Genetics, Structure, Function, Mode of Actions and ...

Send Orders for Reprints to [email protected] Anti-Cancer Agents in Medicinal Chemistry, 2013, 13, 000-000

1

Genetics, Structure, Function, Mode of Actions and Role in Cancer Development of CYP17 Tatyana. A Sushko, Andrei A. Gilep* and Sergey A. Usanov Institute of Bioorganic Chemistry NASB, Belarus, 220141 Minsk, Belarus, Kuprevicha str. 5/2 Abstract: Most prostate and breast cancers are hormone dependent. The inhibition of steroid 17
-hydroxylase/17,20- lyase (CYP17), which is a crucial enzyme for steroid hormone biosynthesis, is widely used to treat androgen-dependent prostate cancer (PC). CYP17 has dual enzymatic activity: 17alpha-hydroxylase activity (utilizing delta4- C21 steroids as substrates) and the 17,20-lyase activity (using delta5- C21 steroids as substrates). The steroid biosynthetic pathway is directed to either the production of corticosteroids or sex hormones depending on the activity of CYP17. In this review, the current information on the genetics, molecular structure, substrate specificity and inhibitors of CYP17 is analyzed and discussed.

Keywords: Cytochrome P450, CYP17, hormone-dependent cancer, inhibitors of androgen biosynthesis, prostate cancer, steroid hormone biosynthesis, steroid 17
-hydroxylase, 17,20-lyase. INTRODUCTION According to the data reported by the World Health Organization, death from cancer is the leading cause of death in the world, and cancer is supposed to cause more than 12 million deaths by the year 2030 [1]. Prostate cancer and breast cancer are the most prevalent types of cancer in men and women, respectively [2]. Most prostate and breast cancers are hormone dependent. Sex steroid hormones play significant role either in the normal development and functioning of these organs or in cancerous growths. Cytochrome P450s (CYPs) are essential enzymes, such as catalyzing the key steps in the biosynthetic pathway of steroid hormones. CYPs are also involved in biotransformation of several precarcinogens and in activation/inactivation of the antineoplastic drugs. Thus, CYPs are crucial for the cancer development and they are the promising targets for anti-cancer therapy. The development of potent inhibitors of aromatase (CYP19) which have been used to treat breast cancer was the first successful example of application of targeting CYP enzymes in anti-cancer therapy. The clinical success of aromatase inhibitors opened up a new direction in developing hormone ablation therapy for estrogendependent cancers and gave rise to similar strategies for CYP17 inhibition, which have become very valuable in treating androgendependent prostate cancer (PC) [3]. Hormone therapy (androgen deprivation therapy) is the main strategy for advanced PC [4], because androgens, including testosterone (T) and dihydrotestosterone (DHT), stimulate the growth of prostate cancer cells by activating the transcription of genes associated with cell proliferation and survival. Androgen suppression therapy is frequently combined with chemical or surgical castration. Due to the castration a decreased production of T and DHT by the testes is observed, but adrenal glands and the cancerous prostate gland itself still continue to produce androgens; it is also suggested that the adipose tissue and skin produce androgens; so as a result, cancer cells continue to grow [5]. Therefore, compounds which have high potencies in inhibiting androgen biosynthesis may prove to be more efficacious than castration in the treatment of PC. CYP17 (steroid 17
hydroxylase/17,20-lyase) plays a crucial role in the steroid hormone biosynthesis. CYP17 is unique because of its ability to catalyze two different types of reactions, the 17alpha -hydroxylase and *Address correspondence to this author at the Institute of Bioorganic Chemistry NASB Belarus, 220141 Minsk, Kuprevicha str. 5/2; Tel/Fax: +375-17-263-7274; E-mail: [email protected]

1871-5206/13 $58.00+.00

17,20-lyase reactions, in one active site,. Furthermore, the ratio of these reactions is physiologically important and may direct steroid hormone biosynthesis towards the production of corticoid or sex hormones. Thus, CYP17 is a highly promising target in the inhibition of androgen biosynthesis. THE CYP17A1 GENE The CYP17A1 gene is typical for the genomes of all Chordata species and encodes highly conserved cytochrome P450. The human gene is located in chromosome 10 at position 10q24.3 and spans over 10 kb [6]. Transcription initiation site is mapped approximately 180 bp upstream of the initiation codon in exon 1 and ~1.7 kb mRNA is produced. Recently, a number of studies have focused on genetic factors that may influence the risk of developing cancer. Much attention has been given to the study of polymorphic variants of the genes encoding enzymes that participate in sex hormone metabolism. It is suggested that variants of CYP17A1 may be associated with increased risk of hormone dependent cancer [7]. By now, more than 100 polymorphic variants of CYP17A1 have been identified; the majority of polymorphisms have been mapped to non-coding regions of the CYP17A1. In order to elucidate if CYP17A1 gene polymorphisms are associated with the risk of developing PC, many case-control studies have been carried out to answer this problem. However, the data obtained were contradictory [7, 8]. In the past years, the polymorphism -34T/C (rs743572) has attracted widespread attention because this polymorphism was postulated to be associated with changes in circulating concentrations of sex hormones and with an increased risk of breast, prostate or endometrial cancer [9]. Polymorphism rs743572, which is mapped to the 5’-untranslated promoter region of CYP17, creates a site of restriction endonuclease cleavage (restriction enzyme MspA1) giving rise to the two allelic variants: A1 (T, wild allele) and A2 (C, variant allele). A recently conducted meta-analysis [7] indicated that the rs743572 polymorphism is associated with increased risk of PC only in Black population. However, there is no significant relationship between rs743572 polymorphism and risk of PC in the general population, so this polymorphism might not be considered as a critical factor for PC susceptibility in humans. This fact supports the hypothesis that PC is a multigenic disease. At the same time, persons carrying A2 allele of CYP17 have a lower risk of developing pancreatic cancer [12]. Furthermore,

© 2013 Bentham Science Publishers

2 Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

polymorphism rs743572 is thought not to be associated with cancer risk only. We have shown that polymorphism rs743572 is associated with alterations in circulating concentrations of cortisol, leading to the changes in physical performance in humans [10]. There are also abundant contradictory results concerning the association of -34T/C polymorphism with polycystic ovary syndrome [11]. Additional studies have shown a large number of polymorphisms of the CYP17A1 gene. It was suggested that the rs619824 (-15830 A/C/G/T) and rs2486758 (-362 A/C/G/T) polymorphisms are associated with PC risk [7]. It was reported, that humans carrying C allele of the polymorphism rs2486758, found in the promoter region of CYP17A1 gene, have a 7% higher risk of developing PC. However, carriers of the A allele of the rs619824 polymorphism, which occurs in the 3’-UTR, show a 5% decreased risk for PC. The polymorphism rs619824 is identified in the region of CYP17A1, which is characterized by a high sequence homology to a transcription regulator CCAAT/enhancer-binding protein. For that reason, it has been hypothesized, that this polymorphism has a direct functional significance, in contrast to the polymorphism rs2486758. A number of single-nucleotide substitutions that were found in the CYP17A1 gene may cause 17alpha-hydroxylase/17,20-lyase deficiency. The biochemical effects of many mutations identified in clinical studies can be explained through molecular modeling experiments and the examination of the recently obtained CYP17 crystal structure [13]. It has been revealed that the mutants H373L, H373G, R440H, D298V, G301I and G111D can not bind heme [14, 15]. As a result of S106P mutation [16], proline is introduced into B-helix adjacently to amino acids which form a substratebinding pocket, consequently it leads to complete loss of the 17alpha -hydroxylase and 17,20-lyase activities of CYP17. Mutations insIle112, R96W and G90D also disturb substrate binding site, lead to the loss of catalytic activity as well. The engineered mutation A105L [17], which is located in the active site facing the face, causes the formation of additional bulk that may restrict the movement of the steroid within the active site, resulting in a strong reduction of minor 16alpha-hydroxylase activity of CYP17. There are some mutations that result in retaining partial catalytic activity only. Thus, substitutions T64S [18] and P342T [19], CYP17 retains 15% and 20% of wild type catalytic activity, respectively. Several patients were reported as having 17,20-lyase deficiency, because of the mutations Q461stop and R496C [20]. The Q461stop mutant is inactive, while mutation R496C leads to the combined partial 17alpha -hydroxylase /17,20-lyase deficiency. Mutations E305G, R347H/C, R358Q and R449A ablate only the 17,20-lyase activity of CYP17 [21, 22]. Residue E305 is crucial for the interaction with natural modulator cytochrome b5. The analysis of structural model let to hypothesize that substitutions R347H and R358Q might change the positive surface charge in the region responsible for the interaction with redox partner [23]. Therefore, deficiency of 17,20-lyase activity for these mutants results from a disturbance of redox partner binding. There is no indication of an association of these mutations with cancer. EXPRESSION OF CYP17 DURING ONTOGENESIS AND CHANGES IN CYP17 EXPRESSION ASSOCIATED WITH DISEASES The ability to synthesize major types of steroid hormones is mainly controlled by the relative expression level of cytochrome P450s. CYP17 expression is regulated by adrenocorticotropic hormone in the adrenal glands and gonadotropic hormone in the testis and ovaries. Transcription factors such as NF1, SF1 and SF3 are essential for the regulation of CYP17A1 expression [24]. Transcriptional factors binding sites in the CYP17A1 gene include nt -107 to -85 and nt -178 to -152 for NF1- 1, and nt -227 to -184 for SF1 and SF3 [24]. Recent studies in our laboratory have

