Synthesis and biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P-450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been targeted against the two components of this enzyme, that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5) (IC50=10.06 microM against 17alpha-OHase and IC50=1.58 microM against lyase) showing equipotent activity against lyase compared to the standard compound, ketoconazole (KTZ) (IC50=3.76+/-0.01 microM against 17alpha-OHase and IC50=1.66+/-0.15 microM against lyase). Furthermore, the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha).     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of 4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

We report the preliminary results of the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of phenyl alkyl imidazole-based compounds as inhibitors of the two components of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results show that N-3-(4-bromophenyl) propyl imidazole (12) (IC50 = 2.95 microM against 17alpha-OHase and IC50 = 0.33 microM against lyase) is the most potent compound within the current study, in comparison to ketoconazole (KTZ) (IC50 = 3.76 microM against 17alpha-OHase and IC50 = 1.66 microM against lyase). Modelling of these compounds suggests that the length of the alkyl chain enhances the interaction between the inhibitor and the area of the active site corresponding to the C3 area of the steroid backbone, thereby increasing potency.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of phenyl alkyl imidazole-based compounds as potent inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity.     

Synthesis and biochemical evaluation of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole-based compounds as potent inhibitors of 17α-hydroxylase/17,20-lyase (P45017α) derived from rat testicular microsomes

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole against the two components of 17α-hydroxylase/17,20-lyase (P450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results show the compounds to be highly potent inhibitors with limited selectivity towards the lyase component [e.g., toluene-4-sulfonic acid 4-imidazol-1-ylmethyl-phenyl ester (4) possessed an IC₅₀ value of 40 nM against 17α-OHase and 30 nM against lyase].     

Inhibition of 17alpha-hydroxylase/C17,20-lyase (CYP17) from rat testis by green tea catechins and black tea theaflavins

In the course of screening for 17alpha-hydroxylase/C17,20-lyase inhibitors from food ingredients, the methanol soluble fraction of green tea and black tea, which were expected to be rich in catechin and theaflavin content, showed potent inhibitory activity. (-)-Epigallocathechin gallate and theaflavin 3-O-gallate with a pirogallol moiety significantly inhibited C17,20-lyase activity on IC50 values of 24.5 microM and 11.5 microM respectively. They had potent cytotoxicity against human prostate cancer LNCaP cells (IC50=28.1 microM and 37.4 microM).     

Synthesis and biological evaluation of 3-(azolylmethyl)-1H-indoles and 3-(alpha-azolylbenzyl)-1H-indoles as selective aromatase inhibitors

This present study identifies a number of azolyl-substituted indoles as potent inhibitors of aromatase. In the sub-series of 3-(azolylmethyl)-1H-indoles, four imidazole derivatives and their triazole analogues were tested. Imidazole derivatives 11 and 14 in which the benzyl moiety was substituted by 2-chloro and 4-cyano groups, respectively, were the most active, with IC50 values ranging between 0.054 and 0.050 microM. In the other sub-series, eight 3-(alpha-azolylbenzyl)-1H-indoles were prepared and tested. Compound 30, the N-ethyl imidazole derivative, proved to be an aromatase inhibitor, showing an IC50 value of 0.052 microM. All target compounds were further evaluated against 17alpha-hydroxylase/C17,20-lyase to determine their selectivity profile.     

Synthesis and biochemical evaluation of a range of sulfonated derivatives of 4-hydroxybenzyl imidazole as highly potent inhibitors of rat testicular 17α-hydroxylase/17,20-lyase (P-45017α)

We report the synthesis and biochemical evaluation of a range of 4-sulfonated derivatives of 4-hydroxybenzyl imidazole which has been targetted against the two components of 17α-hydroxylase/17,20-lyase (P-450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are highly potent inhibitors possessing excellent selectivity towards the lyase component.     

A novel molecular modelling study of inhibitors of the 17alpha-hydroxylase component of the enzyme system 17alpha-hydroxylase/17,20-lyase (P-450(17alpha)).

The enzyme 17alpha-hydroxylase/17,20-lyase (P-450(17alpha) has recently become the focus of research into the fight against hormone dependent prostate cancer. However, the specific nature of this enzyme, in particular, the dual role of its active site, remains unknown. In our drive to elucidate further information regarding P-450(17alpha), and in light of our experience of other cytochrome P-450 enzymes, we chose to consider each part of this complex enzyme separately (i.e. the 17alpha-hydroxylase (17alpha-OHase) and the 17,20-lyase components). We therefore initiated a series of molecular modelling studies involving the construction of a 'substrate heme complex' for each of the two components. Here, we consider the construction and use of the complex for the 17alpha-OHase component of this enzyme. Using this approach, we have successfully considered: the binding of steroidal and non-steroidal reversible inhibitors: the structural features necessary for potent inhibition: and, rationalised the mode of action of a number of compounds whose inhibitory activity has not been previously explained, for example aminoglutethimide (an inhibitor of another related cytochrome P-450 enzyme, aromatase AR). The study concludes that the ability of the inhibitors of 17alpha-OHase to undergo polar polar interaction with the active site and for the compounds to closely mimic the substrate plane is a major factor in determining potency. Factors such as log P (log of the partition coefficient value for the distribution of a compound between octanol and water) would then appear to determine the extent of overall inhibitory activity. Overall, the study suggests that the novel substrate-heme complex approach has provided a good approximation of the 17alpha-OHase active site and has proved to be a useful tool in drug design and discovery.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

