Antibiotics Trigger Host Innate Immune Response via Microbiota-Brain Communication in C. elegans.

Besides their direct bactericidal effect, antibiotics have also been suggested to stimulate the host immune response to defend against pathogens. However, it remains unclear whether any antibiotics may stimulate the host immune response by affecting bacterial activity. In this study, reasoning that genetic mutations inhibit bacterial activities and, thereby, may mimic the effects of antibiotics, we performed genome-wide screening and identified 77 E. coli genes whose inactivation induces C. elegans cyp-14A4, representing an innate immune and detoxification response. Further analyses reveal that this host immune response can clearly be induced through either inactivating the E. coli respiratory chain via the bacterial cyoB mutation or using the antibiotic Q203, which is able to enhance host survival when encountering the pathogen Pseudomonas aeruginosa. Mechanistically, the innate immune response triggered by both the cyoB mutation and Q203 is found to depend on the host brain response, as evidenced by their reliance on the host neural gene unc-13, which is required for neurotransmitter release in head neurons. Therefore, our findings elucidate the critical involvement of the microbiota-brain axis in modulating the host immune response, providing mechanistic insights into the role of antibiotics in triggering the host immune response and, thus, facilitating host defense against pathogens.

Repurposing of Tuberculosis Drug Candidates for the Treatment of Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M. tuberculosis, classical lineage M. ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M. ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.

Safety, Tolerability, and Pharmacokinetics of Telacebec (Q203), a New Antituberculosis Agent, in Healthy Subjects.

Telacebec (Q203) is a potent drug candidate under clinical development for the treatment of drug-naïve and drug-resistant tuberculosis. The first-in-human randomized, placebo-controlled, double-blind, dose-escalation Phase 1A trial (Q203-TB-PI-US001) was conducted to evaluate the safety, tolerability, and pharmacokinetics of telacebec. A total of 56 normal, healthy, male and female subjects (42 active and 14 placebo) were enrolled in the study. The doses of telacebec were 10 mg (Cohort 1), 30 mg (Cohort 2), 50 mg (Cohort 3), 100 mg (Cohort 4), 200 mg (Cohort 5), 400 mg (Cohort 6), and 800 mg (Cohort 7) in a fasted state. Subjects participating in Cohort 4 were also enrolled in Cohort 8 to investigate the food effect on the pharmacokinetics of telacebec after a high-fat meal. In all subjects dosed with telacebec (10 to 800 mg), telacebec was well tolerated and did not lead to any significant or serious adverse events. Following a single oral administration of telacebec (10 to 800 mg), telacebec plasma concentration reached the maximal plasma concentration (Cmax) in average 2.0 to 3.5 h and showed multi-exponential decline thereafter. The area under the plasma concentration versus time curve (AUC) was approximately dose-proportional. A significant increase in plasma concentrations was observed in the fed condition compared with the fasted condition with the geometric mean ratio of 3.93 for Cmax. Moderate delay in Tmax (4.5 h) was also observed in the fed condition. These results, combined with the demonstrated activity against drug-sensitive and multidrug-resistant Mycobacterium tuberculosis, support further investigation of telacebec for the treatment of tuberculosis.

Targeting the cytochrome bc1 complex for drug development in M. tuberculosis: review.

The terminal oxidases of the oxidative phosphorylation pathway play a significant role in the survival and growth of M. tuberculosis, targeting these components lead to inhibition of M. tuberculosis. Many drug candidates targeting various components of the electron transport chain in M. tuberculosis have recently been discovered. The cytochrome bc1-aa3 supercomplex is one of the most important components of the electron transport chain in M. tuberculosis, and it has emerged as the novel target for several promising candidates. There are two cryo-electron microscopy structures (PDB IDs: 6ADQ and 6HWH) of the cytochrome bc1-aa3 supercomplex that aid in the development of effective and potent inhibitors for M. tuberculosis. In recent years, a number of potential candidates targeting the QcrB subunit of the cytochrome bc1 complex have been developed. In this review, we describe the recently identified inhibitors that target the electron transport chain's terminal oxidase enzyme in M. tuberculosis, specifically the QcrB subunit of the cytochrome bc1 complex.

