Deazaflavin-dependent Nitroreductase Research Articles

Phenotypic-Based Discovery and Exploration of a Resorufin Scaffold with Activity against Mycobacterium tuberculosis.

Tuberculosis remains a leading cause of death by infectious disease. The long treatment regimen and the spread of drug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) necessitates the development of new treatment options. In a phenotypic screen, nitrofuran-resorufin conjugate 1 was identified as a potent sub-micromolar inhibitor of whole cell Mtb. Complete loss of activity was observed for this compound in Mtb mutants affected in enzyme cofactor F420 biosynthesis (fbiC), suggesting that 1 undergoes prodrug activation in a manner similar to anti-tuberculosis prodrug pretomanid. Exploration of the structure-activity relationship led to the discovery of novel resorufin analogues that do not rely on the deazaflavin-dependent nitroreductase (Ddn) bioactivation pathway for their antimycobacterial activity. These analogues are of interest as they work through an alternative, currently unknown mechanism that may expand our chemical arsenal towards the treatment of this devastating disease.

Both Nitro Groups Are Essential for High Antitubercular Activity of 3,5-Dinitrobenzylsulfanyl Tetrazoles and 1,3,4-Oxadiazoles through the Deazaflavin-Dependent Nitroreductase Activation Pathway.

3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.

Discovery and characterization of antimycobacterial nitro-containing compounds with distinct mechanisms of action and in vivo efficacy.

Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F420 machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F420-dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus. Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1. Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro, but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model.

Synthesis and Anti-Mycobacterium tuberculosis Activity of Imidazo[2,1-b][1,3]oxazine Derivatives against Multidrug-Resistant Strains.

The emergence of multidrug-resistant strains of M. tuberculosis has raised concerns due to the greater difficulties in patient treatment and higher mortality rates. Herein, we revisited the 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine scaffold and identified potent new carbamate derivatives having MIC90 values of 0.18-1.63 μM against Mtb H37Rv. Compounds 47-49, 51-53, and 55 exhibited remarkable activity against a panel of clinical isolates, displaying MIC90 values below 0.5 μM. In Mtb-infected macrophages, several compounds demonstrated a 1-log greater reduction in mycobacterial burden than rifampicin and pretomanid. The compounds tested did not exhibit significant cytotoxicity against three cell lines or any toxicity to Galleria mellonella. Furthermore, the imidazo[2,1-b][1,3]oxazine derivatives did not show substantial activity against other bacteria or fungi. Finally, molecular docking studies revealed that the new compounds could interact with the deazaflavin-dependent nitroreductase (Ddn) in a similar manner to pretomanid. Collectively, our findings highlight the chemical universe of imidazo[2,1-b][1,3]oxazines and their promising potential against MDR-TB.

Nitroimidazopyrazinones with Oral Activity against Tuberculosis and Chagas Disease in Mouse Models of Infection.

Tuberculosis and parasitic infections continue to impose a significant threat to global public health and economic growth. There is an urgent need to develop new treatments to combat these diseases. Here, we report the in vitro and in vivo profiles of a new bicyclic nitroimidazole subclass, namely, nitroimidazopyrazinones, against mycobacteria and Trypanosoma cruzi. Derivatives with monocyclic side chains were selective against Mycobacterium tuberculosis and were able to reduce the bacterial load when dosed orally in mice. We demonstrated that deazaflavin-dependent nitroreductase (Ddn) could act effectively on nitroimidazopyrazinones, indicating the potential of Ddn as an activating enzyme for these new compounds in M. tuberculosis. Oral administration of compounds with extended biaryl side chains (73 and 74) was effective in suppressing infection in an acute T. cruzi-infected murine model. These findings demonstrate that active nitroimidazopyrazinones have potential to be developed as orally available clinical candidates against both tuberculosis and Chagas disease.

