ChemComm COMMUNICATION
Cite this: Chem. Commun., 2017, 53, 3118 Received 28th October 2016, Accepted 16th February 2017
Structure-based optimisation of non-steroidal cytochrome P450 17A1 inhibitors† Morten Larsen,a Cecilie H. Hansen,b Tobias B. Rasmussen,b Julie Islin,b Bjarne Styrishave,b Lars Olsena and Flemming Steen Jørgensen*a
DOI: 10.1039/c6cc08680b rsc.li/chemcomm
Five new non-steroidal inhibitors for cytochrome P450 17A1 (CYP17A1) were identified by structure-based optimisation from a recently identified selective CYP17A1 inhibitor. The compounds are nanomolar inhibitors of steroidogenesis measured in recombinant CYP17A1 and in H295R cells.
Cytochrome P450 17A1 (CYP17A1) is an enzyme involved in human steroidogenesis by converting pregnenolone to dehydroepiandrosterone and progesterone to androstenedione by two subsequent reactions, 17a-hydroxylase and C17,20-lyase reactions. Both the CYP17A1 mediated hydroxylase and lyase reactions are essential for the biosynthesis of androgens and oestrogens.1 In castration-resistant prostate cancer (CRPC), androgens produced by the tumour and/or the adrenal gland drive disease progression.2,3 Thus, CYP17A1 inhibition represents an obvious strategy for the treatment of hormone-dependent tumours such as prostate cancer.4–6 Presently, a handful of compounds are in different stages of clinical development,7 but only one compound, abiraterone, has until now been approved by FDA for the treatment of CRPC. It is anticipated that several of the undesirable side effects of abiraterone are caused by its steroid-like structure and accordingly possible interaction with other receptors.8 Recently, we identified two novel non-steroidal and selective CYP17A1 inhibitors by virtual screening.9 Here, we report the structural optimisation of one of these inhibitors, compound 1, by structure-based approaches, yielding five new inhibitors 2–6 (see Fig. 1). The 3D structures of human CYP17A1 in complex with the inhibitors abiraterone and galeterone have been determined experimentally. Abiraterone and galeterone both contain a steroidal scaffold with a pyridin-3-yl and 1H-benzimidazole-1yl moiety, respectively, in position 17 that coordinates to the heme iron.10 Compound 1, which also contains a pyridin-3-yl
a
Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark. E-mail:
[email protected] b Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark † Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc08680b
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Fig. 1
CYP17A1 inhibitors.
moiety, inhibits CYP17A1 with IC50 values of 230 nM and 500 nM for the 17a-hydroxylase and C17,20-lyase reactions, respectively.9 The binding mode of compound 1 was determined by docking and further refined by QM/MM optimization (see Fig. 2).9 Compound 1 is a relatively non-polar compound with no hydrogen-bonding possibilities and, accordingly, no polar enzyme–inhibitor interactions were observed. In order to explore the CYP17A1 active site in detail and identify additional interaction possibilities for new inhibitors, we performed a SiteMap analysis11 of CYP17A1 to identify favourable regions for hydrogen bonding atoms (donors as well as acceptors) and hydrophobic groups. This analysis revealed that it would be favourable to place hydrogen bonding donors and acceptors close to the ASP-298 and ARG-239 side chains as well as to the ALA-105 backbone carbonyl oxygen. The analysis also showed that hydrophobic substituents could be favourably added ‘‘on-the-other-side’’ of compound 1, i.e. in positions 2, 3 and 4 on the phenyl ring, and in position 5 on the pyridin-3-yl moiety.
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Fig. 2 SiteMap of the binding pocket of CYP17A1 with the residues ALA-105, ARG-239 and ASP-298 shown as sticks and with C atoms of compound 1 in cyan. The hydrogen bond acceptor and donor sites are contoured at 12 kJ mol 1 and the hydrophobic sites at 1 kJ mol 1, respectively.
