Deazaflavin-dependent Nitroreductase Research Articles

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles.

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.

Structure–Activity Relationships of Antitubercular Nitroimidazoles. 3. Exploration of the Linker and Lipophilic Tail of ((S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-Amino PA-824).

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 μM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.

Zircon ages, Sr-Nd-Hf isotopic compositions, and geochemistry of granitoids associated with the northern ophiolite melange of Central Cuba: Tectonic implication for Late Cretaceous magmatism in the Northwestern Caribbean

<h3>Abstract</h3> Tuberculosis remains one of the leading causes of death from a single infectious agent, surpassing both AIDS and malaria. In recent years, two bicyclic nitroimidazole drugs, delamanid and pretomanid have been approved to treat this airborne infection. This has spurred a renewed interest in developing new and improved nitroimidazole analogs. We have previously identified a new bicyclic heteroaromatic subclass, the nitroimidazopyrazinones, with substituted analogs showing promising activity against <i>Mycobacterium tuberculosis</i> under both aerobic and hypoxic environments. A second generation of nitroimidazopyrazinones with extended biaryl side chain also possessed good antiparasitic activity against <i>Trypanosoma brucei brucei</i> and <i>Trypanosoma cruzi</i>, suggesting the utility of this new scaffold for development into potential candidates against both tuberculosis and the kinetoplastid parasites which cause neglected tropical diseases. In this study, we further evaluated the properties of nitroimidazopyrazinone derivatives by assessing their selectivity against different mycobacterial species, measuring their reduction potential, and determining the kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn), which is the activating enzyme of delamanid and pretomanid in <i>M. tuberculosis</i>. We also conducted an <i>in vivo</i> evaluation of a lead compound, MCC8967 that demonstrated a favorable pharmacokinetic profile, with good oral bioavailability and efficacy in an acute <i>M. tuberculosis</i> infection model. Two other promising compounds MCC9481 and MCC9482, with good <i>in vitro</i> activity (IC₅₀ = 0.016 and 0.10 µM, respectively) against <i>T. cruzi</i>, the causative agent for Chagas diseases, were similarly tested for <i>in vivo</i> activity. These compounds also exhibited good oral bioavailability, and transiently reduced the acute-stage parasite burden by &gt;98‒99% at doses of 50 mg/kg once or twice daily, similar to benznidazole at 100 mg/kg once daily. Overall, we have demonstrated that active nitroimidazopyrazinones have potential to be developed as clinical candidates against both tuberculosis and Chagas disease. <h3>Author Summary</h3> Tuberculosis and parasitic infections continue to impose a significant threat to public health and economic growth worldwide. Most of the efforts to control these diseases still rely on drug treatments with limited effectiveness and significant side effects. There is now an urgent need to develop new treatments to combat these infections. Here, we report the <i>in vitro</i> and <i>in vivo</i> profile of a new bicyclic nitroimidazole subclass, namely nitroimidazopyrazinones, against mycobacteria and <i>Trypanosoma cruzi</i>. We found that derivatives with monocyclic side chains are selective against <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, but not active against other nontuberculosis mycobacteria. In an acute mouse model, they were able to reduce the bacterial load in lungs via oral administration. From a biochemistry perspective, 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 <i>M. tuberculosis</i>. We also showed that derivatives with extended biaryl side chain were effective in suppressing infection in an acute <i>T. cruzi</i> infected murine model, with satisfactory oral bioavailability. These findings improve the understanding of the biological profile of nitroimidazopyrazinones for further development as potential antitubercular and antiparasitic agents.

The Nitroimidazooxazines (PA-824 and Analogs): Structure–Activity Relationship and Mechanistic Studies

PA-824 is an experimental anti-tubercular agent that has a novel mechanism of action. It is effective against both active and persistent forms of the disease and has recently shown early bactericidal activity in a Phase II clinical trial. This review summarizes recent studies on the mode of action of PA-824 and outlines successful efforts to prepare more effective second-generation analogs. PA-824 displays unusual chemistry following both enzymatic and radiolytic reduction, which is clearly related to its activity as an anti-tubercular agent. The nitroreductase enzyme deazaflavin-dependent nitroreductase, reduces PA-824 with loss of the nitro group, generating reactive nitrogen species such as nitric oxide, which appear important in mediating the activity of the drug. Bioreductive drugs such as PA-824 hold the promise of shorter treatment regimens.

Unexpected Abundance of Coenzyme F 420 -Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F(420), which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F(420) biosynthesis nominated many actinobacterial proteins as candidate F(420)-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5'-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F(420)-producing species. The LLM and PPOX families were observed in F(420)-producing species as well as species lacking F(420) but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism's ability to make F(420) allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F(420)-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F(420)-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F(420) biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F(420) for its redox reactions. This dependence and the cofactor's rarity may make F(420)-related proteins promising drug targets.

Structure−Activity Relationships of Antitubercular Nitroimidazoles. 1. Structural Features Associated with Aerobic and Anaerobic Activities of 4- and 5-Nitroimidazoles

The 4-nitroimidazole PA-824 is active against aerobic and anaerobic Mycobacterium tuberculosis (Mtb) while 5-nitroimidazoles like metronidazole are active against only anaerobic Mtb. We have synthesized analogues of both 4- and 5-nitroimidazoles and explored their antitubercular activities. The nitro group is required for both activities in all compounds. The key determinants of aerobic activity in the 4-nitroimidazoles include the bicyclic oxazine, the lipophilic tail, and the 2-position oxygen. For the 5-nitroimidazoles, neither the corresponding bicyclic analogue nor addition of a lipophilic tail conveyed aerobic activity. Incorporation of a 2-position oxygen atom into a rigid 5-nitroimidazooxazine provided the first 5-nitroimidazole with aerobic activity. Across both series, anaerobic and aerobic activities were not correlated and Mtb mutants lacking the deazaflavin-dependent nitroreductase (Ddn) retained anaerobic sensitivity to some compounds. Aerobic activity appears to be correlated with efficiency as a substrate for Ddn, suggesting a means of structure-based optimization of improved nitroimidazoles.

PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release.

Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biological function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivatives of PA-824 were used, the amount of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compounds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system.