Upregulation, Functional Association, and Correlated Expressions of TRPV1 and TRPA1 During Telmisartan-Driven Immunosuppression of T Cells.

TRPV1 and TRPA1, are known to be functionally expressed in T cells, where these two channels differentially regulate effector immune responses. Telmisartan (TM), an anti-hypertension drug, has been recently repurposed to suppress various inflammatory responses. However, the possible involvement of TRP channels during TM-driven suppression of T cells responses has not been explored yet. In this study, we investigated the potential role of TRPV1 and TRPA1 during TM-driven immunosuppression of T cells in vitro. We observed a significant elevation of both TRPV1 and TRPA1 during TM-induced immunosuppression of T cells.We found that TRPA1 activation-driven suppression of T cell activation and effector cytokine responses during TM treatment is partially, yet significantly overridden by TRPV1 activation. Moreover, the expressions of TRPV1 and TRPA1 were highly correlated in various conditions of T cell. Mechanistically, it might be suggested that TRPV1 and TRPA1 are differentially involved in regulating T cell activation despite the co-elevation of both these TRP channels' expressions in the presence of TM. T cell activation was delineated by CD69 and CD25 expressions along with the effector cytokine levels (IFN-γ and TNF) in TM-driven suppression of T cell. These findings could have broad implications for designing possible future immunotherapeutic strategies, especially in the repurposing of TM for T cell-TRP-directed immune disorders.

TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) channels are known to be actively involved in various pathophysiological conditions, including neuronal inflammation, neuropathic pain, and various immunological responses. Heat shock protein 90 (Hsp90), a cytoplasmic molecular chaperone, is well-reported for various cellular and physiological processes. Hsp90 inhibition by various molecules has garnered importance for its therapeutic significance in the downregulation of inflammation and are proposed as anti-cancer drugs. However, the possible role of TRPA1 in the Hsp90-associated modulation of immune responses remains scanty. RESULTS: Here, we have investigated the role of TRPA1 in regulating the anti-inflammatory effect of Hsp90 inhibition via 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) in lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) stimulation in RAW 264.7, a mouse macrophage cell lines and PMA differentiated THP-1, a human monocytic cell line similar to macrophages. Activation of TRPA1 with Allyl isothiocyanate (AITC) is observed to execute an anti-inflammatory role via augmenting Hsp90 inhibition-mediated anti-inflammatory responses towards LPS or PMA stimulation in macrophages, whereas inhibition of TRPA1 by 1,2,3,6-Tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7 H-purine-7-acetamide,2-(1,3-Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7 H-purin-7-yl)-N-(4-isopropylphenyl)acetamide (HC-030031) downregulates these developments. LPS or PMA-induced macrophage activation was found to be regulated by TRPA1. The same was confirmed by studying the levels of activation markers (major histocompatibility complex II (MHCII), cluster of differentiation (CD) 80 (CD80), and CD86, pro-inflammatory cytokines (tumor necrosis factor (TNF) and interleukin 6 (IL-6)), NO (nitric oxide) production, differential expression of mitogen-activated protein kinase (MAPK) signaling pathways (p-p38 MAPK, phospho-extracellular signal-regulated kinase 1/2 (p-ERK 1/2), and phosphor-stress-activated protein kinase/c-Jun N-terminal kinase (p-SAPK/JNK)), and induction of apoptosis. Additionally, TRPA1 has been found to be an important contributor to intracellular calcium levels toward Hsp90 inhibition in LPS or PMA-stimulated macrophages. CONCLUSION: This study indicates a significant role of TRPA1 in Hsp90 inhibition-mediated anti-inflammatory developments in LPS or PMA-stimulated macrophages. Activation of TRPA1 and inhibition of Hsp90 has synergistic roles towards regulating inflammatory responses associated with macrophages. The role of TRPA1 in Hsp90 inhibition-mediated modulation of macrophage responses may provide insights towards designing future novel therapeutic approaches to regulate various inflammatory responses.

