Structural and Functional Evaluation of Clinically Relevant Inhibitors of Steroidogenic Cytochrome P450 17A1

Abstract

Human steroidogenic cytochrome P450 17A1 (CYP17A1) is a bifunctional enzyme that performs both hydroxylation and lyase reactions, with the latter required to generate androgens that fuel prostate cancer proliferation. The steroid abiraterone, the active form of the only CYP17A1 inhibitor approved by the Food and Drug Administration, binds the catalytic heme iron, nonselectively impeding both reactions and ultimately causing undesirable corticosteroid imbalance. Some nonsteroidal inhibitors reportedly inhibit the lyase reaction more than the preceding hydroxylase reaction, which would be clinically advantageous, but the mechanism is not understood. Thus, the nonsteroidal inhibitors seviteronel and orteronel and the steroidal inhibitors abiraterone and galeterone were compared with respect to their binding modes and hydroxylase versus lyase inhibition. Binding studies and X-ray structures of CYP17A1 with nonsteroidal inhibitors reveal coordination to the heme iron like the steroidal inhibitors. ()-seviteronel binds similarly to both observed CYP17A1 conformations. However, ()-orteronel and ()-orteronel bind to distinct CYP17A1 conformations that differ in a region implicated in ligand entry/exit and the presence of a peripheral ligand. To reconcile these binding modes with enzyme function, side-by-side enzymatic analysis was undertaken and revealed that neither the nonsteroidal seviteronel nor the ()-orteronel inhibitors demonstrated significant lyase selectivity, but the less potent ()-orteronel was 8- to 11-fold selective for lyase inhibition. While active-site iron coordination is consistent with competitive inhibition, conformational selection for binding of some inhibitors and the differential presence of a peripheral ligand molecule suggest the possibility of CYP17A1 functional modulation by features outside the active site.

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1

“…A role for multiple P450 17A1 conformations, indicated by NMR spectroscopy (13) and X-ray crystallography (42,44), is supported by some of our own results. As indicated, we were unable to fit the substrate-binding data with only a simple twostate system (Fig.…”

“…We believe that multiple conformations of P450 17A1 (Fig. 13) are necessary to explain several of the kinetic results, in support of NMR spectroscopic (22) and X-ray crystallographic evidence (42,44) for the existence of multiple conformations of the protein.…”

“…X-ray crystallography and spectroscopy work of Scott and coworkers (22,42,44). Using a model in which both the ligandfree and ligand-bound forms of the enzyme have multiple, inter-convertible conformations, we were able to fit the raw data files for binding of pregnenolone, 17␣-OH pregnenolone, and DHEA to P450 17A1 ( Fig.…”

“…Some selectivity for lyase Ͼ 17␣hydroxylation activity has been reported for abiraterone (36), orteronel (Takeda TAK-700) (40), the secosteroid (S)-seviteronel (VT-464) (41), and two other non-steroidal pyridine derivatives (36). X-ray crystal structures of human P450 bound to abiraterone (6) and to both enantiomers of orteronel (42) have been published by Scott and co-workers (42). A completely processive reaction should not have differential sensitivities to a single inhibitor.…”

“…A completely processive reaction should not have differential sensitivities to a single inhibitor. Even in a dissociative mechanism, the question arises as to how a drug can occupy a single site on the enzyme and have different K i values for two reactions (42,43).…”