Sushko et al.

demonstrated that human CYP17A1 expression level is also regulated by the coding sequence of exon I. If this sequence was transfected to the promoter region in a reporter system in nonsteroidogenic cells, the expression of CYP17A1 was abolished [25]. A comparative study of the expression profiles of the steroidogenic enzymes in ontogenesis has been conducted [26]. Large amounts of steroid hormone precursors have been detected in the fetal circulation. Quantification of CYP17A1 mRNA shows that CYP17A1 gene is expressed at a high level in adrenal glands and testis (608 ± 116 and 333 ± 120 attomol/ μg CYP17A1 18S rRNA, accordingly), while a lower expression level is detected in the ovary and placenta (4.5 ± 1.1 and 7.18 ± 1.6 attomol/ μg18S rRNA, accordingly). Similar results were obtained for CYP17A1 mRNA level in the fetal aorta (4.7 ± 1.3 attomol/ μg 18S rRNA). All nonsteroidogenic tissues are characterized by much lower CYP17A1 mRNA level (0.0034 - 0.61 attomol/ μg 18S rRNA).No expression of the CYP17A1 gene was reported in the lung. The overall abundance of CYP17A1 mRNA was not significantly different between the fetal and adult adrenal glands [26]. In contrast to a nonsignificant difference in 17alphahydroxylase/17,20lyase mRNA abundance, cytochrome b5 and NADPH:ferricytochrome oxidoreductase (CPR) were expressed 2.3-fold and 2.0-fold higher, respectively, in the fetal than the adult adrenal glands. Both cytochrome b5 and CPR may upregulate 17alpha-hydroxylase/17,20-lyase activity and, consequently, dehydroepiandrosterone sulfate (DHEAS) formation in the human fetal adrenal glands as well. Therefore, the alterations in the expression patterns of the enzymes involved in steroid hormone biosynthesis and cofactors involved in gene expression might account for some of the phenotypic differences between the fetal and adult adrenal glands [27]. Changes in the expression level of steroidogenic enzymes may be associated with different diseases. Autonomous androgen production in prostate cancer may cause the tumor growth and progression in patients who undergo ablation therapy. Androgens exert their actions on prostate cells through different mechanisms, including stimulation of proliferation, promotion of differentiation and inhibition of apoptosis, programmed cell death. Experimental study suggests that the level of CYP17A1 mRNA and protein expression might be associated with disease stage and recurrence [28, 29]. Metastatic lymph node 64 (MLN64) and CYP17 are upregulated in PC, and their expression is strictly associated with PC stage. High expression levels of MLN64 and CYP17A1 are typical for later stages of PC, and the abnormal expression level of either MLN64 or CYP17A1 is correlated with poor survival of PC patients [29]. The excess production of aldosterone or cortisol, which is characteristic of primary aldosteronism or Cushing’s syndrome, respectively, has profound effects on cardiovascular function and also impacts other major organ systems. The leading cause of primary aldosteronism is aldosterone-producing adenoma (APA). Similarly, elevated levels of corticosteroids can cause Cushing’s syndrome and cortisol-producing adenoma (CPA). A decreased expression level of CYP17A1 has been reported in aldosterone-producing adrenal samples compared with the normal adrenal gland. Contrarily, there was a significant increase in CYP17A1 transcript levels in the cortisol-producing adrenal samples compared with normal adrenal samples. These results are consistent with the selectively increased aldosterone production observed in APA and cortisol production observed in CPA. The CYP17 protein carries out 17alpha-hydroxylase and 17,20-lyase activities, which are required for cortisol, but not aldosterone, biosynthesis. However, low level of cytochrome b5 mRNA was detected in normal adrenals, APA and CPA [30]. Adrenarche is an early sexual maturation stage in humans and some Old World monkeys. Adrenarche is caused by an increase in the level of androgens secretion (dehydroepiandrosterone (DHEA)

Genetics, Structure, Function, Mode of Actions and Role in Cancer

and DHEAS) by adrenals; adrenarche can usually be detected between 6 and 8 years of age. Whereas only two cytochrome P450s are necessary for the biosynthesis of DHEA from cholesterol (Fig. 1), during adrenarche changes in other steroid metabolizing enzymes and cofactors also result in the alterations in adrenal androgen secretion. Adrenarche is characterized by an elevated level of the 17,20-lyase activity of CYP17, and, consequently, by a dramatic increase in DHEA formation from 17alpha-hydroxypregnenolone (17OHP5). The regulation of CYP17 expression seems not to contribute to adrenarche. In contrast to CYP17 expression, during adrenarche an increase in cytochrome b5 expression, which predominately stimulates the 17,20-lyase activity of CYP17, is observed as a consequence of expansion of the adrenal zona reticularis [31]. THE CYP17 ENZYME The human CYP17A1 mRNA encodes a protein of 508 amino acids (EC 1.14.99.9, NCBI accession no. NP_000093.1) which is able to catalyze the 17alpha -hydroxylase and 17,20-lyase reactions utilizing different C21-steroids as substrates (Fig. 2). Furthermore, the ratio of these reactions is physiologically important and may direct steroid hormone biosynthesis towards the production of corticoid or sex hormones [32]. In humans, the endoplasmic reticular CYP17, which is found in the zona fasciculata and zona reticularis of the adrenal glands and in gonadal tissues, is a key branch-point enzyme in the biosynthesis of steroid hormones. Abnormalities in CYP17 activity are associated with many endocrine-related human diseases, such as polycystic ovary syndrome [33-35], Cushing’s syndrome [36], congenital adrenal hyperplasia [37] and prostate cancer [38, 39]. CYP17 enzymes from different species share 46-98% sequence homology, which depends on the evolutionary distance between the organisms. The CYP17s from various mammalian species are characterized by a relatively high homology with regard to their amino acid sequences, whereas there are a lot of differences in substrate specificity for steroid17,20-lyase reaction and in requirement for cytochrome b5 for maximal activity. The transformation of progesterone (P4) and pregnenolone (P5) by CYP17 was investigated using microsomal fractions from the steroidogenic tissues (adrenal gland and gonadal tissues) of various species or using heterologously expressed proteins, and significant alterations in the products formed were elucidated [40-42]. Comprehensive study of the catalytic activities of the CYP17s allows for the categorization of all known CYP17s into three main groups, 5, 4, and 4,5 types . CYP17s, belonging to 5-type (human, primate, cat, bovine, sheep, goat, bison), are characterized by the lack or very low levels of 17,20-lyase activity with 17alpha-hydroxyprogesterone (17OHP4) [18, 43-45]. Human CYP17, belonging to 5-type CYP17, effectively catalyzes the 17alpha-hydroxylation of P4 and P5 and the subsequent transformation of 17OHP5 to DHEA. 17OHP4 is a poor substrate for 5-type CYP17s, with a negligible amount of androstenedione (AND) formed. Both P4 and 17OHP4 can be subsequently used

Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

3

as substrates for 21-hydroxylase reaction catalyzed by CYP21 with the production of the precursors of mineralocorticoids and glucocorticoids. Alternatively, the product of the 17,20-lyase reaction is a precursor of sex hormones. CYP17s, belonging to 4type (guinea pig), are characterized by the lack or very low levels of 17,20-lyase activity with 17OHP5 [46]. 4-type CYP17s effectively catalyze the 17alpha-hydroxylase reaction with P4 and P5 and the subsequent transformation of 17OHP4 to AND. 17OHP5 is a poor substrate for 4-type CYP17s, with a negligible amount of DHEA formed. 4,5-type CYP17s (pig, hamster, horse, rat, nonmammalian species), catalyze the 17alpha –hydroxylase reaction with P4 and P5 and the 17,20-lyase reaction with 17OHP4 and 17OHP5 [45, 47]. CYP17 is a unique enzyme because of its ability to catalyze different types of reactions in a single active site. The structural features of the active site that provide this ability are under investigation. Analysis of the recently obtained crystal structure of CYP17 with inhibitors [13] led to the prediction of the orientations of the native CYP17 substrates. It should be noted that the topology of the active site might vary when the substrate enters the active site. Thus, it is important to determine the 3D structure of CYP17 with its substrates. Results of docking experiments have shown that P4 maintains the N202 hydrogen bond [13]. It was revealed that C17 atom of bound ligand is 3.7 from the catalytic oxygen, C16 atom of ligand is 3.9 from the catalytic oxygen. These data are in accordance with experimental data on the catalysis of 16alpha-hydroxylation and 17alpha-hydroxylation of P4 by CYP17. The P5 3-OH hydrogen bonds to N202, and the distance observed between C17 atom and Compound I oxygen is 3.6 [13]. Interestingly, the binding and orientation of the substrate and inhibitors in the crystal structure differs substantially from those reported in studies using molecular modeling techniques [13, 48, 49]. Compared to the cytochrome P450s with a known crystal structure, which are involved in steroid hormone biosynthesis (CYP19, CYP11A1 and CYP46) [50-52], CYP17 orients steroids in the opposite direction. The steroid substrates are located near the K–L loop and oriented towards the 1-sheet, instead of being oriented towards helices F and G [13]. Aromatase is a promising target in the treatment of hormone-dependent breast cancer. Recently obtained crystal structure of CYP19 [50] clarified the structural basis for its ligand specificity and role in the biosynthesis of estrogens. CYP19 differs from CYP17 in that a distortion in the I-helix of the CYP19, which results in an approximately 3.5-Å rearrangement of the helix axis, is important for the formation of ligand -binding pocket which is specific for androgens. Despite the fact that CYP21 and CYP17 are evolutionarily closely related enzymes and have a common substrate, analysis of the recently obtained crystal structure of CYP21 [53] has shown a number of features in active site topology of CYP21 that differ from those of CYP17. It has been reported that two molecules of