Telmisartan Restricts Chikungunya Virus Infection In Vitro and In Vivo through the AT1/PPAR-γ/MAPKs Pathways.

Chikungunya virus (CHIKV) has reemerged as a global public health threat. The inflammatory pathways of the renin-angiotensin system (RAS) and peroxisome proliferator-activated receptor-gamma (PPAR-γ) are usually involved in viral infections. Thus, telmisartan (TM), which is known to block the angiotensin 1 (AT1) receptor and activate PPAR-γ, was investigated for activity against CHIKV. The anti-CHIKV effect of TM was investigated in vitro (Vero cells, RAW 264.7 cells, and human peripheral blood mononuclear cells [hPBMCs]) and in vivo (C57BL/6 mice). TM was found to abrogate CHIKV infection efficiently (50% inhibitory concentration (IC50) of 15.34 to 20.89 µM in the Vero cells and RAW 264.7 cells, respectively). Viral RNA and proteins were reduced remarkably. Additionally, TM interfered in the early and late stages of the CHIKV life cycle with efficacy during pretreatment and posttreatment. Moreover, the agonist of the AT1 receptor and an antagonist of PPAR-γ increased CHIKV infection, suggesting that the antiviral potential of TM occurs through modulating host factors. In addition, reduced activation of all major mitogen-activated protein kinases (MAPKs), NF-κB (p65), and cytokines by TM occurred through the inflammatory axis and supported the fact that the anti-CHIKV efficacy of TM is partly mediated through the AT1/PPAR-γ/MAPKs pathways. Interestingly, at a human equivalent dose, TM abrogated CHIKV infection and inflammation significantly, leading to reduced clinical scores and complete survival of C57BL/6 mice. Additionally, TM reduced infection in hPBMC-derived monocyte-macrophage populations in vitro. Hence, TM was found to reduce CHIKV infection by targeting both viral and host factors. Considering its safety and in vivo efficacy, it can be a suitable candidate in the future for repurposing against CHIKV.

Unsatisfactory quality of E. coli asparaginase biogenerics in India: Implications for clinical outcomes in acute lymphoblastic leukaemia.

BACKGROUND: The biotherapeutic asparaginase is a cornerstone of therapy in acute lymphoblastic leukaemia (ALL). With limited access to the original native Escherichia coli-derived asparaginase (EcASNase), a variety of EcASNase biogenerics are used in low-middle-income countries (LMICs). The variable quality of these biogenerics potentially influences clinical outcomes. PROCEDURE: Seven biogeneric EcASNases (P1-P7) marketed widely in India were evaluated, with P2 as an exemplar for in vivo monitoring. Therapeutic activity of P2 (10,000 IU/m2 /dose, intramuscular, every 72 hours) was monitored during induction therapy, and drug-related toxicities recorded. Molecular identity, purity and in vitro drug activity of seven biogenerics were characterised using multimodal analyses, and findings compared with reference EcASNase (R). RESULTS: In patients (N = 62) receiving P2, subtherapeutic asparaginase activity (<100 U/L) was observed in 66% (46/70) of trough timepoints (72 hours postdose) during induction. Twelve patients (19%), 11 with high-risk ALL, developed hypersensitivity. Isoforms of EcASNase were identified in all seven biogenerics. All generic products contained impurities with batch-to-batch variability. These included high levels of protein aggregates and host cell protein contamination. In vitro assays of EcASNase activity and leukaemia cell line cytotoxicity were not discriminatory. CONCLUSIONS: Our findings confirm widespread concerns over the unsatisfactory quality and therapeutic activity of native EcASNase biogenerics marketed in LMICs. Appropriate use of these products requires monitored studies to identify clinical suitability and determine appropriate dosing and schedule. For large parts of the world, assured access to high-quality asparaginases remains an unmet therapeutic need.

Syntheses, structures, and stabilities of aliphatic and aromatic fluorous iodine(I) and iodine(III) compounds: the role of iodine Lewis basicity.

