Structure-Based Design of Inhibitors with Improved Selectivity for Steroidogenic Cytochrome P450 17A1 over Cytochrome P450 21A2

Abstract

Inhibition of androgen biosynthesis is clinically effective for treating androgen-responsive prostate cancer. Abiraterone is a clinical first-in-class inhibitor of cytochrome P450 17A1 (CYP17A1) required for androgen biosynthesis. However, abiraterone also causes hypertension, hypokalemia, and edema, likely due in part to off-target inhibition of another steroidogenic cytochrome P450, CYP21A2. Abiraterone analogs were designed based on structural evidence that B-ring substituents may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs increased selectivity of CYP17A1 inhibition up to 84-fold compared with 6.6-fold for abiraterone. Cocrystallization with CYP17A1 validated the intended new contacts with CYP17A1 active site residues. Docking these analogs into CYP21A2 identified steric clashes that likely underlie decreased binding and CYP21A2 inhibition. Overall, these analogs may offer a clinical advantage in the form of reduced side effects.

Discovery of Novel Non-Steroidal Cytochrome P450 17A1 Inhibitors as Potential Prostate Cancer Agents

“…Further optimization of 1c is required to eliminate off-target effects on the steroidogenic human CYP21A2 enzyme to prevent disruption of glucocorticoid and mineralocorticoid biosynthesis in vivo. This might be achieved by adding substituents compatible with the CYP17A1 active site topography forming interactions with the bordering Arg239 and/or Asp298 residues, but incompatible with the more spatially constrained CYP21A2 active site, as has been accomplished for abiraterone [ 17 ].…”

“…In subsequent assays the range of inhibitor concentrations was optimized to best define the IC 50 of individual compounds. Reactions were run and the 17α -hydroxyprogesterone product measured by LC-MS as previously published [ 17 ].…”

“…The CYP2D6 dextrometorphan (purchased from Sigma-Aldrich, St. Louis, MO, USA) assays were carried out with 100 µM DXM. Inhibition of steroidogenic cytochrome P450 21A2 progesterone 21-hydroxylation was evaluated as reported [ 17 ].…”

Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes

“…1H). The K d and IC 50 values for abiraterone are in the nanomolar range (17,22), but an abiraterone analog without the nitrogen that coordinates iron has an IC 50 increased by over 3 orders of magnitude (22). Thus, two changes were made to compensate for the likely reduction in loss of abiraterone affinity to the various diamagnetic metals.…”

“…Specifically, the 17A1⌬19H construct is as described (17). The CYP21A2dH construct omitted the N-terminal 2-19 residues, substituted the sequence coding for 20 NWWKLRSLH 28 with nucleotide coding for AKKTSSKGK, and added codons for a C-terminal four-histidine tag immediately before the stop codon (22). The CYP3A4⌬23H construct deleted N-terminal residues 3-23 (25) and similarly added a C-terminal four-histidine tag.…”