Elucidating the Substrate Specificity of Cytochrome P450 Enzymes: Insights into N- and S-Containing Small-Molecule Metabolism

Cytochrome P450 enzymes (P450s or CYPs) are the primary metabolic contributors to the absorption, distribution, metabolism, and excretion (ADME) properties of small-molecule drugs. These enzymes can catalyze various types of reactions, including metabolic reactions that occur at nitrogen (N) and sulfur (S) sites of small molecules. In this review, we conducted a comprehensive statistical analysis of 294 P450s-mediated small-molecule substrates, among which more than 47% substrates contained N and S. The purpose of the analysis is to elucidate the broad-spectrum cross-reactivity and specificity between these substrates and various CYP isoforms across five reaction types. Our findings reveal that substrates with molecular weights greater than 500 Da or less than 200 Da are predominantly governed by the dominant effect of the CYP isoform’s active sites. In contrast, small- to medium-sized molecules with molecular weights ranging from 200 to 400 Da exhibit a stronger dependence on the types of heteroatoms they contain, with the size of the enzyme’s catalytic site (cavity) playing a negligible role in determining substrate specificity. This review starts from the metabolic mechanisms of P450s-mediated N- and S-containing compounds, and systematically analyzes the structural characteristics of substrates involved in N-dealkylation, N-oxidation, and S-oxidation, as well as their metabolic interactions with P450s. These analyses provide a new perspective for improving the existing understanding of the relationship between the P450s substrate specificity and substrate structural characteristics, and offer a valuable perspective for enhancing drug design and predicting metabolic stability based on the P450s-catalyzed reaction framework.