Abstract

K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl) is a potent, irreversible cysteine protease inhibitor. Its therapeutic targets are cruzain, a cysteine protease of the protozoan parasiteTrypanosoma cruzi, and cathepsins B and L, which are associated with cancer progression. We evaluated the metabolism of K11777 by human liver microsomes, isolated cytochrome P450 (CYP) enzymes, and flavin-containing monooxygenase 3 (FMO3) in vitro. K11777 was metabolized by human liver microsomes to three major metabolites:N-oxide K11777 (apparentK_m = 14.0 ± 4.5 μM and apparentV_max = 3460 ± 3190 pmol · mg⁻¹ · min⁻¹, n = 4), β-hydroxy-homoPhe K11777 (K_m = 16.8 ± 3.5 μM and V_max = 1260 ± 1090 pmol · mg⁻¹ · min⁻¹, n = 4), andN-desmethyl K11777 (K_m = 18.3 ± 7.0 μM and V_max = 2070 ± 1830 pmol · mg⁻¹ · min⁻¹, n = 4). All three K11777 metabolites were formed by isolated CYP3A and their formation by human liver microsomes was inhibited by the CYP3A inhibitor cyclosporine (50 μM, 54–62% inhibition) and antibodies against human CYP3A4/5 (100 μg of antibodies/100 μg microsomal protein, 55–68% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl metabolite with an apparent K_m (9.2 ± 1.4 μM) lower than for CYP3A4 (25.0 ± 4.0 μM) and human liver microsomes. The apparent K_m forN-oxide K11777 formation by cDNA-expressed FMO3 was 109 ± 11 μM. Based on the intrinsic formation clearances and the results of inhibition experiments (CYP2D6, 50 μM bufuralol; FMO3 mediated, 100 mM methionine) using human liver microsomes, it was estimated that CYP3A contributes to >80% of K11777 metabolite formation. K11777 was a potent (IC₅₀ = 0.06 μM) and efficacious (maximum inhibition 85%) NADPH-dependent inhibitor of human CYP3A4 mediated 6′β-hydroxy lovastatin formation, suggesting that K11777 is not only a substrate but also a mechanism-based inhibitor of CYP3A4.