Fig. (1). Human CYP17A1 gene and protein. SNPs which are proved to be associated with PC risk are noted above the gene.

4 Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

Sushko et al.

Fig. (2). Human steroid biosynthesis pathway.

17OHP4 are bound to CYP21; the distal molecule of 17OHP4 is located at the entrance of the substrate access channel and the proximal one is located in the active site. The key substrate recognition residues are located not only around the heme but also along the substrate access channel. For CYP11A1 the active site organization contributes to releasing of reaction products through various routes depending on their role in metabolism [51]. P5, a highly hydrophobic product of CYP11A1 catalyzed reaction, that undergoes subsequent conversions outside of the mitochondria, could throw over the mitochondria using the access channel for the cholesterol substrate. The smaller and more hydrophilic isocapronic aldehyde can be released through the water channel. Analysis of the molecular structure of CYP17 [13] suggests a similar way of reaction product release as CYP11A1. Hydroxylated products (17OHP4 and 17OHP5) and DHEA are released through the substrate access channel toward the membrane, and acetic acid can exit to the cytosol through the egress channel formed by the parts of K-helix, L-helix and I-helix of CYP17 (Fig. 3A). Notably, residues R347, R358 and R449, which are shown to participate in the proteinprotein interaction with cytochrome b5 [21], surround exit of this egress channel. We propose that mutations in these residues, which eliminate only the 17,20-lyase activity of CYP17 [21], change the structure of this channel. In summary, CYP17 has structural features that are not similar to other cytochrome P450s with known crystal structures, which are involved in steroid hormone biosynthesis. Knowledge of the distinctive features of the catalytic mechanism and molecular organization of CYP17 can be used to develop highly specific and effective inhibitor-based drugs for prostate cancer treatment.

INVOLVEMENT OF CYP17 IN ALTERNATIVE STEROID BIOSYNTHESIS CYP17 is an essential enzyme, catalyzing not only the production of sex steroid precursors but also involved in the biosynthesis of 16-ene steroids (including androstenol). CYP17 catalyzes the reaction of androstadienol production, which is a precursor in the 16-androstene pathway. 16alpha-Hydroxylase activity is a specific feature of human CYP17 [54, 55]. P4 (but not P5) is utilized in the reaction of 16alpha –hydroxylation catalyzed by human CYP17. Experimental studies of the different aspects of P4 biotransformation have shown that CYP17 catalyzes 17alpha-hydroxylase and 16alphahydroxylase reactions at the same active center [45]. Additionally, human CYP17 catalyzes a cytochrome b5-dependent 16-enesynthase reaction, converting P5 to the
5,16-steroid, androstadienol [56]. It has been presumed that the main steps of the steroid biosynthesis in mammals, involving the chemical structures of products and intermediates, are designated in details. Recently a number of studies focusing on alternative steroids metabolism have been conducted. It has been uncovered that CYP11A1 efficiently catalyzes reaction of cleavage of the side chain of 7dehydrocholesterol (7dhC), which serve as a precursor to cholesterol, with 7-dehydropregnenolone (7dhP5) formation [57, 58]. This fact may indicate on existence of a novel pathway for biosynthesis of steroids containing unsaturated B ring. Smith-Lemli-Opitz syndrome (SLOS) is an inborn defect resulting from a deficiency in 3- hydroxysteroid-
7–reductase, which catalyzes the reduction of the double bond on 7dhC to form

Genetics, Structure, Function, Mode of Actions and Role in Cancer

Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

5

Fig. (3). A. Access and egress channels to/from the active site of CYP17 calculated using CAVER [106]. Amino acids involved in interaction with cytochrome b5 are labeled. Structural figure was prepared with the program PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. B. Model of the tertiary structure of CYP17 (PDB ID: 3RUK). Amino acids involved in interaction with redox partners are labeled on the proximal surfaces of the enzyme. Structural figure was prepared with the program UCSF Chimera [107].

6 Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

unesterified endogenous cholesterol [59]. More than 50% of steroids circulating in fetus with inborn SLOS are ring-B unsaturated equine-type steroids. Ring-B unsaturated steroids are precursors in vitamin D3 biosynthesis, and thus, it makes sense that 7- steroids are also detected in skin [60]. There are some indications on antiproliferative activity of the some 7- steroids in skin [61]. The role of ring-B unsaturated estrogens, such as equilin, which are foreign to the human body, is unknown. Conjugated equine estrogens isolated from the urine of mares are a commonly prescribed medication for menopausal women. At least nine estrogenic compounds have been identified in the urine of pregnant mares; six are known as ring-B unsaturated estrogens [62]. A large number of observational studies suggested that women who underwent hormone replacement therapy had a higher incidence of breast and endometrial cancer [63-65]. Thus it is important to elucidate the possibility of the involvement of CYPs in the metabolism of 7-dehydrosteroids. Sequential metabolism of 7dhC has been studied ex vivo using adrenal glands and formation of the 5-7 -dienal intermediates was detected [60]. Recently, we have demonstrated in vitro the ability of human CYP17 to utilize effectively 5 - 7 -type of steroids as substrates [25] (Fig. 2). CYP17 converts 7dhP5 to 7-dehydro-17- hydroxypregnenolone in a 17alpha-hydroxylase reaction. 7-Dehydrodehydroepiandrosterone is formed from 17- hydroxy-7-dehydropregnenolone in a 17,20-lyase reaction. Therefore, the existence of an efficient estrogen biosynthesis pathway using 7-steroids in humans can be hypothesized. Human CYP17 effectively metabolizes 5-reduced C21 steroids [66]. However, the human CYP17 catalyzes only 17alpha –hydroxylase reaction of the biotransformation of 5- pregnan-3,20-dione. The product of the 17alpha – hydroxylation, the 5 -pregnan- 17 -ol-3,20-dione, could not serve as substrate for the 17,20-lyase reaction. On the contrary, CYP17 effectively converts 5 -pregnan- 3 -ol- 20-one to 5 -pregnan3,17 -diol-20-one, which may be rapidly converted to androsterone by 17,20-lyase reaction. The formation of the less polar product 16,( 5)- androsten- 3 –ol has been detected (without requiring cytochrome b5). Moreover, compared to 17OHP5, 5pregnan- 3,17-diol-20-one appeared to be a better substrate for the 17,20-lyase reaction of CYP17. Accordingly, reduced 3 hydroxy-C21 steroids could serve as substrates for human CYP17, in spite of the existence of the alternative pathway for 5dihydrotestosterone biosynthesis in the testis of fetal or neonatal animals [66]. Neurosteroid biosynthesis is of great interest to investigators. The presence of DHEA and DHEAS in the brain in humans and rats has been reported [67]. It is postulated that these steroids might be formed de novo in the nervous system by CYP11A1 and, possibly, CYP17. The neurotrophic function of DHEA and DHEAS has been demonstrated for rats, but their role in humans needs to be clarified. It is assumed that expression of CYP17 in the rat embryo might influence the brain development [68]. THE MECHANISM OF THE REACTIONS CATALYZED BY CYP17 AND THE EFFECT OF CYTOCHROME B 5 AND OTHER ENDOGENOUS EFFECTORS ON CYP17 ACTIVITY CYP17 is a multifunctional enzyme that catalyzes the conventional hydroxylation reaction of the pregnene nucleus at position 17alpha and an acyl-carbon cleavage. In addition, this enzyme promotes the formation 16alpha-steroids. Both its reactions of hydroxylation and acyl-carbon bond cleavage occur sequentially at the same active center. Both CYP17 activities require electron supply; CYP17 receives two electrons from NADPH via flavoprotein NADPH -cytochrome P450 reductase (CPR). The mechanism of the hydroxylation reaction is clearly defined [69]. The resting form of the enzyme is the ligand-free ferric heme with loosely coordinated water. Substrate displaces the heme water

