Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6′,7′-Dihydroxybergamottin

Human cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme. Some drugs and natural compounds can act as suicide (mechanism-based) inactivators of CYP3A4, leading to unanticipated drug-drug interactions, toxicity and therapeutic failures. Despite significant clinical and toxicological implications, the mechanism-based inactivation remains incompletely understood. This study provides the first direct insights into the interaction of CYP3A4 with three suicide substrates: mibefradil, an antihypertensive drug quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6′,7′-dihydroxybergamottin. Novel structural findings help better understand the suicide substrate binding and inhibitory mechanism, and can be used to improve the predictability of the binding ability, metabolic sites and inhibitory/inactivation potential of newly developed drugs and other chemicals relevant to public health.

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Insights into the activity control of the kallikrein-related peptidase 6: small-molecule modulators and allosterism

The activity of kallikrein-related peptidase 6 (KLK6) is deregulated in various diseases such as cancer and neurodegenerative diseases. KLK6 is thus considered as an attractive therapeutical target. In this short report, we depict some novel findings on the regulation of the KLK6 activity. Namely, we identified mechanism-based inhibitors (suicide substrates) from an in-house library of 6-substituted coumarin-3-carboxylate derivatives. In addition, a molecular dynamics study evidenced the allosteric behavior of KLK6 similar to that previously observed for some trypsin-like serine proteases. This allosteric behavior together with the coumarinic scaffold bring new opportunities for the design of KLK6 potent activity modulators, useful as therapeutics or activity-based probes.