Matthias Negri

17β-Hydroxysteroid dehydrogenases (17β-HSDs) as therapeutic targets: protein structures, functions, and recent progress in inhibitor development.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) are oxidoreductases, which play a key role in estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Up to now, 14 different subtypes have been identified in mammals, which catalyze NAD(P)H or NAD(P)(+) dependent reductions/oxidations at the 17-position of the steroid. Depending on their reductive or oxidative activities, they modulate the intracellular concentration of inactive and active steroids. As the genomic mechanism of steroid action involves binding to a steroid nuclear receptor, 17β-HSDs act like pre-receptor molecular switches. 17β-HSDs are thus key enzymes implicated in the different functions of the reproductive tissues in both males and females. The crucial role of estrogens and androgens in the genesis and development of hormone dependent diseases is well recognized. Considering the pivotal role of 17β-HSDs in steroid hormone modulation and their substrate specificity, these proteins are promising therapeutic targets for diseases like breast cancer, endometriosis, osteoporosis, and prostate cancer. The selective inhibition of the concerned enzymes might provide an effective treatment and a good alternative to the existing endocrine therapies. Herein, we give an overview of functional and structural aspects for the different 17β-HSDs. We focus on steroidal and non-steroidal inhibitors recently published for each subtype and report on existing animal models for the different 17β-HSDs and the respective diseases. Article from the Special issue on Targeted Inhibitors.

Design, Synthesis, Biological Evaluation and Pharmacokinetics of Bis(hydroxyphenyl) substituted Azoles, Thiophenes, Benzenes, and Aza-Benzenes as Potent and Selective Nonsteroidal Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1)

17beta-Estradiol (E2), the most potent female sex hormone, stimulates the growth of mammary tumors and endometriosis via activation of the estrogen receptor alpha (ERalpha). 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which is responsible for the catalytic reduction of the weakly active estrogen estrone (E1) into E2, is therefore discussed as a novel drug target. Recently, we have discovered a 2,5-bis(hydroxyphenyl) oxazole to be a potent inhibitor of 17beta-HSD1. In this paper, further structural optimizations were performed: 39 bis(hydroxyphenyl) azoles, thiophenes, benzenes, and aza-benzenes were synthesized and their biological properties were evaluated. The most promising compounds of this study show enhanced IC 50 values in the low nanomolar range, a high selectivity toward 17beta-HSD2, a low binding affinity to ERalpha, a good metabolic stability in rat liver microsomes, and a reasonable pharmacokinetic profile after peroral application. Calculation of the molecular electrostatic potentials revealed a correlation between 17beta-HSD1 inhibition and the electron density distribution.

New Insights into the SAR and Binding Modes of Bis(hydroxyphenyl)thiophenes and -benzenes: Influence of Additional Substituents on 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1) Inhibitory Activity and Selectivity

17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is responsible for the catalytic reduction of weakly active E1 to highly potent E2. E2 stimulates the proliferation of hormone-dependent diseases via activation of the estrogen receptor alpha (ERalpha). Because of the overexpression of 17beta-HSD1 in mammary tumors, this enzyme should be an attractive target for the treatment of estrogen-dependent pathologies. Recently, we have reported on a series of potent 17beta-HSD1 inhibitors: bis(hydroxyphenyl) azoles, thiophenes, and benzenes. In this paper, different substituents are introduced into the core structure and the biological properties of the corresponding inhibitors are evaluated. Computational methods and analysis of different X-rays of 17beta-HSD1 lead to identification of two different binding modes for these inhibitors. The fluorine compound 23 exhibits an IC(50) of 8 nM and is the most potent nonsteroidal inhibitor described so far. It also shows a high selectivity (17beta-HSD2, ERalpha) and excellent pharmacokinetic properties after peroral application to rats.

Novel Aldosterone Synthase Inhibitors with Extended Carbocyclic Skeleton by a Combined Ligand-Based and Structure-Based Drug Design Approach

Pharmacophore modeling of a series of aldosterone synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)-N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors (e.g., 17, IC50 = 2.7 nM) with pronounced selectivity toward the most important steroidogenic and hepatic CYP enzymes.

Novel CYP17 inhibitors: synthesis, biological evaluation, structure-activity relationships and modelling of methoxy- and hydroxy-substituted methyleneimidazolyl biphenyls.