The title molecules are sought in connection with various synthetic applications. The aliphatic fluorous alcohols Rfn CH2OH (Rfn = CF3(CF2) n-1; n = 11, 13, 15) are converted to the triflates Rfn CH2OTf (Tf2O, pyridine; 22-61%) and then to Rfn CH2I (NaI, acetone; 58-69%). Subsequent reactions with NaOCl/HCl give iodine(III) dichlorides Rfn CH2ICl2 (n = 11, 13; 33-81%), which slowly evolve Cl2. The ethereal fluorous alcohols CF3CF2CF2O(CF(CF3)CF2O) x CF(CF3)CH2OH (x = 2-5) are similarly converted to triflates and then to iodides, but efforts to generate the corresponding dichlorides fail. Substrates lacking a methylene group, Rfn I, are also inert, but additions of TMSCl to bis(trifluoroacetates) Rfn I(OCOCF3)2 appear to generate Rfn ICl2, which rapidly evolve Cl2. The aromatic fluorous iodides 1,3-Rf6C6H4I, 1,4-Rf6C6H4I, and 1,3-Rf10C6H4I are prepared from the corresponding diiodides, copper, and Rfn I (110-130 °C, 50-60%), and afford quite stable Rfn C6H4ICl2 species upon reaction with NaOCl/HCl (80-89%). Iodinations of 1,3-(Rf6)2C6H4 and 1,3-(Rf8CH2CH2)2C6H4 (NIS or I2/H5IO6) give 1,3,5-(Rf6)2C6H3I and 1,2,4-(Rf8CH2CH2)2C6H3I (77-93%). The former, the crystal structure of which is determined, reacts with Cl2 to give a 75:25 ArICl2/ArI mixture, but partial Cl2 evolution occurs upon work-up. The latter gives the easily isolated dichloride 1,2,4-(Rf8CH2CH2)2C6H3ICl2 (89%). The relative thermodynamic ease of dichlorination of these and other iodine(I) compounds is probed by DFT calculations.

A novel F(420) -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 F(420) -dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F(420) -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, F(420) (-) 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 F(420) H(2) -dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F(420) H(2) -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 F(420) biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents.

Salicylic Acid Conjugate of Telmisartan Inhibits Chikungunya Virus Infection and Inflammation.

There is no approved antiviral for the management of the Chikungunya virus (CHIKV). To develop an antiviral drug that can manage both CHIKV and arthritis induced by it, an ester conjugate of telmisartan (TM) and salicylic acid (SA) was synthesized (DDABT1). It showed higher potency (IC50 of 14.53 µM) and a good selectivity index [(SI = CC50/IC50) > 33]. On post-treatment of DDABT1, CHIKV infection was inhibited significantly by reducing CPE, viral titer, viral RNA, and viral proteins. Further, the time of addition experiment revealed >95% inhibition up to 4hpi indicating its interference predominantly in the early stages of infection. However, the late stages were also affected. This conjugate of SA and TM was found to increase the antiviral efficacy, and this might be partly attributed to modulating angiotensin II (Ang II) receptor type 1 (AT1). However, DDABT1 might have other modes of action that need further investigation. In addition, the in vivo experiments showed an LD50 of 5000 mg/kg in rats and was found to be more effective than TM, SA, or their combination against acute, subacute, and chronic inflammation/arthritis in vivo. In conclusion, DDABT1 showed remarkable anti-CHIKV properties and the ability to reduce inflammation and arthritis, making it a very good potential drug candidate that needs further experimental validation.

Understanding the role of Ca2+ via transient receptor potential (TRP) channel in viral infection: Implications in developing future antiviral strategies.

