Cytochrome P450 17A1 (P450c17) catalyzes the biosynthesis of androgens in humans1. Since prostate cancer cells proliferate in response to androgen steroids2,3, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer4, but drug development has been hampered by the lack of a CYP17A1 structure. Here we report the only known structures of CYP17A1, which contain either abiraterone, a first-in-class steroidal inhibitor recently approved by the FDA for late-stage prostate cancer5, or TOK-001, another inhibitor in clinical trials4,6. Both bind the heme iron forming a 60° angle above the heme plane, packing against the central I helix with the 3β-OH interacting with N202 in the F helix. Importantly, this binding mode differs substantially from those predicted by homology models or from steroids in other cytochrome P450 enzymes with known structures, with some features more similar to steroid receptors. While the overall CYP17A1 structure provides a rationale for understanding many mutations found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate better understanding of the enzyme’s dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.

Background: Structural information for substrate binding to human steroidogenic cytochrome P450 17A1 (CYP17A1) is unavailable. Results: Within a common overall orientation, different steroids adopt subtly different positions. Conclusion: Steric and hydrogen-bonding modulation of lateral/vertical orientation controls CYP17A1-mediated steroid oxidation. Significance: Understanding the CYP17A1 mechanism provides opportunities for better targeted drug design.

Background: Cytochromes P450 2A13 and 2A6 are involved in nicotine metabolism and tobacco-related lung cancer initiation. Results: CYP2A13 and CYP2A6 structures were solved with nicotine and CYP2A13 with NNK. Conclusion: Structure series reveals basis for nicotine and NNK selectivity and access to buried active site. Significance: Understanding CYP2A binding and oxidation of pharmacological substrates can aid in drug development efforts.

Summary Human xenobiotic-metabolizing cytochrome P450 (P450) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally a single ligand can interact with multiple cytochrome P450 enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the P450 superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human cytochrome P450 enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key amino acids involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 Å), CYP2A13 (3.0 Å), CYP2E1 (2.35 Å), and a CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 Å), have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual amino acids lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine. Hyperlinking to databases The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) with the following codes: CYP2A6 with pilocarpine (3T3R), CYP2A6 I208S/I300F/G301A/S369G with pilocarpine (3T3Q), CYP2A13 with pilocarpine (3T3S), and CYP2E1 with pilocarpine (3T3Z).

ABSTRACT:Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K D ) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K D values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structurebased quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu 370 .Xenobiotic-metabolizing cytochrome P450 (P450) enzymes act on chemically diverse small molecules, often with overlapping specificities, but with the formation of distinct metabolites and/or metabolic rates. The only active human cytochromes P450 in the 2A subfamily are CYP2A13 and CYP2A6. CYP2A13 is expressed primarily throughout the respiratory system including nasal mucosa, trachea, and lung (Su et al., 2000;Zhu et al., 2006), whereas CYP2A6 is primarily a hepatic enzyme (Yamano et al., 1990;Fernandez-Salguero et al., 1995). Coumarin 7-hydroxylation is a characteristic activity for the P450 2A subfamily. However, reports disagree on whether the catalytic efficiency for coumarin is 10-fold higher for CYP2A6 compared with that for CYP2A13 (He et al., 2004b) or similar (von Weymarn and Murphy, 2003). A number of other substrates are metabolized very differently by these two enzymes, including nicotine and the nicotine-derived compounds cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CYP2A13 metabolizes nicotine and cotinine with 23-and 15-fold higher catalytic efficiency (k cat /K m ) than CYP2A6, respectively (Bao et al., 2005). In addition, the metabolism of NNK by CYP2A13 occurs at a catalytic efficiency 215-fold greater th...