Molecular dynamic assisted investigation on impact of mutations in deazaflavin dependent nitroreductase against pretomanid: a computational study

In the past decade, TB drugs belonging to the nitroimidazole class, pretomanid and delamanid, have been authorised to treat MDR-TB and XDR-TB. With a novel inhibition mechanism and a reduction in the span of treatment, it is now being administered in various combinations. This approach is not the ultimate remedy since the target protein Deazaflavin dependent nitroreductase (Ddn) has a high mutation frequency, and already pretomanid resistant clinical isolates are reported in various studies. Ddn is essential for M.tuberculosis to emerge from hypoxia, and point mutations in critical residues confer resistance to Nitro-imidazoles. Among the pool of available mutants, we have selected seven mutants viz DdnL49P, DdnY65S, DdnS78Y, DdnK79Q, DdnW88R, DdnY133C, and DdnY136S, all of which exhibited resistance to pretomanid. To address this issue, through computational study primarily by MD simulation, we attempted to elucidate these point mutations’ impact and investigate the resistance mechanism. Hence, the DdnWT and mutant (MT) complexes were subjected to all-atom molecular dynamics (MD) simulations for 100 ns. Interestingly, we observed the escalation of the distance between cofactor and ligand in some mutants, along with a significant change in ligand conformation relative to the DdnWT. Moreover, we confirmed that mutations rendered ligand instability and were ejected from the binding pocket as a result. In conclusion, the results obtained provide a new structural insight and vital clues for designing novel inhibitors to combat nitroimidazole resistance Communicated by Ramaswamy H. Sarma

2-((3,5-Dinitrobenzyl)thio)quinazolinones: Potent Antimycobacterial Agents Activated by Deazaflavin (F420)-Dependent Nitroreductase (Ddn).

Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4, ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26. Structure-activity relationship (SAR) studies indicated a crucial role for both meta-nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding (63c, 63d). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S-substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F420 biosynthesis (fbiC), cofactor reduction (fgd), or deazaflavin-dependent nitroreductase activity (rv3547), indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.

Comparison of in vitro Susceptibility of Mycobacteria Against PA-824 to Identify Key Residues of Ddn, the Deazoflavin-Dependent Nitroreductase from Mycobacterium tuberculosis.

ObjectivePA-824 (Pretomanid), a bicyclic nitroimidazole drug, exhibits significant bactericidal activity toward Mycobacterium tuberculosis (MTB) in vitro and in vivo, but not against Mycobacterium smegmatis. Through catalytic bioreduction, deazaflavin-dependent nitroreductase (Ddn) within MTB directly converts PA-824 to potent bactericidal products. This study aimed to identify key MTB Ddn residues involved in PA-824 conversion toward development of in vitro surrogate markers for detection of mycobacterial resistance to PA-824.MethodsWe evaluated in vitro activity of PA-824 toward MTB and nontuberculous mycobacterial species using antimicrobial susceptibility testing. Ddn amino acid sequence alignments and phylogenetic analysis revealed putative key enzyme active site residues. Candidate MTB Ddn residues required for PA-824 conversion activity were evaluated for loss-of-function using recombinantly cloned Ddn mutant proteins expressed in Mycobacterium smegmatis.ResultsPA-824 minimum inhibitory concentrations of 90% of bacterial growth (MIC90s) against MTB and Mycobacterium kansasii were 0.12 mg/L and 8 mg/L, respectively, but >32 mg/L for Mycobacterium spp. M. avium, M. intracellulare, M. abscessus and M. fortuitum. MTB Ddn and M. kansasii Ddn homologous sequences shared the greatest similarity (89.3% amino acid identity). M. smegmatis expressing Ddn proteins with Y65L, A76V or Y133F substitutions (but not V75L, Q125K or V148I) were resistant to PA-824.ConclusionOur data demonstrated that PA-824 exhibited excellent and moderate levels of in vitro activity against MTB and M. kansasii, respectively. Substitutions of Ddn residues Y65, A76 or Y133 conferred mycobacterial resistance to PA-824.

Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering.

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.

Pretomanid for the treatment of pulmonary tuberculosis.

Discovering novel drugs active against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is currently one of the most unmet medical needs. In this context, pretomanid (PA-824), a novel nitroimidazole prodrug that targets both replicating and nonreplicating cells, is being developed by TB Alliance under license from Novartis. In replicating Mtb, pretomanid inhibits mycolic acid biosynthesis, which is an important building block of Mtb cell wall. Under nonreplicating conditions, pretomanid is reduced by deazaflavin-dependent nitroreductase, leading to generation of reactive nitrogen species exhibiting potent antimycobacterial activity. The U.S. Food and Drug Administration (FDA) has approved pretomanid under the Limited Population Pathway for Antibacterial and Antifungal Drugs (LPAD pathway) for treatment of adult patients with treatment-intolerant or nonresponsive multidrug-resistant TB and extensively drug-resistant TB in combination with bedaquiline and linezolid as part of the oral.

Designing of Nitroimidazole Derivatives as a Promising Target for Treatment of Tuberculosis

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, with high level of mortality worldwide, currently with approximately 10 million cases of tuberculosis. These rate of incidence are due to several factors such as bacterial resistance, AIDS, latent tuberculosis that reoccur in patient. Deazaflavin dependent nitroreductase (Ddn) is an emerging target in the field of antitubercular agent. Ddn catalyses the reduction of nitroimidazoles resulting in intra-cellular release of lethal reactive nitrogen species. Nitroimidazole class drug- delamanid and pretonamid are used in the treatment of MDR-TB. In this present study, 26 new nitroimidazole derivatives were designed and docked into Ddn enzyme. In docking study, compounds 3, 5, 15, 16, 17, 18 and 21 showed similar interaction with amino acid residues such as Tyr 65, Ser 78, Tyr 136 as pretonamid reference drug and highest docking score and better ADMET compatibility. The ADMET prediction docking study of new designed compound revealed that the compounds 3, 16, 17 and 21 showed good binding with Ddn. In future it may be good and effective lead for development of antitubercular agent.

3D QSAR, Docking, Molecular dynamics simulations and MM-GBSA studies of Extended Side Chain of the Antitubercular Drug (6S) 2-Nitro-6-{[4-(trifluoromethoxy) benzyl] oxy}-6,7-dihydro-5H-imidazo[2,1-b] [1,3] oxazine.

PA-824 analogues have been proposed on a promising approach for treating MDR/XDR tuberculosis. In order to understand the structural requirement of reported extended side chain analogues were studied to get insight into their structural requirements responsible for high affinity as a ligand-based pharmacophore, 3D-QSAR model have been developed. Docking and molecular dynamics studies revealed the better binding interaction of inhibitor binding pocket of deazaflavin dependent nitroreductase (Ddn) with cofactor F420 crystal. For pharmacophore generation and atom-based 3D-QSAR analysis, a dataset of 84 compounds were selected which were carried out using PHASE. The docking studies were performed using Glide module consists of five steps protein preparation, ligand preparation, receptor grid generation, actual docking procedure and finally viewing the docking results using the poseviewer. QikProp provides ranges for comparing a particular molecule's properties with those of 95% of known drugs. Molecular dynamics (MD) simulations for docking complex of deazaflavin dependent nitroreductase (Ddn) with molecule 63 were performed using Desmond. Prime Molecular Mechanics/Generalized-Born/Surface Area (MM-GBSA) was used for the calculation of binding free energy for the docked complexes. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of R2 = 0.8988 for training set compounds, higher variance ratio F= 127.3 and the model generated showed excellent predictive power, with a correlation coefficient of structure to analyses Q2= 0.8543 for a randomly chosen test set of 17 compounds. The binding position of most active molecule 63 is shown in figure 4. Several favorable interactions between ligand and enzyme clearly observed; H-bond showed between O atom presence as spacer in C-6- 2-Nitro-6-{[4-(trifluoromethoxy) benzyl] oxy}-6,7-dihydro-5H-imidazo[2,1-b] [1,3] oxazine and Asn 62. Weak hydrogen bonding observed between N1 atom in imidazole ring and Asn91. The binding of imidazole nucleus occurs at site, which has extensive hydrophobic interaction with Arg60 residues. All these pharmacokinetic parameters within the acceptable range defined for human use, thereby indicating their potential as drug- like molecule. Stability of deazaflavin dependent nitroreductase (Ddn) with molecule 63 complex was evaluated through 100 ns molecular dynamic simulations. Main contributions to the tight binding of molecule 63 to Ddn are the exceptionally electrostatics (dG_bind_Coulomb) and enhance hydrogen bond interactions (dG_bind_Hbond). Docking, MM-GBSA, MD stimulation, pharmacophore model and 3D-QSAR studies as well as QikProp pharmacokinetic analysis presented in this paper is hoped to be a primer towards the development of various novel PA-824 with different chemical scaffolds and further its biological activity predictions to invent novel, potent, selective and safe PA-824 analogues for the treatment of MDR/XDR tuberculosis. Moreover, further use of contemporary experimental and computational techniques to data presented here may widen its scope and applicability.