Transient receptor potential (TRP) channels are a superfamily of cation-specific permeable channels primarily conducting Ca2+ions across various membranes of the cell. The perturbation of the Ca2+ homeostasis is the hallmark of viral infection. Viruses hijack the host cell Ca2+ signaling, employing tailored Ca2+ requirements via TRP channels to meet their own cellular demands. This review summarizes the importance of Ca2+ across diverse viruses based on the Baltimore classification and focuses on the associated role of Ca2+-conducting TRP channels in viral pathophysiology. More emphasis has been given to the role of the TRP channel in viral life-cycle events such as viral fusion, viral entry, viral replication, virion maturation, and egress. Additionally, this review highlights the TRP channel as a store-operated channel which has been discussed vividly. The TRP channels form an essential aspect of host-virus interaction by virtue of its Ca2+ permeability. These channels are directly involved in regulating the viral calcium dynamics in host cells and thereby affect the viral infection. Considering its immense potential in regulating viral infection, the TRP channels may act as a target for antiviral therapeutics.

TLR4 is one of the receptors for Chikungunya virus envelope protein E2 and regulates virus induced pro-inflammatory responses in host macrophages.

Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and in vivo mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, in vitro. Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, in vitro. These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, in vitro and supported by molecular docking analysis, in silico. TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.

Pyridoxal phosphate: biosynthesis and catabolism.

Vitamin B(6) is an essential cofactor that participates in a large number of biochemical reactions. Pyridoxal phosphate is biosynthesized de novo by two different pathways (the DXP dependent pathway and the R5P pathway) and can also be salvaged from the environment. It is one of the few cofactors whose catabolic pathway has been comprehensively characterized. It is also known to function as a singlet oxygen scavenger and has protective effects against oxidative stress in fungi. Enzymes utilizing vitamin B(6) are important targets for therapeutic agents. This review provides a concise overview of the mechanistic enzymology of vitamin B(6) biosynthesis and catabolism. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Elevation of TRPV1 expression on T-cells during experimental immunosuppression.

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermo-sensitive, polymodal cation channel. An increase in intracellular calcium (Ca2+) is essential for T-cell responses. Similarly, various immunosuppressive agents are also reported to induce Ca2+ influx. However, the possible involvement of TRPV1 during immunosuppression has not been studied yet. Here, we investigated the possible functional role of TRPV1 in FK506 or B16F10-culture supernatant (B16F10-CS)-driven experimental immunosuppression in T-cells. Intriguingly, it was found that TRPV1 surface expression was further significantly elevated during immunosuppression compared with concanavalin A (ConA) or TCR-activated T-cells. Moreover, in B16F10 tumor-bearing mice, TRPV1 expression was upregulated on splenic T-cells as compared with T-cells derived from control mice. We also observed an immediate increase in intracellular Ca2+ levels in FK506 (marked increase) and B16F10-CS treatment (modest increase) or in combination with T-cell activation as compared with resting and activated T-cells. Likewise, in B16F10 tumor-bearing mice, the basal intracellular calcium level was upregulated in T-cells as compared with controls. The elevated Ca2+ level(s) were found to be significantly downregulated by 5'-iodoresiniferatoxin (50-IRTX) (a TRPV1-specific inhibitor), suggesting an important role of TRPV1 during immune activation and immunosuppression. The current study may have implications for immunosuppressive diseases along with inflammatory disorders associated with the coordinating role of TRPV1 and Ca2+ influx.

Correction to: Elevation of TRPV1 expression on T-cells during experimental immunosuppression.

Correction to: J. Biosci. (2022) 47:42 https://doi.org/10.1007/s12038-022-00279-2 In the Journal of Biosciences article titled ''Elevation of TRPV1 expression on T-cells during experimental immunosuppression'' by P Sanjai Kumar et al. (https://doi.org/10.1007/s12038-022-00279-2; Vol. 47, Art. ID 42), published in July 2022, the affiliation of the authors has been incompletely mentioned as: School of Biological Sciences, National Institute of Science Education and Research, Bhubaneswar 752050, India The correct affiliation should read as: School of Biological Sciences, National Institute of Science Education and Research, an Off-campus Centre (OCC) of Homi Bhabha National Institute, Bhubaneswar 752050, India.

Structure determination and characterization of the vitamin B6 degradative enzyme (E)-2-(acetamidomethylene)succinate hydrolase.