Sushko et al.

ligand. Then ferrous protein binds molecular oxygen to produce the FeII-O2 complex. A second electron transfer to this intermediate leads to the formation of the ferric peroxy anion, which is converted by protonation to the ferric hydroperoxo complex. The ferric hydroperoxo intermediate fragments give a ferryl intermediate interacting with substrate to form the hydroxylated product. The last step in hydroxylation reaction is product release, returning the P450 to the resting ferric state. The mechanism of the 17,20-lyase reaction catalyzed by CYP17 is under investigation. This mechanism has been proposed to be similar to the third reaction catalyzed by aromatase [70]. The key step of the 17,20lyase reaction is the nucleophilic attack by the ferric-peroxyanion FeIII-O-O-. The FeIII-O-O - species react with the carbonyl group at C-20 position of 17alpha- hydroxyprogestogen to yield the tetrahedral intermediate, which decays giving the acetic acid. Cytochrome b5 is crucial for regulating 17,20-lyase activity of CYP17 [56, 71] and is an important physiological modulator of sex hormone biosynthesis. While it does not affect steroid 17alphahydroxylation, cytochrome b5 causes a 5- to10-fold increase in the 17,20-lyase activity catalyzed by CYP17. The mechanism by which cytochrome b5 selectively stimulates only 17,20-lyase activity of CYP17 is unknown. There is evidence that cytochrome b5 induces conformational changes in CYP17 without directly transferring electrons to P450 [72]. This assumption is based on the fact that heme- free cytochrome b5 (apo- b5) might increase the 17,20-lyase activity of CYP17. Nevertheless, it has been reported that the reconstruction of holo- b5 might occur in the reconstituted system due to the heme transfer from denatured P450 to apo- b5 [73]. Subsequent studies with mutant CYP17s which had no 17,20lyase activity provided strong evidence that cytochrome b5 is not involved directly in the electron transfer [21]. It is hypothesized that the nucleophilic attack of FeIII–O–O
on the target atom of substrate is stimulated in the presence of cytochrome b5. The binding of cytochrome b5 to the CYP17 induces conformational changes that direct the iron-oxygen ligand of CYP17 from C-17 towards C-20. Consequently, it promotes a nucleophilic attack of the peroxide anion on the carbonyl carbon [70]. Detailed understanding of the reaction mechanism and elucidation of the role of cytochrome b5 has been impeded in absence of crystal structure of CYP17 in the past. Analysis of the recently obtained crystal structure of CYP17 [13] allows us to consider another possible mechanism of the action of cytochrome b5. Products of 17alpha-hydroxylase reaction are released from the active site through the substrate access channel, and acetic acid, the side product of 17,20-lyase reaction, probably exits to the cytosol through the egress channel (Fig. 3A). We propose that cytochrome b5 may mediate the structure of egress channel for the acetic acid providing a quick release of the acetic acid. Mutations in the residues surrounding egress channel may eliminate only 17,20lyase activity as a result of preventing the free exit of the acetic acid. Recently, another form of cytochrome b5, outer mitochondrial cytochrome b5, that stimulates androgen biosynthesis has been identified in cells in the testis [74]. The data obtained in our laboratory show that the effect of microsomal and mitochondrial cytochromes b5 on the catalytic activity of CYP17 is strongly dependent on the CYP17 type (4, 4-5, or 5 type) [25]. Interestingly, a recently described regulatory protein, inner zone antigen (IZA, progesterone receptor membrane component-1 protein (mPGRMC1p), damage-associated response protein (Dap1p)), which has a folding similar to cytochrome b5 and contains heme prosthetic group, couldn’t increase the level of the catalytic activity of CYP17 [75]. This fact indicates a high specificity of interaction between cytochrome b5 and CYP17. In summary of the role of cytochrome b5 in CYP17-catalyzed reactions, it is important to underline that the interaction of

Genetics, Structure, Function, Mode of Actions and Role in Cancer

cytochrome b5 with CYP17 is characterized by a high specificity. Cytochrome b5 lacking hydrophobic membrane-binding domain or the heme group couldn’t stimulate reactions catalyzed by CYP17, indicating that these regions of the cytochrome b5 are important for this interaction. Cytochrome b5 is a highly specific protein that manages sex hormone production by regulating 17,20-lyase reaction of CYP17. Therefore, cytochrome b5 or the region of CYP17 responsible for interaction with cytochrome b5 may be a promising target to develop novel drugs for hormone-dependent cancer therapy. CYP17-DEPENDENT XENOBIOTIC METABOLISM It is generally accepted that microsomal cytochrome P450s are divided into two main groups according to the substrate specificity: xenobiotic metabolizing P450s and cytochrome P450s involved in the biosynthesis of endogenous substances. However, further investigations demonstrated that this division is too simple. Recently there has been a rise in reported cases of the involvement of xenobiotic metabolizing P450s in the biotransformation of the endogenous compounds [76-78]. There are some indications that CYP17 might be involved in aminopyrine N-demethylation. Moreover, this activity is stimulated (1.5-fold) in the presence of cytochrome b5 [79]. It has been elucidated, that CYP17 is able to catalyze reaction using different xenobiotics with pro-carcinogenic properties as substrates. Thus, it has been shown that DMBA (7,12-dimethylbenz[a]anthracene) and aflatoxin B1 might be metabolized by CYP17. The formation of the epoxide of aflatoxin B1, which displays genotoxic properties is observed during the oxidation of aflatoxin B1 catalyzed by CYP17 [25]. Subsequent investigations are needed to determine the role of CYP17 in the activation of pro-carcinogenic chemicals. The involvement of CYP17 in bioactivation of pro-carcinogenic compounds is of great interest, because CYP17 is found in ovary, placenta, testis, adrenal glands, skin and kidney, where the bioactivation of pro-carcinogenic chemicals might result in the stimulation of carcinogenesis. Furthermore, the findings suggest that it is important to screen the drugs for their effect on CYP17 and on their ability to be metabolized by CYP17 with the production of genotoxic or cytotoxic reactive compounds. Accordingly, the druginduced changes in steroid hormone biosynthesis might be considered as CYP17 –drug interactions. Furthermore, it is very important to take these interactions into account in the prenatal

Fig. (4). Inhibitors of CYP17. A – ketoconazole, B – abiraterone, C- TOK-001.

Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

7

period, where CYP17–drug interactions might exert unpredictable effects. CYP17 INHIBITORS CYP17 catalyzes key steps in all sex steroid hormone biosynthesis, therefore inhibition of catalytic activity of CYP17 is a powerful treatment against PC and other androgen-dependent disorders. The evolution of PC treatment has progressed from surgery to receptor antagonists to hormone synthesis inhibitors [8082]. Inhibitors of CYP17 are commonly classified as steroidal and non-steroidal inhibitors. The majority of steroidal inhibitors of CYP17 contains heterocycle at the 17-position (usually imidazole or pyridine), which forms a coordination bond with the heme iron. According to the putative mechanism of action, steroidal inhibitors can be divided into competitive inhibitors, irreversible inhibitors, and mechanism-based inhibitors. Suicides, or mechanism-based inhibitors, are preferable in drug design due to their high specificity, prolonged action, and low toxicity [83]. Ketoconazole has been prescribed to treat advanced PC; but the clinical application of ketokonazole has been restricted due to the unwanted side effects resulted from the nonselective inhibitory effect on different cytochrome P450s. A large number of more selective inhibitors of CYP17 had been designed (Fig. 4) [84, 85]. The recently developed CYP17 inhibitor, abiraterone acetate, acts as a mechanism-based steroidal inhibitor. The distinctive molecular features of abiraterone acetate, which lead to its high efficacy and selectivity, are pyridin-3-yl substituent and a double bond between carbon atoms C16 and C17 of the molecule [86, 87]. Abiraterone was approved by the US Food and Drug Administration in 2011 to treat patients with late-stage PC [88]. It should be mentioned that abiraterone acetate monotherapy causes a secondary hyperaldosteronism [89]. To reduce these symptoms, abiraterone acetate should be prescribed in combination with prednisone. Another promising CYP17 inhibitor is TOK-001 (androsta5,16-dien-3- ol, 17-(1H-benzimidazol-1-yl)-, (3)-), a novel
1617-azolylsteroid, which inhibits the catalytic activity of CYP17, and also acts as an androgen receptor antagonist and disrupts androgen signaling through multiple targets [90]. TOK-001 is currently in clinical trials. The design of CYP17 inhibitors has been based on molecular modeling techniques, the results of site directed mutagenesis, and information from comparative interspecies studies because the