4-pregnene-3-one-20 beta-carboxaldehyde: a potent inhibitor of 17 alpha-hydroxylase/C17,20-lyase and of 5 alpha-reductase.

The pregnene derivative, 4-pregnene-3-one-20 beta-carboxaldehyde (22-A) was evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and of 5 alpha-reductase in human prostatic homogenates. The effect of the compound in vivo was studied in adult male rats. The 22-A demonstrated potent and competitive inhibition of 17 alpha-hydroxylase and C17,20-lyase with Ki values 8.48 and 0.41 microM, respectively, significantly below the Km values for these two enzymes (33.75 and 4.55 microM). This compound also showed potent inhibition of 5 alpha-reductase with a Ki value of 15.6 nM (Km for this enzyme is 50 nM). By comparison, ketoconazole, a currently studied 17 alpha-hydroxylase/C17,20-lyase inhibitor for the treatment of prostatic cancer, showed less potent inhibition of 17 alpha-hydroxylase (Ki 39.5 microM) and C17,20-lyase (Ki 3.6 microM) and did not inhibit 5 alpha-reductase. Progesterone which has been reported to inhibit the 17 alpha-hydroxylase/C17,20-lyase, did not significantly reduce the production of testosterone by rat testes in vitro in comparison to controls, while the same concentration of 22-A demonstrated a 42% reduction of testosterone biosynthesis. When the adult male rats were injected s.c. with 22-A at 50 mg/day/kg for a 2 week period, the testosterone concentrations in the rat sera were significantly lower than control values (P less than 0.05), whereas serum corticosterone levels did not change. These results suggest that 22-A is a selective potent inhibitor for 17 alpha-hydroxylase and C17,20-lyase, but is more potent for the C17,20-lyase. The compound also inhibits 5 alpha-reductase, and therefore may reduce biosynthesis of testosterone and dihydrotestosterone effectively. Thus, 22-A may be useful in the treatment of problems associated with the androgen excess and prostatic cancer.     

Inhibitory effect of some imidazole antifungal compounds on the synthesis of 16-ene-C19-steroid catalyzed by pig testicular microsomes

The activity of the enzyme (16-ene-C19-steroid synthesizing enzyme) responsible for the conversion of C21-steroids to 16-ene-C19-steroids, which was localized on pig testicular microsomes, was inhibited by some typical imidazole antifungal compounds such as clotrimazole, econazole, miconazole and ketoconazole which are known to be universal inhibitors of cytochrome P-450-dependent enzymes. The 50% inhibitory concentrations of clotrimazole, econazole and miconazole were 0.29, 0.36 and 1.25 microM, respectively for 16-ene-C19-steroid synthesizing enzyme activity. Clotrimazole was the most powerful inhibitor of all the compounds examined, which shows the competitive inhibition for 16-ene-C19-steroid synthesizing enzyme activity. The Ki-value was 0.26 microM for its activity. The degree of the inhibition by these imidazole compounds was very similar to the inhibition of 17 alpha-hydroxylase and C17,20-lyase activities on pig testicular microsomes.     

17,20-lyase inhibitors. Part 4: design, synthesis and structure-activity relationships of naphthylmethylimidazole derivatives as novel 17,20-lyase inhibitors

A novel series of naphthylmethylimidazole derivatives and related compounds have been investigated as selective 17,20-lyase inhibitors. Optimization of the substituent at the 6-position on the naphthalene ring was performed to yield a methylcarbamoyl derivative, which exhibited potent inhibitory activity against human 17,20-lyase and promising selectivity (>200-fold) for 17,20-lyase over CYP3A4. Further modifications of the methylcarbamoyl derivative led to the discovery of the corresponding tricyclic compound, which showed highly potent activity against human 17,20-lyase (IC(50) 19 nM) and good selectivity (>1000-fold) for inhibition of 17,20-lyase over CYP3A4. Additional biological evaluation revealed that the tricyclic compound had potent in vivo efficacy in monkeys and favorable pharmacokinetic profiles when administered in rats. Asymmetric synthesis of the selective tricyclic inhibitor was also achieved using a chiral α-hydroxy ketone.     