The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B(6) and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 A using SAD phasing. E-2AMS hydrolase is a member of the alpha/beta hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

Catalysis of a flavoenzyme-mediated amide hydrolysis.

A new pyrimidine catabolic pathway (the Rut pathway) was recently discovered in Escherichia coli K12. In this pathway, uracil is converted to 3-hydroxypropionate, ammonia, and carbon dioxide. The seven-gene Rut operon is required for this conversion. Here we demonstrate that the flavoenzyme RutA catalyzes the initial uracil ring-opening reaction to give 3-ureidoacrylate. This reaction, while formally a hydrolysis reaction, proceeds by an oxidative mechanism initiated by the addition of a flavin hydroperoxide to the C4 carbonyl. While peroxide-catalyzed amide hydrolysis has chemical precedent, we are not aware of a prior example of analogous chemistry catalyzed by flavin hydroperoxides. This study further illustrates the extraordinary catalytic versatility of the flavin cofactor.

Structure of the PLP degradative enzyme 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti MAFF303099 and its mechanistic implications.

A vitamin B(6) degradative pathway has recently been identified and characterized in Mesorhizobium loti MAFF303099. One of the enzymes on this pathway, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO), is a flavin-dependent enzyme and catalyzes the oxidative ring-opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid to form E-2-(acetamino-methylene)succinate. The gene for this enzyme has been cloned, and the corresponding protein has been overexpressed in Escherichia coli and purified. The crystal structure of MHPCO has been solved to 2.1 A using SAD phasing with and without the substrate MHPC bound. These crystal structures provide insight into the reaction mechanism and suggest roles for active site residues in the catalysis of a novel oxidative ring-opening reaction.

CuO nanoparticles catalyzed C-N, C-O, and C-S cross-coupling reactions: scope and mechanism.

CuO nanoparticles have been studied for C-N, C-O, and C-S bond formations via cross-coupling reactions of nitrogen, oxygen, and sulfur nucleophiles with aryl halides. Amides, amines, imidazoles, phenols, alcohols and thiols undergo reactions with aryl iodides in the presence of a base such as KOH, Cs(2)CO(3), and K(2)CO(3) at moderate temperature. The procedure is simple, general, ligand-free, and efficient to afford the cross-coupled products in high yield.

PLP catabolism: identification of the 2-(Acetamidomethylene)succinate hydrolase gene in Mesorhizobium loti MAFF303099.

The function of the mlr6787 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 2-(acetamidomethylene)succinate hydrolase, an enzyme involved in the catabolism of pyridoxal 5'-phosphate (vitamin B 6). This enzyme was overexpressed in Escherichia coli, purified to homogeneity, and characterized. 2-(Acetamidomethylene)succinate hydrolase catalyzes the hydrolysis of 2-(acetamidomethylene)succinate to yield succinic semialdehyde, acetic acid, carbon dioxide, and ammonia. The k cat and K M for this reaction were 0.6 s (-1) and 143 microM, respectively. The enzyme was shown to utilize the E isomer of 2-(acetamidomethylene)succinate.

Asymmetric magnetoresistance in an exchange bias Co/CoO bilayer.

Magnetoresistance (MR) measurements are carried out on a Co(8 nm)/CoO(3.5 nm) bilayer in the exchange bias (EB) state prepared by molecular beam epitaxy. With the applied magnetic field parallel to the current, the EB MR curves show an asymmetric behavior about the minimum, in contrast to the symmetric one for non-EB systems. We generalize a well-known analytical expression used for the field dependence of the MR of paramagnets. Our generalization incorporates coercivity and EB in a new phenomenological MR expression. Excellent fits of the latter to the experimental MR data are achieved, showing the way to use MR techniques for the quantitative characterization of EB systems. Furthermore, the temperature dependence of the EB field obtained from MR loops can be described with a power law, which yields a value of 96.6 K for the EB blocking temperature, which is significantly below the Néel temperature of 293 K for bulk CoO.

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.