An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 µM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 µM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

Neither dapsone hydroxylation nor cortisol 6beta-hydroxylation detects the inhibition of CYP3A4 by HIV-1 protease inhibitors.

OBJECTIVE: This study examined the use of dapsone N-hydroxylation and cortisol 6beta-hydroxylation, well accepted in vivo probes of cytochrome P4503A4 (CYP3A4) activity, on defining the effect of three HIV protease inhibitors on CYP3A4 activity. METHODS: Subjects from University Hospital Infectious Disease Clinic about to be started on indinavir, and subjects from two clinical studies, one using ritonavir and the other using amprenavir, were recruited to participate in the study. Subjects received dapsone 100 mg p.o. followed by an 8-h urine collection for dapsone, dapsone N-hydroxylamine, cortisol, and 6beta-hydroxycortisol concentrations before HIV protease inhibitor administration, and 3 4 weeks into receiving HIV protease inhibitors. RESULTS: None of the HIV protease inhibitors demonstrated statistically significant alterations in dapsone recovery ratio and 6beta-hydroxycortisol/cortisol ratio. In fact, with ritonavir, the dapsone recovery ratio tended to increase rather than decrease, suggesting induction. These negative results were found despite evidence of CYP3A4 inhibition by these three HIV protease inhibitors via published drug-drug interactions with drugs that are substrates for CYP3A4. CONCLUSIONS: These in vivo assays used to probe CYP3A4 activity are suboptimal, most likely because of the presence of extrahepatic sites of metabolism for both dapsone and cortisol, and multiple CYP isozymes involved in dapsone N-hydroxylation.