The mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by <italic>tert</italic>-butyl acetylenes: Characterization of a novel reversible inactivation mechanism and a role for the conserved T303 in proton delivery to the 2E1 active site.

Rabbit cytochromes P450 2E1 and P450 2E1 T303A were inactivated by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) in a time-, concentration- and NADPH-dependent manner with KI values in the millimolar range. Losses in the enzymatic activity, the P450 CO spectrum and native P450 heme were accompanied by the formation of two different tBA- or tBMP-modified heme products as detected by ESI-LC-MS analysis (m/z of 661 or 705 Da, respectively). Only the tBA-inactivated P450 2E1 revealed a tBA adduct to the apoprotein. Surprisingly, losses in the activity, the CO spectrum and native heme of the tBA-inactivated T303A mutant were completely restored following dialysis, suggesting a reversible heme alkylation. Investigations into the mechanism for the novel reversible inactivation of P450 2E1 T303A by tBA demonstrated that the activity and CO spectral losses were restored following overnight incubation. The reversibility was time-dependent, required an intact P450 enzyme, and was independent of NADPH and tBA. ESI-LC-MS/MS analysis under non-denaturing conditions of a pre-acidified tBA-inactivated T303A sample yielded two tBA adducts (m/z 661 Da) that were absent in non-acidified samples. In contrast, both non- and pre-acidified tBA-inactivated wild-type 2E1 samples were able to form the two tBA adducts, suggesting that the T303A mutant was deficient in the delivery of protons to the enzyme active site. Substrate binding analysis of tBA and tBMP with the P450s 2E1 yielded KS values in the micromolar range. Spectral examination of the tBA-inactivated T303A mutant revealed the formation of a novel, reversible acetylene-iron intermediate with an absorption maximum at 485 nm. The artificial oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of P450 2E1 by tBA, but only tBHP supported the inactivation of 2E1 T303A, suggesting a disruption in proton delivery in this enzyme. The tBHP- and CHP-supported inactivations resulted in the formation of tBA-adducted heme products and were completely irreversible with dialysis. In support of these data, models of the P450s 2E1 with tBA bound in the active site suggest a role for the conserved T303 residue in proton delivery and in the inactivation of the 2E1 P450s by small acetylenic compounds.Ph.D.BiochemistryHealth and Environmental SciencesPharmacologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124631/2/3150163.pd

The 2′-Trifluoromethyl Analogue of Indomethacin Is a Potent and Selective COX‑2 Inhibitor

Indomethacin is a potent, time-dependent, nonselective inhibitor of the cyclooxygenase enzymes (COX-1 and COX-2). Deletion of the 2′-methyl group of indomethacin produces a weak, reversible COX inhibitor, leading us to explore functionality at that position. Here, we report that substitution of the 2′-methyl group of indomethacin with trifluoromethyl produces CF₃–indomethacin, a tight-binding inhibitor with kinetic properties similar to those of indomethacin and unexpected COX-2 selectivity (IC₅₀ mCOX-2 = 267 nM; IC₅₀ oCOX-1 > 100 μM). Studies with site-directed mutants reveal that COX-2 selectivity results from insertion of the CF₃ group into a small hydrophobic pocket formed by Ala-527, Val-349, Ser-530, and Leu-531 and projection of the methoxy group toward a side pocket bordered by Val-523. CF₃–indomethacin inhibited COX-2 activity in human head and neck squamous cell carcinoma cells and exhibited in vivo anti-inflammatory activity in the carrageenan-induced rat paw edema model with similar potency to that of indomethacin

Sterol 14α-Demethylase Structure-Based Design of VNI ((R)- N-(1-(2,4-Dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)) Derivatives to Target Fungal Infections: Synthesis, Biological Evaluation, and Crystallographic Analysis

Because of the increase in the number of immunocompromised patients, the incidence of invasive fungal infections is growing, but the treatment efficiency remains unacceptably low. The most potent clinical systemic antifungals (azoles) are the derivatives of two scaffolds: ketoconazole and fluconazole. Being the safest antifungal drugs, they still have shortcomings, mainly because of pharmacokinetics and resistance. Here, we report the successful use of the target fungal enzyme, sterol 14α-demethylase (CYP51), for structure-based design of novel antifungal drug candidates by minor modifications of VNI [(R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)], an inhibitor of protozoan CYP51 that cures Chagas disease. The synthesis of fungi-oriented VNI derivatives, analysis of their potencies to inhibit CYP51s from two major fungal pathogens (Aspergillus fumigatus and Candida albicans), microsomal stability, effects in fungal cells, and structural characterization of A. fumigatus CYP51 in complexes with the most potent compound are described, offering a new antifungal drug scaffold and outlining directions for its further optimization