Based on the SiteMap analysis, we searched compound databases12,13 for compounds (Fig. 1) with substituents containing different hydrogen bonding groups on the phenyl ring to interact with ASP-298, ARG-239 and/or ALA-105. After docking these compounds into the active site of CYP17A1, three compounds, 2, 5 and 6, were selected for further analysis (Fig. 3). Compound 2, with an amino group in the meta position, acting as a hydrogen bond donor interacts with the negatively charged side chain of ASP-298 and with the backbone carbonyl oxygen of ALA-105. Compounds 5 and 6 both contain two hydroxy groups, but only the meta and ortho hydroxy groups form hydrogen bonds with the ASP-298 side chain. The SiteMap analysis also revealed the possibility of adding hydrophobic substituents on the phenyl ring. One obvious possibility was to increase the existing substituents on the original inhibitor, compound 1, by replacing the methyl groups with methoxy groups, yielding compound 4. In the crystal structures of abiraterone (3ruk) and galeterone (3swz) complexes, the C2 methylene and the 19-methyl groups of the steroid part of the
Fig. 3
ChemComm
molecules fill this hydrophobic pocket. We considered expanding the phenyl ring with a substituted napthyl moiety, but since adding aromatic rings generally reduces bioavailability,14 we settled for a heterocyclic system, the 1H-indol-3-yl moiety, yielding compound 3. The binding modes of the five compounds, 2–6, were determined by docking into the active site of CYP17A1 using the abirateroneCYP17A1 structure (3ruk).10 The docking was performed using the GOLD program15 by applying the scoring function developed for heme-containing proteins.16 We have previously used thresholds of about 50 kJ mol 1 (or better) for the ChemScore and less than 13 kJ mol 1 in strain to identify CYP17A1 inhibitors.9 Compound 2 has a more favourable ChemScore compared to the other two compounds with hydrophilic substituents (5 and 6) and binds in a nearly strain free conformation (see Table 1). Compound 3 had a ChemScore comparable to the best of the hydrogen bonding compounds, compound 2, and a low conformational energy, whereas compound 4 displayed less favourable energies (see Table 1). The five compounds, 2–6, were subjected to short (20–40 ns) molecular dynamics (MD) simulations to allow the enzyme to adapt to the ligand and vice versa. The RMSD values for all five ligands were less than 1 Å compared to the initial pose, indicating that the ligands maintain the binding mode identified by docking. The RMSD values for the protein backbone were in the range of 3–4 Å, as also observed previously by Xiao et al.17 in an MD simulation of the CYP17A1–galeterone complex. The compounds with hydrogenbonding possibilities, 2, 5 and 6, all maintain the direct hydrogen bonding found in the docking poses and, additionally, establish hydrogen bonding to ASN-202 (6), ALA-105 and ASP-298 (5) (see Table 1), thereby enabling both hydroxyl groups to participate in hydrogen bonding with the protein. Water-mediated hydrogen bonds were also observed, but were always present in less than 50% of the MD simulation (see the ESI† for further details). The direct effects of compounds 2–6 on both the 17a-hydroxylase and C17,20-lyase reactions were determined and compared to compound 1 using a purified enzyme system. The IC50 values for the 17a-hydroxylase reaction were determined using progesterone as a substrate and for the C17,20-lyase reaction with both progesterone and 17-hydroxyprogesterone as substrates (Table 2). All five compounds, 2–6, inhibited both the 17a-hydroxylase
Compounds (2, 5 and 6) docked into CYP17A1. Hydrogen bonds with residues ALA-105, ARG-239 and ASP-298 are highlighted.