Inhibition of human adrenal steroidogenic enzymes in vitro by imidazole drugs including ketoconazole

The effect of several imidazole containing drugs including keto on human adrenal 17 alpha-hydroxylase, 17,20-lyase, 21-hydroxylase, 11 beta-hydroxylase and 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) activities was studied in vitro. The order of decreasing inhibitory potency as determined from ID50 values for both 17 alpha-hydroxylase (ID50 values ranged from 1.13-4.17 mumol/l) and 17,20-lyase (0.57-1.95 mumol/l) activities was: bifon greater than clot greater than keto greater than micon greater than econ greater than isocon greater than tiocon. Using [3H]progesterone (5.50-12.25 mumol/l) as the substrate for the 21-hydroxylase activity the order of decreasing inhibitory potency was: clot greater than bifon greater than isocon greater than micon greater than tiocon greater than econ greater than tiocon greater than keto. For the 11 beta-hydroxylation of [3H]deoxycortisol (1.48-2.34 mumol/l) the order of decreasing inhibitory potency was keto greater than bifon greater than clot greater than micon greater than econ greater than isocon greater than tiocon. The cytochrome P-450 dependent enzyme most sensitive to inhibition was 17,20-lyase and the least sensitive was 21-hydroxylase whereas the imidazole drugs were without effect on the cytochrome P-450 independent 3 beta-HSD-I activity. In agreement with previous results a common structural feature of the imidazole drugs having an inhibitory effect was the presence of aromatic rings on the N-1 substituent of the imidazole ring.     

Synthesis of 6- or 4-functionalized indoles via a reductive cyclization approach and evaluation as aromatase inhibitors

Two new series of benzonitrile derivatives on position 6 or 4 of indole ring were successfully synthesized via a Leimgruber-Batcho reaction. All the compounds were evaluated in vitro on the inhibition of aromatase (CYP19) and 17alpha-hydroxylase-C17,20-lyase (CYP17). The racemate, 4-[(1H-imidazol-1-yl)(1H-indol-4-yl)methyl]benzonitrile 9, showed high level of inhibitory activity towards CYP19 (IC(50)=11.5 nM).     

Triphenyltin and Tributyltin inhibit pig testicular 17beta-hydroxysteroid dehydrogenase activity and suppress testicular testosterone biosynthesis

We previously reported that tributyltin chloride (TBT) and triphenyltin chloride (TPT) powerfully suppressed human chorionic gonadotropin- and 8-bromo-cAMP-stimulated testosterone production in pig Leydig cells at concentrations that were not cytotoxic [Nakajima Y, Sato Q, Ohno S, Nakajin S. Organotin compounds suppress testosterone production in Leydig cells from neonatal pig testes. J Health Sci 2003;49:514-9]. This study investigated the effects of these organotin compounds on the activity of enzymes involved in testosterone biosynthesis in pig testis. At relatively low concentrations of TPT, 17beta-hydroxysteroid dehydrogenase (17beta-HSD; IC(50)=2.6microM) and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (IC(50)=117microM) activities were inhibited, whereas cholesterol side-chain cleavage cytochrome P450 and 3beta-HSD/Delta(4)-Delta(5) isomerase activities were less sensitive. Overall, TPT was more effective than TBT. TPT also inhibited both ferredoxin reductase and P450 reductase activities at concentrations over 30microM; however, TBT had no effect, even at 100microM. The IC(50) values of TPT were estimated to be 25.7 and 22.8microM for ferredoxin reductase and P450 reductase, respectively. The inhibitory effect of TPT (30microM) on microsomal 17beta-HSD activity from pig testis was eliminated by pretreatment with the reducing agents dithiothreitol (1mM) and dithioerythritol (1mM). On the other hand, TPT (0.03microM) or TBT (0.1microM) exposure suppressed the testosterone production from androstenedione in pig Leydig cells indicating that these organotins inhibit 17beta-HSD activity in vivo as well as in vitro, and the IC(50) values of TPT and TBT for 17beta-HSD activity were estimated to be 48 and 114nM, respectively. Based on these results, it appears possible that the effects of TBT and TPT are largely due to direct inhibition of 17beta-HSD activity in vivo.     

Imidazole substituted biphenyls: a new class of highly potent and in vivo active inhibitors of P450 17 as potential therapeutics for treatment of prostate cancer.