Design of 2-Nitroimidazooxazine Derivatives as Deazaflavin-Dependent Nitroreductase (Ddn) Activators as Anti-Mycobacterial Agents Based on 3D QSAR, HQSAR, and Docking Study with In Silico Prediction of Activity and Toxicity.

Deazaflavin-dependent nitroreductase (Ddn) is an emerging target in the field of anti-tuberculosis agents. In the present study, 2-nitroimidazooxazine derivatives as Ddn activators were aligned for CoMFA, CoMSIA and HQSAR analysis. The best CoMFA and CoMSIA model were generated with leave-one-out correlation coefficients (q2) of 0.585 and 0.571, respectively. Both the CoMFA and CoMSIA models were also validated by a test set of 11 compounds with satisfactory [Formula: see text] value of 0.701 and 0.667, respectively. Results of 3D QSAR and HQSAR study were used for the designing of novel and potent nitroimidazooxazine derivatives as Ddn activators. 21 novel compounds were designed, and docked into the Ddn enzyme. In docking study compound ng11 showed interaction with key amino acid residues such as Tyr65 and Tyr133, and also showed better ADMET compatibility. The ADMET prediction, docking study and the predicted activity of novel designed compounds revealed that compound ng11 showed good potential as Ddn activators for the treatment of tuberculosis.

Hydrophobic Shielding Drives Catalysis of Hydride Transfer in a Family of F420H2-Dependent Enzymes.

A family of flavin/deazaflavin-dependent oxidoreductases (FDORs) from mycobacteria has been recently characterized and found to play a variety of catalytic roles, including the activation of prodrugs such as the candidate anti-tuberculosis drug pretomanid (PA-824). However, our understanding of the catalytic mechanism used by these enzymes is relatively limited. To address this, we have used a combination of quantum mechanics and molecular dynamics calculations to study the catalytic mechanism of the activation of pretomanid by the deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis. The preferred pathway involves an initial hydride transfer step from the deprotonated cofactor (i.e., F420H-), with subsequent protonation, before a series of spontaneous intramolecular reactions to form the final reactive nitrogen species. The most likely proton source is a hydroxonium ion within the solvent accessible active site. Intriguingly, catalysis of the rate-determining hydride transfer step is aided by three tyrosine residues that form a hydrophobic barrier around the active site that, upon reaction, is then disrupted to allow increased water accessibility to facilitate the subsequent proton transfer step. The catalytic mechanism we propose is consistent with previous experimental observations of the Ddn enzyme and will inform the design of improved prodrugs in the future.

