Synthesis of avicequinone c and analogs toward the study of 5α-reductase inhibitory activity

Abstract

Steroid 5α-reductase (5α-R) or 3-oxo-5α-steroid ∆4-reductase, is an enzyme having 3 isozymes, which involves in steroid metabolism. It converts testosterone (T) to dihydrotestosterone (DHT) with the aid of NADPH as a cofactor. 5α-R plays an important role in several human diseases such as prostate cancer, benign prostatic hyperplasia, acne, hirsutism and androgenic alopecia. To date, the three-dimensional structure of 5α-R has not been available due to structural unstability during the protein crystallization. In this study, we performed the in silico three-dimensional 5α-R1 and 5α-R2 homology modeling using SWISS-MODEL and the isoprenylcysteine carboxyl methyltransferase (ICMT, PDB code: 4A2N) was selected as a homologous protein template. The catalytic site of 5α-R was verified by molecular docking simulation with 5α-R inhibitors including the known drugs such as finasteride and dutasteride and a series of reported 5α-R inhibitors. According to the previous study, avicequinone C, which is a furanonaphthoquinone, showed 5α-R1 inhibitory activity with IC50 = 9.94 µg/ml. Therefore, we hypothesized that furanonaphthoquinone moiety potentially an essential pharmacophore controlling 5α-R inhibitory activity. A series of avicequinone C and analogs and natural napthoquinones were studied to evaluate for 5α-R1 inhibiting potency by molecular docking simulations using Autodock Vina in conjugation with the resulting in silico three-dimensional 5α-R1 and 5α-R2. The results showed that avicequinone C displayed the best protein-ligand binding interactions at 5α-R catalytic region. Next, avicequinone C and analogs were synthesized by chemical process for in vitro 5α-R inhibitory assay along with natural naphthoquinones. HaCaT cell line was used for 5α-R inhibitory activity investigation. The results showed that 2-hydroxy-1,4-naphthoquinone was the most potent 5α-R inhibitor. It exhibited 82.73 ± 14.42 %inhibition at 40 µM with IC50 = 23.93 µM. Importantly, 2-hydroxy-1,4-naphthoquinone showed stronger 5α-R inhibition than avicequinone C. Considering molecular docking together with the in vitro 5α-R inhibitory assay toward HaCaT cell line, we found that the naphthoquinone motif is crucial for 5α-R inhibitory activity. Moreover, the resulting in silico three-dimensional 5α-R1 would be used for virtual screening and development of 5α-reductase (5α-R) in the future.