Response of Mycobacterium smegmatis to the Cytochrome bcc Inhibitor Q203.

For the design of next-generation tuberculosis chemotherapy, insight into bacterial defence against drugs is required. Currently, targeting respiration has attracted strong attention for combatting drug-resistant mycobacteria. Q203 (telacebec), an inhibitor of the cytochrome bcc complex in the mycobacterial respiratory chain, is currently evaluated in phase-2 clinical trials. Q203 has bacteriostatic activity against M. tuberculosis, which can be converted to bactericidal activity by concurrently inhibiting an alternative branch of the mycobacterial respiratory chain, cytochrome bd. In contrast, non-tuberculous mycobacteria, such as Mycobacterium smegmatis, show only very little sensitivity to Q203. In this report, we investigated factors that M. smegmatis employs to adapt to Q203 in the presence or absence of a functional cytochrome bd, especially regarding its terminal oxidases. In the presence of a functional cytochrome bd, M. smegmatis responds to Q203 by increasing the expression of cytochrome bcc as well as of cytochrome bd, whereas a M. smegmatisbd-KO strain adapted to Q203 by increasing the expression of cytochrome bcc. Interestingly, single-cell studies revealed cell-to-cell variability in drug adaptation. We also investigated the role of a putative second cytochrome bd isoform postulated for M. smegmatis. Although this putative isoform showed differential expression in response to Q203 in the M. smegmatisbd-KO strain, it did not display functional features similar to the characterised cytochrome bd variant.

Impact of Dose, Duration, and Immune Status on Efficacy of Ultrashort Telacebec Regimens in Mouse Models of Buruli Ulcer.

Telacebec (Q203) is a new antituberculosis drug in clinical development that has extremely potent activity against Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU). The potency of Q203 has prompted investigation of its potential role in ultrashort, even single-dose, treatment regimens for BU in mouse models. However, the relationships of Q203 dose, dose schedule, duration, and host immune status to treatment outcomes remain unclear, as does the risk of emergence of drug resistance with Q203 monotherapy. Here, we used mouse footpad infection models in immunocompetent BALB/c and immunocompromised SCID-beige mice to compare different Q203 doses, different dosing schedules, and treatment durations ranging from 1 day to 2 weeks, on long-term outcomes. We also tested whether combining Q203 with a second drug can increase efficacy. Overall, efficacy depended on total dose more than on duration. Total doses of 5 to 20 mg/kg rendered nearly all BALB/c mice culture negative by 13 to 14 weeks posttreatment, without selection of Q203-resistant bacteria. Addition of a second drug did not significantly increase efficacy. Although less potent in SCID-beige mice, Q203 still rendered the majority of footpads culture negative at total doses of 10 to 20 mg/kg. Q203 resistance was identified in relapse isolates from some SCID-beige mice receiving monotherapy but not in isolates from those receiving Q203 combined with bedaquiline or clofazimine. Overall, these results support the potential of Q203 monotherapy for single-dose or other ultrashort therapy for BU, although highly immunocompromised hosts may require higher doses or durations and/or combination therapy.

The QcrB Inhibitors TB47 and Telacebec Do Not Potentiate the Activity of Clofazimine in Mycobacterium abscessus.

The antituberculosis 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 not only is ineffective against M. abscessus in vitro but also does not potentiate the activity of clofazimine.

Telacebec (Q203): Is there a novel effective and safe anti-tuberculosis drug on the horizon?

