Structure of human cortisol-producing cytochrome P450 11B1 bound to the breast cancer drug fadrozole provides insights for drug design

Abstract

Human cytochrome P450 11B1 (CYP11B1) is responsible for the final step generating the steroid hormone cortisol, which controls stress and immune responses and glucose homeostasis. CYP11B1 is a promising drug target to manage Cushing's disease, a disorder arising from excessive cortisol production. However, the design of selective inhibitors has been hampered because structural information for CYP11B1 is unavailable and the enzyme has high amino acid sequence identity (93%) to a closely related enzyme, the aldosterone-producing CYP11B2. Here we report the X-ray crystal structure of human CYP11B1 (at 2.1 Å resolution) in complex with fadrozole, a racemic compound normally used to treat breast cancer by inhibiting estrogen-producing CYP19A1. Comparison of fadrozole-bound CYP11B1 with fadrozole-bound CYP11B2 revealed that despite conservation of the active-site residues, the overall structures and active sites had structural rearrangements consistent with distinct protein functions and inhibition. Whereas fadrozole binds to both CYP11B enzymes by coordinating the heme iron, CYP11B2 binds to the R enantiomer of fadrozole, and CYP11B1 binds to the S enantiomer, each with distinct orientations and interactions. These results provide insights into the cross-reactivity of drugs across multiple steroidogenic cytochrome P450 enzymes, provide a structural basis for understanding human steroidogenesis, and pave the way for the design of more selective inhibitors of each human CYP11B enzyme.

Keywords: 11β-hydroxylase; CYP11B1; CYP11B2; Cushing's disease; X-ray crystallography; aldosterone; aldosterone synthase; cortisol; cytochrome P450; enzyme inhibitor; fadrozole; membrane enzyme; membrane protein; steroidogenesis.

Figure 1.

CYP11B1 structure overview (A) and active sites (B). In both, the enzyme is shown in rainbow colors from blue at the N terminus to red at the C terminus. The heme is shown as black sticks with the iron as a rust sphere. The inhibitor fadrozole (structure inset) is shown in cyan sticks. In B, water is shown as a small red sphere with atoms within hydrogen bonding distance indicated by black dashed lines.

Figure 2.

Structural alignments of CYP11B1 and CYP11B2 structures, both with the inhibitor fadrozole. A, overview of CYP11B1 (shown as in Fig. 1A) and CYP11B2 (gray ribbons with magenta fadrozole, PDB code 4FDH). B, active site showing repositioning of conserved active-site residues to selectively bind (S)-fadrozole in CYP11B1 (cyan) and (R)-fadrozole (magenta) in CYP11B2 (PDB code 4FDH).

Figure 3.

CYP11B1 (light cyan) and CYP11B2 (gray) structure comparison. A, nonidentical amino acids (darker sticks with red for oxygen atoms and blue for nitrogen atoms) are relatively distant from the active site and/or face outward, like residue 112 just above fadrozole. B, differences in the I helix conformation occur adjacent to residue 320, which is Val in CYP11B1 and Ala in CYP11B2 (PDB code 4FDH).

Figure 4.

CYP11B1 channel. A, the active site is connected to a channel (gray mesh) all the way to the protein surface. B, the opening of this channel is flanked by the A′ helix, the B/B′ loop, and the F/G′ region.