8 Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

crystal structure was not available for CYP17. Recently, X-ray crystal structures of CYP17 with the inhibitors abiraterone and TOK-001 have been reported [13]. It is interesting to mention that substrate and inhibitor binding and orientation in the crystal structures differ substantially from those previously reported in molecular modeling studies [13, 48, 49]. According to the structural analysis, important features that make abiraterone and TOK-001 very effective inhibitors were elucidated. The heterocyclic nitrogen of abiraterone and TOK-001 binds the heme iron, making an angle of 60° above the plane of the heme ring. Notably, the binding of these inhibitors differs substantially from the binding predicted by homology models based on cytochrome P450s with determined tertiary structures [13]. These interactions are similar to the interactions in the steroid receptors [91]. This fact may explain why TOK-001 not only inhibits CYP17 but also acts as an androgen receptor antagonist. Studying the mechanisms of CYP17 inhibition is required for oncology drug development as well as for the screening of chemicals, which might disrupt endocrine function. Analysis of the cytochrome P450 inhibition is a promising strategy for the investigation of the possible side effects of drugs and commonly used pesticides. We tested several azole-containing compounds, including non- CYP17 targeted drugs and widely used pesticides. The data obtained in our laboratory demonstrate that azolecontaining pesticides might bind to human CYP17 with different affinities: flusilazole with Kd-1.7 μM, propiconazole with Kd-3.6 μM, triadimenol with Kd-5.9 μM and bitertanol with Kd-7.6 μM. Tebuconazole is characterized by the highest affinity for human CYP17 (Kd-0.3 μM). Azole antifungal agents have high affinities for CYP17 too, e.g., fluconazole (Kd-0.18 μM) and ketoconazole (Kd-0.38 μM); while itraconazole has no effect on human CYP17 [25]. Nevertheless, the use of CYP17 inhibitors in hormonedependent cancer therapy is restricted due to the side effects related to the inhibition of 17alpha-hydroxylase activity, which is important for the production of corticoid hormones in addition to 17,20-lyase activity. It is essential to design a new class of inhibitors that might eliminate only 17,20-lyase activity of CYP17. Cytochrome b5 modulates predominantly 17,20-lyase activity, functioning as a fine tuning for sex hormones biosynthesis regulation. It is a promising strategy to develop inhibitors that may disturb the interaction between CYP17 and cytochrome b5 to inhibit only the 17,20-lyase activity responsible for sex hormone biosynthesis. ELECTRON DONOR SYSTEM CPR (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) is crucial for the electron transfer from NADPH to microsomal cytochrome P450s. CPR is a 78-kDa membrane-associated flavoprotein using FAD and FMN as cofactors [92, 93]. CPR contains four major domains, the FMN-binding domain and the FAD-binding domain, the connecting domain, and the NADP-binding domain. During a catalytic cycle, CPR is capable of transferring electrons accepted from NADPH (as a hydride ion) via FAD and FMN to the heme iron of cytochrome P450 [94]. It has been demonstrated that electrostatic interactions play a significant role in the interaction of CPR with cytochrome P450s; in addition, there are also indications involving hydrophobic interactions. Residues of the Lhelix, C-helix and the heme-binding loop have been reported to be involved in the interaction of microsomal CYPs with CPR (Fig 3B) [95]. The functional complex formed between cytochrome P450 and CPR is necessary for productive electron transfer [96]. The availability of electrons received from CPR is a limiting factor for the most reactions catalyzed by microsomal cytochrome P450s. The amount of cytochrome P450s in the liver and steroidogenic tissues significantly exceeds the amount of CPR (approximately 20:1) [97].

Sushko et al.

The CPR concentration in adrenal microsomes is 4 times higher than that in testicular microsomes [98]. Because CPR is the obligate redox partner for all known microsomal cytochrome P450s, it is postulated that the lack of CPR activity would results in significant alterations in metabolism. CPR deficiency in humans leads to the various disorders: primary amenorrhea, ambiguous genitalia, adrenal insufficiency and skeletal malformation [99]. Catalytic activity of CYP17, CYP21 and CYP19 might be altered by specific CPR mutations. Thus, the A287P variant of CPR decreases the catalytic activity of CYP17 by approximately 70%, while there were no changes in the catalytic activity of CYP19 and CYP21 associated with this mutation [100]. Hence, various CPR mutations exert different effect on cytochrome P450 activities, leading a wide range of clinical manifestations. From the other side, it is possible that some mutations in CPR gene may cause hyperproduction of CPR and, consequently, result in considerably increased cytochrome P450s catalytic activity and increase the risk of cancer developing. It was previously postulated that individual partners in the redox coupling in P450-depending monooxygenase systems are highly specific. Further investigations have found that flavodoxin (FMN-containing bacterial analog of the CPR) and NADHflavodoxin reductase or ferredoxin and NADH-ferredoxin reductase are able to support the catalytic activities of different microsomal cytochrome P450s in vitro [101, 102]. Removal of the highly hydrophobic N-terminal sequence of microsomal P450s might influence the catalytic activity, leads to the changes in their preference for the electron donor, and even affect subcellular distribution of CYPs [101, 103, 104]. These data is in accordance with our results that the pair of mitochondrial electron donors (adrenodoxin and adrenodoxin reductase) might support catalytic activity of CYP17 and CYP21. Furthermore, full-length and truncated human CYP17 and truncated human CYP21 are able to receive electrons from adrenodoxin and adrenodoxin reductase, while CYP19 demonstrates a high specificity for electron donor proteins. Truncated and full-length human CYP17 demonstrates different preference for redox partners; truncated CYP17 has higher catalytic activity with adrenodoxin in pair with adrenodoxin reductase than with CPR [105]. CONCLUSIONS CYP17 is an essential enzyme, which participate in a variety of very important physiological, pharmacological and toxicological processes. It is involved in the key step of steroid hormone biosynthesis and may be responsible for the changes in cells induced by xenobiotic metabolism. Major functions of CYP17 are involvement in glucocorticoid biosynthesis by catalyzing steroid17alpha-hydroxylase reaction and sex hormone biosynthesis via steroid 17,20-lyase reaction. 17,20-Lyase reaction is regulated by the direct influence of cytochrome b5 on CYP17. Inhibition of CYP17 proved to be an instrument for the treatment of hormonedependent prostate cancer caused by excessive androgens. However, undesirable side effect of existing drugs (e.g., inhibitors of CYP17) enforces the development of more specific and effective medications. Discovering new selective inhibitors targeting only the 17,20-lyase part of CYP17 enzyme activity is paramount important. Most promising strategy might be to target the CYP17 structural elements involved in cytochrome b5 interaction or the part of enzyme in which dynamic changes occurs upon cytochrome b5 binding. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Declared none.

Genetics, Structure, Function, Mode of Actions and Role in Cancer

Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

REFERENCES [1]

[2] [3] [4] [5]

[6] [7] [8]

[9]

[10]

[11] [12]

[13]

[14]

[15]

[16] [17]

[18]

[19]

[20]

[21]

