However, two of the HTS molecules (compounds 1 and 3) were calculated to bind twice per CYP126A1 monomer at 5:1 ligand/protein ratios. high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands moonlight as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in remains a major global cause of mortality as the infectious bacterium that causes tuberculosis (TB)8 (1). Recent data from the World Health Organization indicate that TB is the leading cause of human death worldwide among infectious diseases (2). The mortality rate in TB victims may be increased by co-infection with the human immunodeficiency virus (HIV). Moreover, the development of strains resistant to leading drugs usually results in extended treatment times (2). Multidrug-resistant (MDR) and extensively drug-resistant strains are resistant to at least the two leading TB drugs (rifampicin and isoniazid) or to both of these drugs as well as to any one of the quinolone drugs and to at least one of the second-line injectable TB drugs amikacin, IL5R capreomycin, and kanamycin (3, 4). Consequently, there is increased need for development of new TB drugs with novel modes of action. This need has been partially met recently by the development of drugs such as delamanid (which inhibits cell wall mycolic acid synthesis) and bedaquiline (an ATPase proton pump inhibitor), both of which have been authorized for use in MDR TB treatment (5). A revelation from the GR148672X first genome sequence of (that for the virulent H37Rv strain) was that 20 different cytochrome P450 (CYP or P450) enzymes were encoded (1). This large number of P450s suggested important functions for these enzymes, and key roles for P450s GR148672X were identified in the metabolism of host cholesterol/cholest-4-en-3-one (CYP125A1 and CYP142A1) and branched chain lipids (CYP124A1), oxidative tailoring of cyclic dipeptides (CYP121A1), hydroxylation of menaquinone (CYP128A1), and sterol demethylation (CYP51B1) (6,C14). The and in the macrophage (7, 8, 15). CYP128A1 is implicated in the synthesis of a virulence-associated sulfolipid (S881) through hydroxylating menaquinone 9, (MK9H2), the sole quinol electron carrier in the respiratory chain. CYP128A1 catalyzes terminal hydroxylation of MK9H2 to enable sulfation at the hydroxyl group by the sulfotransferase Stf3 encoded by the gene (1, 12). The first P450 to be structurally and biochemically characterized was CYP51B1, the first member of the (sterol demethylase) gene family identified in a prokaryote (13, 16, 17). The CYP51B1 FeII-CO complex is unstable and collapses from the cysteine thiolate-coordinated P450 form to the thiol-coordinated P420 state. However, the thiolate-coordinated form is stabilized by binding of estriol (14). Later studies on the cholesterol hydroxylase CYP142A1 and the epothilone C/D epoxidase EpoK showed that binding of substrates (cholest-4-en-3-one and epothilone D, respectively) regenerated the P450 state when added to the FeII-CO P420 forms (8, 18). Importantly, the soluble CYP51B1 enzyme catalyzes oxidative 14-demethylation of lanosterol, 24,25-dihydrolanosterol, and the plant sterol obtusifoliol and also binds azole drugs used clinically to inhibit fungal CYP51 enzymes (13, 17). These findings inspired research to examine the potency of azole drugs against mycobacteria. studies revealed that several azoles had good MIC values against H37Rv, albeit with higher MIC values (8 g/ml for both drugs) (19, 20). This is possibly due to lower azole penetration into cells or to drug efflux (21). Studies in mice also showed that econazole reduced bacterial burden by 90% in lungs and spleen and was also effective against MDR strains (22, 23). Thus, regardless of issues surrounding cross-reactivity of azole drugs with human P450s, various azoles are clearly potent inhibitors of P450s and are important tools for GR148672X characterization of these enzymes (13, 24). Several of the P450s remain structurally uncharacterized. Among.