Structure–activity relationships for inhibition of human 5α-reductases by polyphenols

Abstract

The enzyme steroid 5α-reductase (EC 1.3.99.5) catalyzes the NADPH-dependent reduction of the double bond of a variety of 3-oxo-Δ⁴ steroids including the conversion of testosterone to 5α-dihydrotestosterone. In humans, 5α-reductase activity is critical for certain aspects of male sexual differentiation, and may be involved in the development of benign prostatic hyperplasia, alopecia, hirsutism, and prostate cancer. Certain natural products contain components that are inhibitors of 5α-reductase, such as the green tea catechin (−)-epigallocatechin gallate (EGCG). EGCG shows potent inhibition in cell-free but not in whole-cell assays of 5α-reductase. Replacement of the gallate ester in EGCG with long-chain fatty acids produced potent 5α-reductase inhibitors that were active in both cell-free and whole-cell assay systems. Other flavonoids that were potent inhibitors of the type 1 5α-reductase include myricetin, quercitin, baicalein, and fisetin. Biochanin A, daidzein, genistein, and kaempferol were much better inhibitors of the type 2 than the type 1 isozyme. Several other natural and synthetic polyphenolic compounds were more effective inhibitors of the type 1 than the type 2 isozyme, including alizarin, anthrarobin, gossypol, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and octyl and dodecyl gallates. The presence of a catechol group was characteristic of almost all inhibitors that showed selectivity for the type 1 isozyme of 5α-reductase. Since some of these compounds are consumed as part of the normal diet or in supplements, they have the potential to inhibit 5α-reductase activity, which may be useful for the prevention or treatment of androgen-dependent disorders. However, these compounds also may adversely affect male sexual differentiation.

Introduction

The microsomal enzyme steroid 5α-reductase (EC 1.3.99.5) catalyzes the NADPH-dependent reduction of the Δ^4,5 double bond of a variety of 3-oxo-Δ⁴ steroids [1], [2], including the conversion of testosterone to 5α-dihydrotestosterone, a process thought to amplify the androgenic response, perhaps because of the higher affinity of the androgen receptor for 5α-dihydrotestosterone than for testosterone [3]. Two different 5α-reductase isozymes have been characterized in humans, monkeys, rats, and mice [4], [5], [6], [7]. The two human isozymes share approximately 50% sequence identity and have different biochemical properties. For example, the type 1 isozyme has a broad basic pH optimum and low affinity for testosterone (K_m>1 μM), while the type 2 isozyme has an acidic pH optimum and high affinity for testosterone (K_m<10 nM) [8].

Studies of mice with genetically engineered 5α-reductase gene knockouts, as well as investigations of natural 5α-reductase deficiencies in humans, have identified some of the roles this enzyme plays in different biological processes [7]. Female mice deficient in the type 1 5α-reductase have impaired cervical ripening, leading to defective parturition [9], [10]. This defect may be due to impaired catabolism of cervical progesterone, which is also a substrate for 5α-reductase. Uterine type 1 5α-reductase is also important for fetal viability, because 5α-reductase activity limits fetal exposure to toxic levels of 17β-estradiol by competing for substrate with aromatase, which catalyzes the synthesis of 17β-estradiol from testosterone [11]. In humans, activity of the type 2 isozyme is critical for differentiation of the prostate and male external genitalia [4], [12], [13]. Based on studies of individuals with inherited deficiencies in 5α-reductase activity as well as laboratory studies of affected tissues, 5α-reductase may also have a role in the development of a variety of human disorders including benign prostatic hyperplasia [14], acne [15], alopecia [16], [17], and hirsutism [18]. 5α-Reductase also has been proposed to have a role in the development of prostate cancer and possibly may be responsible for differences in prostate cancer mortality among different racial groups [19]. A common missense mutation (V89L) that decreases the activity of the type 2 5α-reductase, is common among Asians, a group that has a lower mortality from prostate cancer compared with African–American men and non-Hispanic whites [20]. Finasteride, a synthetic 5α-reductase inhibitor, is currently used to treat benign prostatic hyperplasia [21] and alopecia [22], and it is also being studied in clinical trials as a chemopreventative for prostate cancer [23].

Diet has an important role in modulating cancer incidence and mortality, and differences in diet may explain geographical differences in prostate cancer mortality [24], [25]. Since androgens regulate the growth and function of the normal prostate and prostate cancer [26], [27], dietary components capable of altering this growth signaling pathway in the prostate may affect prostate cancer development and progression. We have shown that green tea contains phytochemicals called catechins that are inhibitors of 5α-reductase [28]. It is not known whether green tea catechins modulate androgenic activity in vivo in humans, but rats injected with the green tea catechin EGCG have a variety of endocrine changes, including lower serum testosterone concentrations and smaller prostates than the controls [29]. Other natural product inhibitors of 5α-reductase that have been identified include polyunsaturated fatty acids, such as γ-linolenic acid [30]; the macrocyclic ellagitannins, oenothein A and B [31], [32]; the flavonoids and lignans, genistein, formononentin, biochanin A, daidzein, coumestrol, equol, and enterolactone [33]; the bisnaphthoquinone, impatienol [34]; and the isoprenylated flavone and stilbene, artocarpin and chlorophorin [35]. Since many of these inhibitors of 5α-reductase are polyphenols, we have investigated the ability of a variety of natural and synthetic polyphenols to inhibit 5α-reductase to determine what structural elements are important for potent inhibition of 5α-reductase by this class of compounds in both the cell-free and whole-cell assay systems.