Hanson, E.D.; Hurley, B.F. Intervening on the side effects of hormone-dependent cancer treatment: the role of strength training. J. Aging. Res., 2011, 2011, 903291. Jemal, A.; Bray, F.; Center, M.M.; Ferlay, J.; Ward, E.; Forman, D. Global cancer statistics. CA Cancer J. Clin., 2011, 61 (2), 69-90. Bruno, R.D.; Njar, V.C. Targeting cytochrome P450 enzymes: a new approach in anti-cancer drug development. Bioorg. Med. Chem., 2007, 15 (15), 5047-60. Huggins, C. Endocrine Control of Prostatic Cancer. Science, 1943, 97 (2529), 541-4. Puche, C.; Jose, M.; Cabero, A.; Meseguer, A. Expression and enzymatic activity of the P450c17 gene in human adipose tissue. Eur. J. Endocrinol., 2002, 146 (2), 223-9. Miller, W.L. Structure of genes encoding steroidogenic enzymes. J. Steroid Biochem., 1987, 27 (4-6), 759-66. Wang, F.; Zou, Y.F.; Feng, X.L.; Su, H.; Huang, F. CYP17 gene polymorphisms and prostate cancer risk: a meta-analysis based on 38 independent studies. Prostate, 2011, 71 (11), 1167-77. dos Santos, A.; Ribeiro, M.L.; Mesquita, J.C.; Carvalho-Salles, A.B.; Hackel, C. No association of the 5' promoter region polymorphism of CYP17 gene with prostate cancer risk. Prostate Cancer Prostatic Dis., 2002, 5 (1), 28-31. Huber, J.C.; Schneeberger, C.; Tempfer, C.B. Genetic modelling of the estrogen metabolism as a risk factor of hormone-dependent disorders. Maturitas, 2002, 42 (1), 1-12. Gilep, I.L.; Ivanchikova, N.N.; Rybina, I.L.; Gilep, A.A. Interrelation of structural (/-34) polymorphism of human gene CYP17A1 with biochemical and bioenergetics characteristics of the organism. Vestnik FFR, 2009, 4, 118-125. Li, Y.; Liu, F.; Luo, S.; Hu, H.; Li, X.H.; Li, S.W. Polymorphism T-->C of gene CYP17 promoter and polycystic ovary syndrome risk: a meta-analysis. Gene, 2012, 495 (1), 16-22. Duell, E.J.; Holly, E.A.; Kelsey, K.T.; Bracci, P.M. Genetic variation in CYP17A1 and pancreatic cancer in a population-based case-control study in the San Francisco Bay Area, California. Int. J. Cancer, 2010, 126 (3), 790-5. DeVore, N.M.; Scott, E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001. Nature, 2012, 482 (7383), 116-9. Monno, S.; Ogawa, H.; Date, T.; Fujioka, M.; Miller, W.L.; Kobayashi, M. Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency. J. Biol. Chem., 1993, 268 (34), 25811-7, Fardella, C.E.; Hum, D.W.; Homoki, J.; Miller, W.L. Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17 alpha-hydroxylase deficiency. J. Clin. Endocrinol. Metab., 1994, 79 (1), 160-4. Lin, D.; Harikrishna, J.A.; Moore, C.C.; Jones, K.L.; Miller, W.L. Missense mutation serine106----proline causes 17 alphahydroxylase deficiency. J. Biol. Chem., 1991, 266 (24), 15992-8. Swart, A.C.; Storbeck, K.H.; Swart, P. A single amino acid residue, Ala 105, confers 16alpha-hydroxylase activity to human cytochrome P450 17alpha-hydroxylase/17, 20 lyase. J. Steroid Biochem. Mol. Biol., 2010, 119 (3-5), 112-20. Imai, T.; Globerman, H.; Gertner, J.M.; Kagawa, N.; Waterman, M.R. Expression and purification of functional human 17 alphahydroxylase/17, 20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alphahydroxylase/17, 20-lyase deficiency. J. Biol. Chem., 1993, 268 (26), 19681-9. Ahlgren, R.; Yanase, T.; Simpson, E.R.; Winter, J.S.; Waterman, M.R. Compound heterozygous mutations (Arg 239----stop, Pro 342----Thr) in the CYP17 (P45017 alpha) gene lead to ambiguous external genitalia in a male patient with partial combined 17 alphahydroxylase/17, 20-lyase deficiency. J. Clin. Endocrinol. Metab., 1992, 74 (3), 667-72. Adachi, M.; Tachibana, K.; Asakura, Y.; Yamamoto, T.; Hanaki, K.; Oka, A. Compound heterozygous mutations of cytochrome P450 oxidoreductase gene (POR) in two patients with AntleyBixler syndrome. Am. J. Med. Genet. A, 2004, 128 (4), 333-9. Lee-Robichaud, P.; Akhtar, M.E.; Wright, J.N.; Sheikh, Q.I.; Akhtar, M. The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the

[22]

[23]

[24]

[25]

[26] [27]

[28]

[29]

[30]

[31] [32]

[33] [34] [35]

[36]

[37] [38]

[39]

[40]

[41]

9

enzyme's cytochrome b5-dependent acyl-carbon cleavage activities. J. Steroid Biochem. Mol. Biol., 2004, 92 (3), 119-30. Tiosano, D.; Knopf, C.; Koren, I.; Levanon, N.; Hartmann, M.F.; Hochberg, Z.; Wudy, S.A. Metabolic evidence for impaired 17alpha-hydroxylase activity in a kindred bearing the E305G mutation for isolate 17, 20-lyase activity. Eur. J. Endocrinol., 2008, 158 (3), 385-92. Auchus, R.J.; Miller, W.L. Molecular modeling of human P450c17 (17alpha-hydroxylase/17, 20-lyase): insights into reaction mechanisms and effects of mutations. Mol. Endocrinol., 1999, 13 (7), 1169-82. Lin, C.J.; Martens, J.W.; Miller, W.L. NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17alpha-hydroxylase/17, 20 lyase) in human adrenal NCI-H295A cells. Mol. Endocrinol., 2001, 15 (8), 1277-93. Gilep, A.A.; Sushko, T.A.; Usanov, S.A. At the crossroads of steroid hormone biosynthesis: the role, substrate specificity and evolutionary development of CYP17. Biochim. Biophys. Acta, 2011, 1814 (1), 200-9. Rainey, W.E.; Carr, B.R.; Wang, Z.N.; Parker, C.R., Jr. Gene profiling of human fetal and adult adrenals. J. Endocrinol., 2001, 171 (2), 209-15. Rehman, K.S.; Carr, B.R.; Rainey, W.E. Profiling the steroidogenic pathway in human fetal and adult adrenals. J. Soc. Gynecol. Investig., 2003, 10 (6), 372-80. Montgomery, R.B.; Mostaghel, E.A.; Vessella, R.; Hess, D.L.; Kalhorn, T.F.; Higano, C.S.; True, L.D.; Nelson, P.S. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth. Cancer Res., 2008, 68 (11), 4447-54. Stigliano, A.; Gandini, O.; Cerquetti, L.; Gazzaniga, P.; Misiti, S.; Monti, S.; Gradilone, A.; Falasca, P.; Poggi, M.; Brunetti, E.; Agliano, A.M.; Toscano, V. Increased metastatic lymph node 64 and CYP17 expression are associated with high stage prostate cancer. J. Endocrinol., 2007, 194 (1), 55-61. Bassett, M.H.; Mayhew, B.; Rehman, K.; White, P.C.; Mantero, F.; Arnaldi, G.; Stewart, P.M.; Bujalska, I.; Rainey, W.E. Expression profiles for steroidogenic enzymes in adrenocortical disease. J. Clin. Endocrinol. Metab., 2005, 90 (9), 5446-55. Nakamura, Y.; Gang, H.X.; Suzuki, T.; Sasano, H.; Rainey, W.E. Adrenal changes associated with adrenarche. Rev. Endocr. Metab. Disord., 2009, 10 (1), 19-26. Hall, P.F. Cytochrome P-450 C21scc: one enzyme with two actions: Hydroxylase and lyase. J. Steroid Biochem. Mol. Biol., 1991, 40 (4-6), 527-32. Qin, K.N.; Rosenfield, R.L. Role of cytochrome P450c17 in polycystic ovary syndrome. Mol. Cell Endocrinol., 1998, 145 (1-2), 111-21. Miller, W.L. Androgen biosynthesis from cholesterol to DHEA. Mol. Cell Endocrinol., 2002, 198 (1-2), 7-14, Strauss, J.F. 3rd, Some new thoughts on the pathophysiology and genetics of polycystic ovary syndrome. Ann. N Y Acad. Sci., 2003, 997, 42-8. Ogo, A.; Haji, M.; Ohashi, M.; Nawata, H. Markedly increased expression of cytochrome P-450 17 alpha-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome. Mol. Cell Endocrinol., 1991, 80 (1-3), 83-9. Maitra, A.; Shirwalkar, H. Congenital adrenal hyperplasia: biochemical and molecular perspectives. Indian J. Exp. Biol., 2003, 41 (7), 701-9. Lunn, R.M.; Bell, D.A.; Mohler, J.L.; Taylor, J.A. Prostate cancer risk and polymorphism in 17 hydroxylase (CYP17) and steroid reductase (SRD5A2). Carcinogenesis, 1999, 20 (9), 1727-31. Madigan, M.P.; Gao, Y.T.; Deng, J.; Pfeiffer, R.M.; Chang, B.L.; Zheng, S.; Meyers, D.A.; Stanczyk, F.Z.; Xu, J.; Hsing, A.W. CYP17 polymorphisms in relation to risks of prostate cancer and benign prostatic hyperplasia: a population-based study in China. Int. J. Cancer, 2003, 107 (2), 271-5. Swart, P.; Swart, A.C.; Waterman, M.R.; Estabrook, R.W.; Mason, J.I. Progesterone 16 alpha-hydroxylase activity is catalyzed by human cytochrome P450 17 alpha-hydroxylase. J. Clin. Endocrinol. Metab., 1993, 77 (1), 98-102. Beaudoin, C.; Lavallee, B.; Tremblay, Y.; Hum, D.W.; Breton, R.; de Launoit, Y.; Belanger, A. Modulation of 17alphahydroxylase/17, 20-lyase activity of guinea pig cytochrome

10 Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0

[42]

[43]