High prevalence and stronger emergency of various forms of drug-resistant tuberculosis (DR-TB), including the multidrug-resistant (MDR-TB) as well as extensively drug-resistant (XDR-TB) ones, caused by variously resistant Mycobacterium tuberculosis pathogens, make first-line anti-tuberculosis (anti-TB) agents therapeutically more and more ineffective. Therefore, there is an imperative to develop novel highly efficient (synthetic) agents against both drug-sensitive-TB and DR-TB. The exploration of various heterocycles as prospective core scaffolds for the discovery, development and optimization of anti-TB drugs remains an intriguing scientific endeavour. Telacebec (Q203; TCB), a molecule containing an imidazo[1,2-a]pyridine-3-carboxamide (IPA) structural motif, is considered a novel very promising anti-TB agent showing a unique mechanism of action. The compound blocks oxidative phosphorylation by inhibiting a mycobacterial respiratory chain due to interference with a specific cytochrome b subunit (QcrB) of transmembrane bc1 menaquinol-cytochrome c oxidoreductase as an essential component for transporting electrons across the membrane from menaquinol to other specific subunit, cytochrome c (QcrC). Thus, the ability of mycobacteria to synthesize adenosine-5´-triphosphate is limited and energy generating machinery is disabled. The TCB molecule effectively fights drug-susceptible, MDR as well as XDR M. tuberculosis strains. The article briefly explains a mechanism of an anti-TB action related to the compounds containing a variously substituted IPA scaffold and is focused on their fundamental structure-anti-TB activity relationships as well. Special consideration is paid to TCB indicating the importance of particular structural fragments for maintaining (or even improving) favourable pharmacodynamic, pharmacokinetic and/or toxicological properties. High lipophilicity of TCB might be regarded as one of the key physicochemical properties with positive impact on anti-TB effect of the drug. In January 2021, the TCB molecule was also involved in phase-II clinical trials focused on the treatment of Coronavirus Disease-19 caused by Severe Acute Respiratory Syndrome Coronavirus 2.

Telacebec (Q203): Is there a novel effective and safe anti-tuberculosis drug on the horizon?

High prevalence and stronger emergency of various forms of drug-resistant tuberculosis (DR-TB), including the multidrug-resistant (MDR-TB) as well as extensively drug-resistant (XDR-TB) ones, caused by variously resistant Mycobacterium tuberculosis pathogens, make first-line anti-tuberculosis (anti-TB) agents therapeutically more and more ineffective. Therefore, there is an imperative to develop novel highly efficient (synthetic) agents against both drug-sensitive-TB and DR-TB. The exploration of various heterocycles as prospective core scaffolds for the discovery, development and optimization of anti-TB drugs remains an intriguing scientific endeavour. Telacebec (Q203; TCB), a molecule containing an imidazo[1,2-a]pyridine-3-carboxamide (IPA) structural motif, is considered a novel very promising anti-TB agent showing a unique mechanism of action. The compound blocks oxidative phosphorylation by inhibiting a mycobacterial respiratory chain due to interference with a specific cytochrome b subunit (QcrB) of transmembrane bc1 menaquinol-cytochrome c oxidoreductase as an essential component for transporting electrons across the membrane from menaquinol to other specific subunit, cytochrome c (QcrC). Thus, the ability of mycobacteria to synthesize adenosine-5´-triphosphate is limited and energy generating machinery is disabled. The TCB molecule effectively fights drug-susceptible, MDR as well as XDR M. tuberculosis strains. The article briefly explains a mechanism of an anti-TB action related to the compounds containing a variously substituted IPA scaffold and is focused on their fundamental structure-anti-TB activity relationships as well. Special consideration is paid to TCB indicating the importance of particular structural fragments for maintaining (or even improving) favourable pharmacodynamic, pharmacokinetic and/or toxicological properties. High lipophilicity of TCB might be regarded as one of the key physicochemical properties with positive impact on anti-TB effect of the drug. In January 2021, the TCB molecule was also involved in phase-II clinical trials focused on the treatment of Coronavirus Disease-19 caused by Severe Acute Respiratory Syndrome Coronavirus 2.

Inhibitors of energy metabolism interfere with antibiotic-induced death in mycobacteria.

