by dominant
mutations in the target gene inhA, encoding an enoyl-ACP reductase, or by recessive mutations in ndh, encoding a type II NADH dehydrogenase (NdhII). To address the mechanism of resistance mediated by the latter, we have isolated
novel ndh mutants of Mycobacterium smegmatis and Mycobacterium bovis BCG. The M. smegmatis ndh mutants were highly resistant to INH and ETH, while the M. bovis BCG mutants had low-level resistance to INH and ETH. All mutants had defects in NdhII activity resulting in an increase in
intracellular NADH/NAD+ ratios. Increasing NADH levels were shown to protect InhA against inhibition by the INH-NAD adduct formed upon INH activation.
We conclude that ndh mutations mediate a novel mechanism of resistance by increasing the NADH cellular concentration, which competitively inhibits
the binding of INH-NAD or ETH-NAD adduct to InhA.
Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD- and pstS-lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc(2)155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K(m) values between 40 and 90 muM P(i)). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.
In previous studies, the diarylquinoline R207910 (also known as TMC207) was demonstrated to have high bactericidal activity
when combined with first- or second-line antituberculous drugs. Here we extend the evaluation of R207910 in the curative model
of murine tuberculosis by assessing the activities of one-, two-, and three-drug combinations containing R207910 and isoniazid
(INH), rifampin (RIF), pyrazinamide (PZA), or moxifloxacin (MXF) in the setting of a high initial bacillary load (7.2 log10 CFU). Two months of treatment with the combinations R207910-PZA, R207910-PZA-INH, R207910-PZA-RIF, or R207910-PZA-MXF resulted
in culture-negative lung homogenates in 70 to 100% of the mice, while mice treated with INH-RIF-PZA (the reference regimen)
or RIF-MXF-PZA remained culture positive. Combinations including R207910 but not PZA (e.g., R207910-INH-RIF and R207910-MXF-RIF)
were less active than R207910-PZA-containing regimens administered either alone or with the addition of INH, RIF, or MXF.
These results reveal a synergistic interaction between R207910 and PZA. Three-drug combinations containing these two drugs
and INH, RIF, or MXF have the potential to significantly shorten the treatment duration in patients, provided that these results
can be confirmed in long-term experiments including periods of relapse.
Significance
Antibiotics generally target one of five essential cellular functions in bacteria, but many of these targets are now compromised through rapidly spreading antibiotic resistance. Bedaquiline (BDQ), a new FDA-approved antitubercular drug, targets energy metabolism: defining cellular energetics as a new target space for antibiotics. This is a relatively unexplored area, as BDQ was only FDA approved in 2012. Several studies have recently found that BDQ stimulates mycobacterial respiration, in addition to inhibiting its molecular target, the F 1 F o -ATP synthase. We show that BDQ is an ionophore, which shuttles H ⁺ and K ⁺ ions across membranes, and propose that this activity may contribute to killing of mycobacteria by BDQ. Combining ionophoric activity with high-affinity membrane protein inhibition may enhance the specificity and potency of antibiotics.
From the war on drug resistance, through cancer biology, even to agricultural and environmental protection: there is a huge demand for rapid and effective solutions to control infections and diseases. The development of small molecule inhibitors was once an accepted “one-size fits all” approach to these varied problems, but persistence and resistance threaten to return society to a pre-antibiotic era. Only five essential cellular targets in bacteria have been developed for the majority of our clinically-relevant antibiotics. These include: cell wall synthesis, cell membrane function, protein and nucleic acid biosynthesis, and antimetabolites. Many of these targets are now compromised through rapidly spreading antimicrobial resistance and the need to target non-replicating cells (persisters). Recently, an unprecedented medical breakthrough was achieved by the FDA approval of the drug bedaquiline (BDQ, trade name Sirturo) for the treatment of multidrug-resistant tuberculosis disease. BDQ targets the membrane-bound F1Fo-ATP synthase, validating cellular energy generating machinery as a new target space for drug discovery. Recently, BDQ and several other FDA-approved drugs have been demonstrated to be respiratory “uncouplers” disrupting transmembrane electrochemical gradients, in addition to binding to enzyme targets. In this review, we summarize the role of bioenergetic systems in mycobacterial persistence and discuss the multi-targeting nature of uncouplers and the place these molecules may have in future drug development.
The phenoxy alkyl benzimidazoles (PABs) have good anti-tubercular activity. We expanded our structure-activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against M. tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells, as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.
Oxidative Phosphorylation as a Target Space for Tuberculosis: Success, Caution, and Future Directions, Page 1 of 2
Abstract
The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, have inspired scientists to seek out new targets for drug development. The Mycobacterium tuberculosis complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth. M. tuberculosis is an obligately aerobic heterotroph that depends on oxidative phosphorylation for growth and survival. However, several studies are redefining the metabolic breadth of the genus. Alternative electron donors and acceptors may provide the maintenance energy for the pathogen to maintain viability in hypoxic, nonreplicating states relevant to latent infection. This hidden metabolic flexibility may ultimately decrease the efficacy of drugs targeted against primary dehydrogenases and terminal oxidases. However, it may also open up opportunities to develop novel antimycobacterials targeting persister cells. In this review, we discuss the progress in understanding the role of energetic targets in mycobacterial physiology and pathogenesis and the opportunities for drug discovery.
A series of pyrazolo[1,5-a]pyridine-3-carboxamide hybrids were designed and evaluated as novel anti-tubercular agents. The representative hybrid 7 exhibited promising in vitro activity against susceptive strain H37Rv and a panel of drug-resistant Mtb strains with MIC values of 0.006 μg/mL and ranged from 0.003 to 0.014 μg/mL, respectively. More importantly, the hybrid 7 also showed very low cytotoxicity, and could significantly reduce the mycobacterial burden in a mouse model infected with autoluminescent H37Ra strain, which may serve as a lead compound for further development of new anti-tubercular agents.