[44] [45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

P450c17 by site-directed mutagenesis. DNA Cell Biol., 1998, 17 (8), 707-15. Cloutier, M.; Fleury, A.; Courtemanche, J.; Ducharme, L.; Mason, J.I.; Lehoux, J.G. Characterization of the adrenal cytochrome P450C17 in the hamster, a small animal model for the study of adrenal dehydroepiandrosterone biosynthesis. DNA Cell Biol., 1997, 16 (3), 357-68. Barnes, H.J.; Arlotto, M.P.; Waterman, M.R. Expression and enzymatic activity of recombinant cytochrome P450 17 alphahydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA, 1991, 88 (13), 5597-601. Zuber, M.X.; Simpson, E.R.; Waterman, M.R. Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells. Science, 1986, 234 (4781), 1258-61. Conley, A.J.; Bird, I.M. The role of cytochrome P450 17 alphahydroxylase and 3 beta-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the delta 5 and delta 4 pathways of steroidogenesis in mammals. Biol. Reprod., 1997, 56 (4), 789-99. Tremblay, Y.; Fleury, A.; Beaudoin, C.; Vallee, M.; Belanger, A. Molecular cloning and expression of guinea pig cytochrome P450c17 cDNA (steroid 17 alpha-hydroxylase/17, 20 lyase): tissue distribution, regulation, and substrate specificity of the expressed enzyme. DNA Cell Biol., 1994, 13 (12), 1199-212. Fevold, H.R.; Lorence, M.C.; McCarthy, J.L.; Trant, J.M.; Kagimoto, M.; Waterman, M.R.; Mason, J.I. Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17, 20-lyase reactions. Mol. Endocrinol., 1989, 3 (6), 968-75. Haider, S.M.; Patel, J.S.; Poojari, C.S.; Neidle, S. Molecular modeling on inhibitor complexes and active-site dynamics of cytochrome P450 C17, a target for prostate cancer therapy. J. Mol. Biol., 2010, 400 (5), 1078-98. Pinto-Bazurco Mendieta, M.A.; Negri, M.; Hu, Q.; Hille, U.E.; Jagusch, C.; Jahn-Hoffmann, K.; Muller-Vieira, U.; Schmidt, D.; Lauterbach, T.; Hartmann, R.W. CYP17 inhibitors. Annulations of additional rings in methylene imidazole substituted biphenyls: synthesis, biological evaluation and molecular modelling. Arch. Pharm. (Weinheim), 2008, 341 (10), 597-609. Ghosh, D.; Griswold, J.; Erman, M.; Pangborn, W. Structural basis for androgen specificity and oestrogen synthesis in human aromatase. Nature, 2009, 457 (7226), 219-23. Strushkevich, N.; MacKenzie, F.; Cherkesova, T.; Grabovec, I.; Usanov, S.; Park, H.W. Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system. Proc. Natl. Acad. Sci. USA, 2011, 108 (25), 10139-43. Mast, N.; White, M.A.; Bjorkhem, I.; Johnson, E.F.; Stout, C.D.; Pikuleva, I.A. Crystal structures of substrate-bound and substratefree cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA, 2008, 105 (28), 9546-51. Zhao, B.; Lei, L.; Kagawa, N.; Sundaramoorthy, M.; Banerjee, S.; Nagy, L.D.; Guengerich, F.P.; Waterman, M.R. A Threedimensional Structure of Steroid 21-Hydroxylase (Cytochrome P450 21A2) with Two Substrates Reveals Locations of Diseaseassociated Variants. J. Biol. Chem., 2012. Nakajin, S.; Takahashi, M.; Shinoda, M.; Hall, P.F. Cytochrome b5 promotes the synthesis of delta 16-C19 steroids by homogeneous cytochrome P-450 C21 side-chain cleavage from pig testis. Biochem. Biophys. Res. Commun., 1985, 132 (2), 708-13. Soucy, P.; Lacoste, L.; Luu-The, V. Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor. Role of recombinant cytochrome b5 and P450 reductase. Eur. J. Biochem., 2003, 270 (6), 1349-55. Lee-Robichaud, P.; Wright, J.N.; Akhtar, M.E.; Akhtar, M. Modulation of the activity of human 17 alpha-hydroxylase-17, 20lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications. Biochem. J., 1995, 308 ( Pt 3), 901-8. Guryev, O.; Carvalho, R.A.; Usanov, S.; Gilep, A.; Estabrook, R.W. A pathway for the metabolism of vitamin D3: unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1). Proc. Natl. Acad. Sci. USA, 2003, 100 (25), 14754-9. Slominski, A.; Zjawiony, J.; Wortsman, J.; Semak, I.; Stewart, J.; Pisarchik, A.; Sweatman, T.; Marcos, J.; Dunbar, C.; R, C.T. A

Sushko et al.

[59]

[60]

[61]

[62] [63] [64] [65] [66]

[67] [68]

[69] [70]

[71] [72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

novel pathway for sequential transformation of 7dehydrocholesterol and expression of the P450scc system in mammalian skin. Eur. J. Biochem., 2004, 271 (21), 4178-88. Waterham, H.R.; Wijburg, F.A.; Hennekam, R.C.; Vreken, P.; PollThe, B.T.; Dorland, L.; Duran, M.; Jira, P.E.; Smeitink, J.A.; Wevers, R.A.; Wanders, R.J. Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene. Am. J. Hum. Genet., 1998, 63 (2), 329-38. Slominski, A.T.; Zmijewski, M.A.; Semak, I.; Sweatman, T.; Janjetovic, Z.; Li, W.; Zjawiony, J.K.; Tuckey, R.C. Sequential metabolism of 7-dehydrocholesterol to steroidal 5, 7-dienes in adrenal glands and its biological implication in the skin. PLoS One, 2009, 4 (2), e4309. Slominski, A.T.; Janjetovic, Z.; Fuller, B.E.; Zmijewski, M.A.; Tuckey, R.C.; Nguyen, M.N.; Sweatman, T.; Li, W.; Zjawiony, J.; Miller, D.; Chen, T.C.; Lozanski, G.; Holick, M.F. Products of vitamin D3 or 7-dehydrocholesterol metabolism by cytochrome P450scc show anti-leukemia effects, having low or absent calcemic activity. PLoS One, 2010, 5 (3), e9907. Bhavnani, B.R. The saga of the ring B unsaturated equine estrogens. Endocr. Rev., 1988, 9 (4), 396-416. Gaspard, U. Risks, benefits and costs of hormone replacement therapy in menopause. Rev. Med. Liege., 1998, 53 (5), 298-304. Cox, D. Should a doctor prescribe hormone replacement therapy which has been manufactured from mare's urine? J. Med. Ethics, 1996, 22 (4), 199-203. Taylor, H. S.; Manson, J. E., Update in hormone therapy use in menopause. J. Clin. Endocrinol. Metab., 2011, 96 (2), 255-64. Gupta, M.K.; Guryev, O.L.; Auchus, R.J. 5alpha-reduced C21 steroids are substrates for human cytochrome P450c17. Arch. Biochem. Biophys., 2003, 418 (2), 151-60. Baulieu, E.E.; Robel, P. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as neuroactive neurosteroids. Proc. Natl. Acad. Sci. USA, 1998, 95 (8), 4089-91. Compagnone, N.A.; Mellon, S.H. Dehydroepiandrosterone: a potential signalling molecule for neocortical organization during development. Proc. Natl. Acad. Sci. USA, 1998, 95 (8), 4678-83. Ortiz de Montellano, P.R. Hydrocarbon hydroxylation by cytochrome P450 enzymes. Chem. Rev., 2010, 110 (2), 932-48. Akhtar, M.; Wright, J.N.; Lee-Robichaud, P. A review of mechanistic studies on aromatase (CYP19) and 17alphahydroxylase-17, 20-lyase (CYP17). J. Steroid Biochem. Mol. Biol., 2011, 125 (1-2), 2-12. Akhtar, M.K.; Kelly, S.L.; Kaderbhai, M.A. Cytochrome b(5) modulation of 17{alpha} hydroxylase and 17-20 lyase (CYP17) activities in steroidogenesis. J. Endocrinol., 2005, 187 (2), 267-74. Auchus, R.J.; Lee, T.C.; Miller, W.L. Cytochrome b5 augments the 17, 20-lyase activity of human P450c17 without direct electron transfer. J. Biol. Chem., 1998, 273 (6), 3158-65. Guryev, O.L.; Gilep, A.A.; Usanov, S.A.; Estabrook, R.W. Interaction of apo-cytochrome b5 with cytochromes P4503A4 and P45017A: relevance of heme transfer reactions. Biochemistry, 2001, 40 (16), 5018-31. Ogishima, T.; Kinoshita, J.Y.; Mitani, F.; Suematsu, M.; Ito, A. Identification of outer mitochondrial membrane cytochrome b5 as a modulator for androgen synthesis in Leydig cells. J. Biol. Chem., 2003, 278 (23), 21204-11. Min, L.; Strushkevich, N.V.; Harnastai, I.N.; Iwamoto, H.; Gilep, A.A.; Takemori, H.; Usanov, S.A.; Nonaka, Y.; Hori, H.; Vinson, G.P.; Okamoto, M. Molecular identification of adrenal inner zone antigen as a heme-binding protein. FEBS J., 2005, 272 (22), 583243. Mast, N.; Norcross, R.; Andersson, U.; Shou, M.; Nakayama, K.; Bjorkhem, I.; Pikuleva, I.A., Broad substrate specificity of human cytochrome P450 46A1 which initiates cholesterol degradation in the brain. Biochemistry, 2003, 42 (48), 14284-92. Lund, B.O.; Lund, J. Novel involvement of a mitochondrial steroid hydroxylase (P450c11) in xenobiotic metabolism. J. Biol. Chem., 1995, 270 (36), 20895-7. Suhara, K.; Fujimura, Y.; Shiroo, M.; Katagiri, M. Multiple catalytic properties of the purified and reconstituted cytochrome P450 (P-450sccII) system of pig testis microsomes. J. Biol. Chem., 1984, 259 (14), 8729-36. Niwa, T.; Sato, R.; Yabusaki, Y.; Ishibashi, F.; Katagiri, M. Contribution of human hepatic cytochrome P450s and steroidogenic