Protonation state of F420H2 in the prodrug-activating deazaflavin dependent nitroreductase (Ddn) from Mycobacterium tuberculosis.

The protonation state of the deazaflavin dependent nitroreductase (Ddn) enzyme bound cofactor F420 was investigated using UV-visible spectroscopy and computational simulations. The reduced cofactor F420H2 was determined to be present in its deprotonated state in the holoenzyme form. The mechanistic implications of these findings are discussed.

3D-QSAR and Cell Wall Permeability of Antitubercular Nitroimidazoles against Mycobacterium tuberculosis

Inhibitory activities of monocyclic nitroimidazoles against Mycobacterium tuberculosis (Mtb) deazaflavin-dependent nitroreductase (DDN) were modeled by using docking, pharmacophore alignment and comparative molecular similarity indices analysis (CoMSIA) methods. A statistically significant model obtained from CoMSIA was established based on a training set using pharmacophore-based molecular alignment. The leave-one out cross-validation correlation coefficients q2 (CoMSIA) were 0.681. The CoMSIA model had a good correlation (/CoMSIA = 0.611) between the predicted and experimental activities against excluded test sets. The generated model suggests that electrostatic, hydrophobic and hydrogen bonding interactions all play important roles for interaction between ligands and receptors. The predicted cell wall permeability (logPapp) for substrates with high inhibitory activity against Mtb were investigated. The distribution coefficient (logD) range was 2.41 < logD < 2.89 for the Mtb cell wall membrane permeability. The larger the polar surface area is, the better the permeability is. A larger radius of gyration (rgry) and a small fraction of rotatable bonds (frtob) of these molecules leads to higher cell wall penetration ability. The information obtained from the in silico tools might be useful in the design of more potent compounds that are active against Mtb.

Bactericidal activity of PA-824 against Mycobacterium tuberculosis under anaerobic conditions and computational analysis of its novel analogues against mutant Ddn receptor

BackgroundThe resurgence of multi-drug resistant tuberculosis (MDR-TB) and HIV associated tuberculosis (TB) are of serious global concern. To contain this situation, new anti-tuberculosis drugs and reduced treatment regimens are imperative. Recently, a nitroimidazole, PA-824, has been shown to be active against both replicating and non-replicating bacteria. It is activated by the enzyme Deazaflavin-dependent nitroreductase (Ddn) present in Mycobacterium tuberculosis which catalyzes the reduction of PA-824, resulting in the release of lethal reactive nitrogen species (RNS) within the bacteria. In this context, PA-824 was analyzed for its activity against latent tuberculosis under anaerobic conditions and compared with rifampicin (RIF) and pyrazinamide (PZA). Recent mutagenesis studies have identified A76E mutation which affects the above mentioned catalysis and leads to PA-824 resistance. Hence, novel analogues which could cope up with their binding to mutant Ddn receptor were also identified through this study.ResultsPA-824 at an optimum concentration of 12.5 μg/ml showed enhanced bactericidal activity, resulting in 0 CFU/ml growth when compared to RIF and PZA at normal pH and anaerobic condition. Further docking studies revealed that a combinatorial structure of PA-824 conjugated with moxifloxacin (ligand 8) has the highest binding affinity with the wild type and mutant Ddn receptor.ConclusionsPA-824 has been demonstrated to have better activity under anaerobic condition at 12.5 μg/ml, indicating an optimized dose that is required for overcoming the detoxifying mechanisms of M. tuberculosis and inducing its death. Further, the development of resistance through A76E mutation could be overcome through the in silico evolved ligand 8.

The R Enantiomer of the Antitubercular Drug PA-824 as a Potential Oral Treatment for Visceral Leishmaniasis

The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.

A novel F420‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2-dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F420H2-specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents.

Structure of Ddn, the Deazaflavin-Dependent Nitroreductase from Mycobacterium tuberculosis Involved in Bioreductive Activation of PA-824

SummaryTuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.