Associate Professor University of Pittsburgh Pittsburgh, Pennsylvania, United States
Cheng Zhang (University of Pittsburgh)| Zhiyi Wei (Southern University of Science and Technology)
Human steroid 5α-reductases (SRD5As) are membrane-embedded NADPH-dependent steroid and lipid reductases with diverse physiological and pathological roles. Among them, SRD5A2 is a critical enzyme in steroid metabolism that catalyzes testosterone to dihydrotestosterone. Mutations on its gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride as SRD5A2 inhibitors are widely used anti-androgen drugs for benign prostate hyperplasia and androgenic alopecia, which have recently been indicated in the treatment of COVID-19. The molecular mechanisms underlying enzyme catalysis and inhibition had remained elusive for SRD5A2 and other eukaryotic integral membrane steroid reductases due to a lack of structural information. We recently solved a crystal structure of human SRD5A2 at 2.8 Å with finasteride using the lipid mesophase (or LCP) crystallization method. The structure reveals a unique structural topology of 7-transmembrane helices and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride, which binds in a largely enclosed binding cavity inside the membrane. To our knowledge, similar NADPH adducts have not been reported previously. Structural analysis together with computational and mutagenesis studies reveals molecular mechanisms for the 5α-reduction of testosterone and the semi-irreversible finasteride inhibition involving residues E57 and Y91 of SRD5A2. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region of SRD5A2 including three cytosolic loops regulating the NADPH/NADP+ exchange and their entry into the transmembrane region of SRD5A2. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. In summary, our results offer critical and unprecedented structural insights into the function of integral membrane steroid reductases and will facilitate drug development.
Figure 1. (A) 5α-reduction reaction of the ∆4,5 double bond of testosterone catalyzed by SRD5A2 to generate dihydrotestosterone (DHT) and two SRD5A2 inhibitors. (B) Structure of SRD5A2. The NADP-DHF adduct was shown as spheres. L1-6 represents 6 loops connecting 7-TMs. The NADP and DHF moieties were colored in light cyan and light pink, respectively. (C) Potential mechanisms for the reduction of finasteride and testosterone.
Figure 2. (A) Conformational dynamics of the cytosolic regions of SRD5A2 revealed by MD simulations. (B) Model for the dynamics of SRD5A2 in one cycle of reaction. The reaction can be inhibited by finasteride by forming a stable adduct with the NADPH cofactor to stabilize the closed conformation of SRD5A2. Testosterone and finasteride were indicated as “T” and “Fina”, respectively. The catalysis of testosterone and the finasteride inhibition are shown with black and red arrows, respectively.