Genetics, Structure, Function, Mode of Actions and Role in Cancer

[80]

[81] [82] [83] [84]

[85] [86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

CYP17 to the N-demethylation of aminopyrine. Xenobiotica, 1999, 29 (2), 187-93. Arth, G.E.; Patchett, A.A.; Jefopoulus, T.; Bugianesi, R.L.; Peterson, L.H.; Ham, E.A.; Kuehl, F.A., Jr.; Brink, N.G. Steroidal androgen biosynthesis inhibitors. J. Med. Chem., 1971, 14 (8), 675-9. Gaunt, R.; Steinetz, B.G.; Chart, J.J. Pharmacologic alteration of steroid hormone functions. Clin. Pharmacol. Ther., 1968, 9 (5), 657-81. Vasaitis, T.S.; Bruno, R.D.; Njar, V.C. CYP17 inhibitors for prostate cancer therapy. J. Steroid Biochem. Mol. Biol., 2011, 125 (1-2), 23-31. Brueggemeier, R.W. Aromatase inhibitors--mechanisms of steroidal inhibitors. Breast Cancer Res. Treat., 1994, 30 (1), 31-42. Schuster, I.; Bernhardt, R. Inhibition of cytochromes p450: existing and new promising therapeutic targets. Drug Metab. Rev., 2007, 39 (2-3), 481-99. Baston, E.; Leroux, F.R. Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development. Rec. Pat. Anticancer Drug Discov., 2007, 2 (1), 31-58. Barrie, S.E.; Haynes, B.P.; Potter, G.A.; Chan, F.C.; Goddard, P.M.; Dowsett, M.; Jarman, M. Biochemistry and pharmacokinetics of potent non-steroidal cytochrome P450(17alpha) inhibitors. J. Steroid Biochem. Mol. Biol., 1997, 60 (5-6), 347-51. Potter, G.A.; Barrie, S.E.; Jarman, M.; Rowlands, M.G., Novel steroidal inhibitors of human cytochrome P45017 alpha (17 alphahydroxylase-C17, 20-lyase): potential agents for the treatment of prostatic cancer. J. Med. Chem., 1995, 38 (13), 2463-71. de Bono, J.S.; Logothetis, C.J.; Molina, A.; Fizazi, K.; North, S.; Chu, L.; Chi, K.N.; Jones, R.J.; Goodman, O.B., Jr.; Saad, F.; Staffurth, J.N.; Mainwaring, P.; Harland, S.; Flaig, T.W.; Hutson, T.E.; Cheng, T.; Patterson, H.; Hainsworth, J.D.; Ryan, C.J.; Sternberg, C.N.; Ellard, S.L.; Flechon, A.; Saleh, M.; Scholz, M.; Efstathiou, E.; Zivi, A.; Bianchini, D.; Loriot, Y.; Chieffo, N.; Kheoh, T.; Haqq, C. M.; Scher, H.I. Abiraterone and increased survival in metastatic prostate cancer. N. Engl. J. Med., 2011, 364 (21), 1995-2005. Danila, D.C.; Morris, M.J.; de Bono, J.S.; Ryan, C.J.; Denmeade, S.R.; Smith, M.R.; Taplin, M.E.; Bubley, G.J.; Kheoh, T.; Haqq, C.; Molina, A.; Anand, A.; Koscuiszka, M.; Larson, S.M.; Schwartz, L.H.; Fleisher, M.; Scher, H.I. Phase II multicenter study of abiraterone acetate plus prednisone therapy in patients with docetaxel-treated castration-resistant prostate cancer. J. Clin. Oncol., 2010, 28 (9), 1496-501. Vasaitis, T.; Belosay, A.; Schayowitz, A.; Khandelwal, A.; Chopra, P.; Gediya, L.K.; Guo, Z.; Fang, H.B.; Njar, V.C.; Brodie, A.M. Androgen receptor inactivation contributes to antitumor efficacy of 17{alpha}-hydroxylase/17, 20-lyase inhibitor 3beta-hydroxy-17(1H-benzimidazole-1-yl)androsta-5, 16-diene in prostate cancer. Mol. Cancer Ther., 2008, 7 (8), 2348-57. Huang, P.; Chandra, V.; Rastinejad, F., Structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics. Annu. Rev. Physiol., 2010, 72, 247-72. Porter, T.D.; Kasper, C.B. NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins. Biochemistry, 1986, 25 (7), 1682-7. Porter, T.D. An unusual yet strongly conserved flavoprotein reductase in bacteria and mammals. Trends Biochem. Sci., 1991, 16 (4), 154-8.

Received: June 06, 2012

Revised: January 31, 2013

Accepted: April 12, 2013

Anti-Cancer Agents in Medicinal Chemistry, 2013, Vol. 13, No. 0 [94]

[95]

[96]

[97]

[98]

[99] [100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

11

Iyanagi, T.; Makino, R.; Anan, F.K., Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase. Biochemistry, 1981, 20 (7), 1722-30. Bridges, A.; Gruenke, L.; Chang, Y.T.; Vakser, I.A.; Loew, G.; Waskell, L. Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase. J. Biol. Chem., 1998, 273 (27), 17036-49. Miwa, G.T.; West, S.B.; Huang, M.T.; Lu, A.Y. Studies on the association of cytochrome P-450 and NADPH-cytochrome c reductase during catalysis in a reconstituted hydroxylating system. J. Biol. Chem., 1979, 254 (13), 5695-700. Estabrook, R.W.; Franklin, M.R.; Cohen, B.; Shigamatzu, A.; Hildebrandt, A.G. Biochemical and genetic factors influencing drug metabolism. Influence of hepatic microsomal mixed function oxidation reactions on cellular metabolic control. Metabolism, 1971, 20 (2), 187-99. Yanagibashi, K.; Hall, P.F. Role of electron transport in the regulation of the lyase activity of C21 side-chain cleavage P-450 from porcine adrenal and testicular microsomes. J. Biol. Chem., 1986, 261 (18), 8429-33. Fluck, C.E.; Pandey, A.V.; Huang, N.; Agrawal, V.; Miller, W.L. P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia. Endocr. Dev., 2008, 13, 67-81. Pandey, A.V.; Kempna, P.; Hofer, G.; Mullis, PE.; Fluck, C.E. Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase. Mol. Endocrinol., 2007, 21 (10), 2579-95. Anandatheerthavarada, H.K.; Addya, S.; Mullick, J.; Avadhani, N.G. Interaction of adrenodoxin with P4501A1 and its truncated form P450MT2 through different domains: differential modulation of enzyme activities. Biochemistry, 1998, 37 (4), 1150-60. Sagara, Y.; Barnes, H.J.; Waterman, M.R. Expression in Escherichia coli of functional cytochrome P450c17 lacking its hydrophobic amino-terminal signal anchor. Arch. Biochem. Biophys., 1993, 304 (1), 272-8. Pernecky, S.J.; Larson, J.R.; Philpot, R.M.; Coon, M.J. Expression of truncated forms of liver microsomal P450 cytochromes 2B4 and 2E1 in Escherichia coli: influence of NH2-terminal region on localization in cytosol and membranes. Proc. Natl. Acad. Sci. USA, 1993, 90 (7), 2651-5. Dong, M.S.; Yamazaki, H.; Guo, Z.; Guengerich, F.P. Recombinant human cytochrome P450 1A2 and an N-terminaltruncated form: construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPHcytochrome P450 reductase. Arch. Biochem. Biophys., 1996, 327 (1), 11-9. Pechurskaya, T.A.; Harnastai, I.N.; Grabovec, I.P.; Gilep, A.A.; Usanov, S.A. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s. Biochem. Biophys. Res. Commun., 2007, 353 (3), 598-604. Bene, P.C.E.; Kozlíková, B.; Pavelka, A.; Strnad, O.; Brezovsk , J.; ustr, V.; Klva a, M.; Szabó, T.; Gora, A.; Zamborsk , M.; Biedermannová, L.; Medek P.; Damborsk , J.; Sochor, J. CAVER 2.1. Sofrware, 2010. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem., 2004, 25 (13), 1605-12.