3- And 4-imidazol-1-yl-methyl substituted biphenyl compounds (named as meta- and para-substituted compounds) were synthesized bearing additional substituents in 3'-/4'-position as inhibitors of P450 17 (17alpha-hydroxylase-C17,20-lyase). P450 17 is the key enzyme of androgen biosynthesis. Its inhibition is a novel therapeutic strategy for treatment of prostate cancer (PC). Twenty-nine compounds were synthesized by Ar-Mg-Br, Negishi or Suzuki aryl-aryl cross coupling and tested toward human and rat enzyme. Most of the compounds showed moderate to excellent activity against one of the enzymes (0.087 microM < or = IC50 < or = 7.7 microM (ketoconazole: 0.74 microM) for the human enzyme, 0.63 microM < or = IC50 < or = 32 microM (ketoconazole: 67 microM) for the rat enzyme). Interestingly, strong species differences were observed. In addition compounds were tested for inhibition toward P450 arom. The 3-imidazol-1-yl-methyl substituted compounds showed good inhibitory activity of P450 arom, while for the 4-substituted compounds negligible inhibition was found. For the most active group of P450 17 inhibitors, (i.e. the 4-imidazol-1-yl-methyl substituted compounds) a QSAR study was performed for inhibition of the human enzyme leading to the result that a hydrophilic substituent in 3'-/4'-position is very important. The most promising compounds (with respect to activity toward both enzymes) were tested in vivo using SD-rats for reduction of plasma testosterone concentrations 2 and 6 h after single i.p. application. The fluorine substituted compound 8c decreased the testosterone plasma concentration to castration level (after 2 h; 5 mg/kg) showing a biological half live of about 6 h.     

Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.     

The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21

Human cytochrome P450c17 (17alpha-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6A apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized the enantiomer of progesterone and determined if ent-progesterone is a substrate or inhibitor of CYP17 and CYP21. Neither enzyme metabolizes ent-progesterone; however, ent-progesterone is a potent competitive inhibitor of CYP17 (K(I)=0.2 microM). The ent-progesterone forms a type I difference spectrum with CYP17, but molecular dynamics simulations suggest different binding orientations for progesterone and its enantiomer. The ent-progesterone also inhibits CYP21, with weaker affinity than for CYP17. We conclude that CYP17 accommodates the stereochemically unnatural ent-progesterone better than CYP21. Enantiomeric steroids can be used to probe steroid binding sites, and these compounds may be effective inhibitors of steroid biosynthesis.     

17,20-Lyase inhibitors. Part 3: Design, synthesis, and structure-activity relationships of biphenylylmethylimidazole derivatives as novel 17,20-lyase inhibitors

A novel series of biphenylylmethylimidazole derivatives and related compounds were synthesized as inhibitors of 17,20-lyase, a key enzyme in the production of steroid hormones, and their biological activities were evaluated. In an attempt to identify potent and selective inhibitors of 17,20-lyase over the related CYP3A4 enzyme, a homology model for human 17,20-lyase was developed using the X-ray crystallographic structure of the mammalian CYP2C5 enzyme. With the aid of molecular modeling, optimization of the biphenyl moiety was performed to give an acetamide derivative, which was resolved by HPLC to give the active (-)-enantiomer. The obtained active enantiomer showed not only potent inhibition of both rat and human 17,20-lyase,with IC(50) values of 14 and 26 nM, respectively, but also excellent selectivity (>300-fold) for inhibition of 17,20-lyase over CYP3A4. Moreover, the active enantiomer significantly reduced both serum testosterone and DHEA concentrations in a monkey model after single oral administration. Asymmetric synthesis of the active enantiomer was also developed via a chiral intermediate using a diastereoselective Grignard reaction.     

Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations.

P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities in the biosynthesis of androgens and estrogens. These two activities are differentially regulated in a tissue-specific and developmentally programmed manner. To visualize the active site topology of human P450c17 and to study the structural basis of its substrate specificity and catalytic selectivity, we constructed a second-generation computer-graphic model of human P450c17. The energetics of the model are comparable to those of the principal template of the model, P450BMP, as determined from its crystallographic coordinates. The protein structure analysis programs PROCHECK, WHATIF, and SurVol indicate that the predicted P450c17 structure is reasonable. The hydrophobic active site accommodates both delta4 and delta5 steroid substrates in a catalytically favorable orientation. The predicted contributions of positively charged residues to the redox-partner binding site were confirmed by site-directed mutagenesis. Molecular dynamic simulations with pregnenolone, 17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the substrate-binding pocket demonstrated that regioselectivity of the hydroxylation reactions is determined both by proximity of hydrogens to the iron-oxo complex and by the stability of the carbon radicals generated after hydrogen abstraction. The model explains the activities of all known naturally occurring and synthetic human P450c17 mutants. The model predicted that mutation of lysine 89 would disrupt 17,20-lyase activity to a greater extent than 17alpha-hydroxylase activity; expression of a test mutant, K89N, in yeast confirmed this prediction. Hydrogen peroxide did not support catalysis of the 17,20-lyase reaction, as would be predicted by mechanisms involving a ferryl peroxide. Our present model and biochemical data suggest that both the hydroxylase and lyase activities proceed from a common steroid-binding geometry by an iron oxene mechanism. This model will facilitate studies of sex steroid synthesis and its disorders and the design of specific inhibitors useful in chemotherapy of sex steroid-dependent cancers.