Recently, the steroidal CYP17 inhibitor Abiraterone entered phase II clinical trial for the treatment of androgen-dependent prostate cancer. As 17alpha-hydroxylase-17,20-lyase (CYP17) catalyzes the last step in androgen biosynthesis, inhibition of this target should affect not only testicular but also adrenal androgen formation. Therefore CYP17 inhibitors should be advantageous over existing therapies, for example with GnRH analogues. However, steroidal drugs are known for side effects which are due to affinities for steroid receptors. Therefore we decided to synthesize non-steroidal compounds mimicking the natural CYP17 substrates pregnenolone and progesterone. The synthesis and biological evaluation of a series of 15 novel and highly active non-steroidal CYP17 inhibitors are reported. The compounds were prepared via Suzuki-cross-coupling, Grignard reaction and CDI-assisted S(N)t-reaction with imidazole and their inhibitory activity was examined with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were further tested for their selectivity against the hepatic enzyme CYP3A4 and the glucocorticoid-forming enzyme CYP11B1. All compounds turned out to be potent CYP17 inhibitors. The most active compounds 7 and 8 were much more active than Ketoconazole showing activity comparable to Abiraterone (IC(50) values of 90 and 52nM vs. 72nM). Most compounds also showed higher selectivities than Ketoconazole, but turned out to be less selective than Abiraterone. Docking studies using our CYP17 protein model were performed with selected compounds to study the interactions between the inhibitors and the amino acid residues of the active site.

Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17α-hydroxylase-17.20-lyase (CYP17)-Part II: Core rigidification and influence of substituents at the methylene bridge

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.

Overcoming Undesirable CYP1A2 Inhibition of Pyridylnaphthalene-Type Aldosterone Synthase Inhibitors: Influence of Heteroaryl Derivatization on Potency and Selectivity

Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats.

Synthesis, Biological Evaluation, and Molecular Modeling of Abiraterone Analogues: Novel CYP17 Inhibitors for the Treatment of Prostate Cancer

Abiraterone, a steroidal cytochrome P450 17alpha-hydroxylase-17,20-lyase inhibitor (CYP17), is currently undergoing phase II clinical trials as a potential drug for the treatment of androgen-dependent prostate cancer. Since steroidal compounds often show side effects attributable to their structure, we have tried to replace the sterane scaffold by nonsteroidal core structures. The design and synthesis of 20 new abiraterone mimetics are described. Their activities have been tested with recombinant human CYP17 expressed in E. coli. Promising compounds were further evaluated for selectivity against CYP11B1, CYP11B2, and the hepatic CYP3A4. Compounds 19 and 20 showed comparable activity to abiraterone (IC50 values of 144 and 64 nM vs 72 nM) and similar or even better selectivity against the other CYP enzymes. Selected compounds were also docked into our homology model, and the same binding modes as for abiraterone were found.

Fine-tuning the selectivity of aldosterone synthase inhibitors: structure-activity and structure-selectivity insights from studies of heteroaryl substituted 1,2,5,6-tetrahydropyrrolo[3,2,1-ij]quinolin-4-one derivatives.

Pyridine substituted 3,4-dihydro-1H-quinolin-2-ones (e.g., 1-3) constitute a class of highly potent and selective inhibitors of aldosterone synthase (CYP11B2), a promising target for the treatment of hyperaldosteronism, congestive heart failure, and myocardial fibrosis. Among these, ethyl-substituted 3 possesses high selectivity against CYP1A2. Rigidification of 3 by incorporation of the ethyl group into a 5- or 6-membered ring affords compounds with a pyrroloquinolinone or pyridoquinolinone molecular scaffold (e.g., 4 and 5). It was found that these molecules are even more potent and selective CYP11B2 inhibitors than their corresponding open-chain analogues. Moreover, pyrroloquinolinone 4 exhibits no inhibition of the six most important hepatic CYP enzymes as well as a bioavailability in the range of the marketed drug fadrozole. The SAR studies disclose that subtle changes in the heterocyclic moiety are responsible for either a strong or a weak inhibition of the highly homologous 11β-hydroxylase (CYP11B1). These results are not only important for fine-tuning the selectivity of CYP11B2 inhibitors but also for the development of selective CYP11B1 inhibitors that are of interest for the treatment of Cushings syndrome and metabolic syndrome.

The Role of Fluorine Substitution in Biphenyl Methylene Imidazole-Type CYP17 Inhibitors for the Treatment of Prostate Carcinoma

It has been established that the growth of most prostate carcinomas depends on androgen stimulation. The inhibition of cytochrome P450‐17 (CYP17) to block androgen biosynthesis is therefore regarded as a promising approach to therapy. Based on our previously identified lead compound Ref 1, a series of fluorine‐substituted biphenyl methylene imidazoles were designed, synthesized, and evaluated as CYP17 inhibitors to elucidate the influence of fluorine on in vitro and in vivo activity. It was found that meta‐fluoro substitution at the C ring improved activity, whereas ortho substitution decreased potency. Docking studies performed with our human CYP17 homology model suggest the presence of multipolar interactions between fluorine and Arg109, Lys231, His235, and Glu305. As expected, introduction of fluorine also prolonged the half‐life in plasma. The SARs obtained confirm the reliability of the protein model; compound 9 (IC50=131 nM) was identified as a strong CYP17 inhibitor, showing potent activity in rat, high bioavailability, and a long plasma half‐life: 12.8 h.