(539.9) Steroid-Derived Isonitriles are Potent Cytochrome P450 Ligands that Should Enable Solution NMR as a Structural Technique to Probe P450 Dynamics

Poster Board Number: B121

Alaina Richard (University of Michigan), Thomas Pochapsky (Brandeis University, Brandeis University), Emily Scott (University of Michigan, University of Michigan, University of Michigan)

Human cytochrome P450 (CYP) enzymes are versatile, dynamic, and essential to many aspects of human health. The catalytic versatility of this superfamily of enzymes is driven by structural dynamics, which are often elusive to static structural techniques. For this reason, solution nuclear magnetic resonance (NMR) is likely to provide useful structural information orthogonal to X-ray crystallography. The challenges of human P450 NMR studies are two-pronged: 1) Human P450 enzymes are membrane-bound, and often aggregate or precipitate in aqueous environments, and 2) P450 enzymes contain a paramagnetic heme iron buried within the active site, leading to decreased intensity of NMR correlations corresponding to nearby amino acid residues. We have overcome both obstacles with the human steroidogenic P450 enzyme CYP17A1 in complex with a novel substrate derivative, 3-formyl pregnane-20-isonitrile1. The steroid skeleton of this ligand is similar to that of CYP17A1 endogenous substrates and therapeutics, but in this case the C20 isonitrile functional group directly coordinates both the oxidized (Fe3+) and the reduced (Fe2+) diamagnetic states of the heme iron. As a result, this compound both stabilizes CYP17A1 and suppresses paramagnetic relaxation and will facilitate application of multidimensional solution NMR to CYP17A1. The dissociation constant (Kd) and inhibition (IC50) of this isonitrile have been determined and compared to the prostate cancer drug abiraterone, which is the tightest-binding CYP17A1 inhibitor to date. Structural characterization of this complex by solution NMR is ongoing, but this strategy should open the door to new methods of structural analysis of dynamic human P450 enzymes.

This work is been funded by NIH grant R01-GM130997 (EES, PI).
1) US patent application 63/237,421 filed 08/26/2021, Selective isonitrile inhibitors of cytochrome P450 subtypes, T.C. Pochapsky, N.R. Wong and R. Sundar.