Energy metabolism has recently gained interest as a target space for antibiotic drug development in mycobacteria. Of particular importance is bedaquiline (Sirturo), which kills mycobacteria by inhibiting the F1F0 ATP synthase. Other components of the electron transport chain such as the NADH dehydrogenases (NDH-2 and NdhA) and the terminal respiratory oxidase bc1:aa3 are also susceptible to chemical inhibition. Because antituberculosis drugs are prescribed as part of combination therapies, the interaction between novel drugs targeting energy metabolism and classical first and second line antibiotics must be considered to maximize treatment efficiency. Here, we show that subinhibitory concentration of drugs targeting the F1F0 ATP synthase and the cytochrome bc1:aa3, as well as energy uncouplers, interfere with the bactericidal potency of isoniazid and moxifloxacin. Isoniazid- and moxifloxacin-induced mycobacterial death correlated with a transient increase in intracellular ATP that was dissipated by co-incubation with energy metabolism inhibitors. Although oxidative phosphorylation is a promising target space for drug development, a better understanding of the link between energy metabolism and antibiotic-induced mycobacterial death is essential to develop potent drug combinations for the treatment of tuberculosis.

Telacebec for Ultrashort Treatment of Buruli Ulcer in a Mouse Model.

Telacebec (Q203) is a new antitubercular drug with extremely potent activity against Mycobacterium ulcerans Here, we explored the treatment-shortening potential of Q203 alone or in combination with rifampin (RIF) in a mouse footpad infection model. The first study compared Q203 at 5 and 10 mg/kg doses alone and with rifampin. Q203 alone rendered most mouse footpads culture negative in 2 weeks. Combining Q203 with rifampin resulted in a relapse-free cure 24 weeks after completing 2 weeks of treatment, compared to a 25% relapse rate in mice receiving RIF with clarithromycin, the current standard of care, for 4 weeks. The second study explored the dose-ranging activity of Q203 alone and with RIF, including the extended activity of Q203 after treatment discontinuation. The bactericidal activity of Q203 persisted for ≥ 4 weeks beyond the last dose. All mice receiving just 1 week of Q203 at 2 to 10 mg/kg were culture negative 4 weeks after stopping treatment. Mice receiving 2 weeks of Q203 at 0.5, 2, and 10 mg/kg were culture negative 4 weeks after treatment. RIF did not increase the efficacy of Q203. A pharmacokinetics substudy revealed that Q203 doses of 2 to 10 mg/kg in mice produce plasma concentrations similar to those produced by 100 to 300 mg doses in humans, with no adverse effect of RIF on Q203 concentrations. These results indicate the extraordinary potential of Q203 to reduce the duration of treatment necessary for a cure to ≤ 1 week (or 5 doses of 2 to 10 mg/kg) in our mouse footpad infection model and warrant further evaluation of Q203 in clinical trials.

Toward a Single-Dose Cure for Buruli Ulcer.

A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection.

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 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 bc1 :aa3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.

Identification of Substituted Amino Acid Hydrazides as Novel Anti-Tubercular Agents, Using a Scaffold Hopping Approach.

Discovery and development of new therapeutic options for the treatment of Mycobacterium tuberculosis (Mtb) infection, particularly drug-resistant strains, are urgently required to tackle the global burden of this disease. Herein, we reported the synthesis of a novel series of N-substituted amino acid hydrazides, utilising a scaffold hopping approach within a library of anti-tubercular agents. Efficacy and selectivity were evaluated against three strains of Mtb (wild-type, isoniazid-resistant and rifampicin-resistant), and cytotoxicity against macrophages in vitro. The antibacterial activity and therapeutic index of these molecules were significantly affected by modifications with the N-substituents. Introduction of a 3,5-dinitroaryl moiety demonstrated enhanced antibacterial activity against all three strains of Mtb. In contrast, the inclusion of an imidazo [1,2-a]pyridine-3-carboxy moiety resulted in enhanced activity towards isoniazid mono-resistant Mtb relative to wild-type Mtb. Consequently, this scaffold hopping approach showed significant promise for exemplification of novel molecules with specific activity profiles against drug-resistant tuberculosis.

Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.

Shortening Buruli Ulcer Treatment with Combination Therapy Targeting the Respiratory Chain and Exploiting Mycobacterium ulcerans Gene Decay.

Buruli ulcer is treatable with antibiotics. An 8-week course of rifampin (RIF) and either streptomycin (STR) or clarithromycin (CLR) cures over 90% of patients. However, STR requires injections and may be toxic, and CLR shares an adverse drug-drug interaction with RIF and may be poorly tolerated. Studies in a mouse footpad infection model showed that increasing the dose of RIF or using the long-acting rifamycin rifapentine (RPT), in combination with clofazimine (CFZ), a relatively well-tolerated antibiotic, can shorten treatment to 4 weeks. CFZ is reduced by a component of the electron transport chain (ETC) to produce reactive oxygen species toxic to bacteria. Synergistic activity of CFZ with other ETC-targeting drugs, the ATP synthase inhibitor bedaquiline (BDQ) and the bc1:aa3 oxidase inhibitor Q203 (now named telacebec), was recently described against Mycobacterium tuberculosis Recognizing that M. tuberculosis mutants lacking the alternative bd oxidase are hypersusceptible to Q203 and that Mycobacterium ulcerans is a natural bd oxidase-deficient mutant, we tested the in vitro susceptibility of M. ulcerans to Q203 and evaluated the treatment-shortening potential of novel 3- and 4-drug regimens combining RPT, CFZ, Q203, and/or BDQ in a mouse footpad model. The MIC of Q203 was extremely low (0.000075 to 0.00015 µg/ml). Footpad swelling decreased more rapidly in mice treated with Q203-containing regimens than in mice treated with RIF and STR (RIF+STR) and RPT and CFZ (RPT+CFZ). Nearly all footpads were culture negative after only 2 weeks of treatment with regimens containing RPT, CFZ, and Q203. No relapse was detected after only 2 weeks of treatment in mice treated with any of the Q203-containing regimens. In contrast, 15% of mice receiving RIF+STR for 4 weeks relapsed. We conclude that it may be possible to cure patients with Buruli ulcer in 14 days or less using Q203-containing regimens rather than currently recommended 56-day regimens.

Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis.

New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3.

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-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 bcc, 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 that 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.

Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

Investigation of protein-ligand binding motions through protein conformational morphing and clustering of cytochrome bc1-aa3 super complex.

Cytochrome b (QcrB) is considered an essential subunit in the electron transport chain that coordinates the action of the entire cytochrome bc1 oxidase. It has been identified as an attractive drug target for a new promising clinical candidate Q203 that depletes the intracellular ATP levels in the bacterium, Mycobacterium tuberculosis. However, single point polymorphism (T313A/I) near the quinol oxidation site of QcrB developed resistance to Q203. In the present study, we analyze the structural changes and drug-resistance mechanism of QcrB due to the point mutation in detail through conformational morphing and molecular docking studies. By morphing, we generated conformers between the open and closed state of the electron transporting cytochrome bc1-aa3 super complex. We clustered them to identify four intermediate structures and relevant intra- and intermolecular motions that may be of functional relevance, especially the binding of Q203 in wild and mutant QcrB intermediate structures and their alteration in developing drug resistance. The difference in the binding score and hydrogen bond interactions between Q203 and the wild-type and mutant intermediate structures of QcrB from molecular docking studies showed that the point mutation T313A severely affected the binding affinity of the candidate drug. Together, the findings provide an in-depth understanding of QcrB inhibition in different conformations, including closed, intermediate, and open states of cytochrome bc1-aa3 super complex in Mycobacterium tuberculosis at the atomic level. We also obtain insights for designing QcrB and cytochrome bc1-aa3 inhibitors as potential therapeutics that may combat drug resistance in tuberculosis.