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Table 1 Calculated characteristics for compounds 1–6. ChemScore and conformational energy (DEconf) values for the best docked pose and percentages of intermolecular hydrogen bonds during 10 ns molecular dynamics simulation. See the ESI for computational details
Compound 1 2 3 4 5 6
ChemScore (kJ mol 1) 48.9 53.2 50.1 46.2 46.2 43.1
DEconf (kJ mol 1)
ALA-105 (%)
ARG-239 (%)
ASP-298 (%)
ASN-202 (%)
3.7 0.7 1.4 9.5 2.3 7.3
— 64 — — 50 —
— — — — 70 —
— 71 — — 99 96
— — — — — 99
Table 2 Biological characterization, IC50 values (nM) and standard deviations, of compounds 1–6 in recombinant human CYP17A1 and in H295R cells. See the ESI for experimental details
System
Recombinant CYP17A1 a
H295R cells a
Compound
Hydroxylase
Lyase
1 2 3 4 5 6
641 999 964 2836 4710 95
1274 738 739 1567 4100 73
26 169 200 770 1697 19
b
Lyase 34 191 273 1411 1279 19
460 230 291 723 261 33
Hydroxylase 22 134 87 337 159 9
830 1480 1053 686 1567 2738
80 f 1378 32 465 93 376
ANc
Lyase 94 132 43 190 775 834
30 f 13 3 119 319 478
67 247 178 106 1332 899
DHEAd
4 7 15 20 347 NA
92 534 235 186 1426 1411
TEe 11 76 11 37 390 538
52 181 151 93 1358 235
a Assay based on conversion of progesterone. b Assay based on conversion of 17-hydroxyprogesterone. c AN = androstenedione. dehydroepiandrosterone. e TE = testosterone. f Data from ref. 9.
and C17,20-lyase reactions with a preference, or selectivity, for the C17,20-lyase reaction by a factor of 3–4. Compound 5 is the most selective, but also the least active. Compound 6 is the most active compound being more than 10 fold more active than compound 1 in inhibiting the C17,20-lyase reaction. Finally, we tested the five compounds, 2–6, in an H295R cell assay. The human H295R adrenocortical carcinoma cell line represents the most complete in vitro system mimicking the biosynthesis of androgens and oestrogens by expressing all the key enzymes and metabolites in adrenal and gonadal steroidogenesis.18 The H295R cell assay allows a simultaneous quantification of several steroid hormones and, thus, determination of the disrupting activities of compounds on the key enzymes involved in steroidogenesis,19,20 and has previously been used for characterising CYP17A1 inhibitors for their potential as drugs against CRPC.21 The in vitro tests of the five compounds, 2–6, in the H295R cell assay showed that all the compounds penetrated the cell membrane and resulted in dose-dependent effects on the steroidogenesis (Fig. 4). Treatment of prostate cancer with abiraterone is due to its ability, via inhibition of CYP17A1, to suppress androgen production.22 All five compounds, 2–6, show increased levels of pregnenolone and progesterone, demonstrating CYP17A1 hydroxylase inhibition, and a clear suppression of the androgens androstenedione and DHEA and, consequently, a decrease in testosterone. The relative changes in the production of the hydroxylated progestagens also reveal significant lyase inhibition. Fig. 4 also shows the relative changes in the product/ substrate ratios for the hydroxylase and lyase reactions, respectively. The inhibition constants for the five compounds are listed in Table 2 and dose–response curves are shown in Fig. 4 and Fig. S1 (ESI†). Compounds 2 and 3 have a 10–20 fold preference for C17,20-lyase inhibition relative to 17a-hydroxylation, whereas a somewhat smaller preference is observed for the remaining
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d
4 22 17 11 320 NA
DHEA =
compounds (cf. Fig. 4). The parent compound 1 had a preference for lyase inhibition.9 The response observed in Fig. 4 is a classic steroidogenesis response to a CYP17 inhibitor. It is due to the fact that the CYP17 carries out two reactions, the 17a-hydroxylase and the C17,20-lyase reactions. For CYP17 inhibitors, the steroids on the hydroxylase axis (pregnenolone to aldosterone) always increase. For very selective hydroxylase inhibitors, 17-hydroxysteroids will then decrease, and androgens will follow, due to sequential inhibition. However, some CYP17 inhibitors are not completely selective towards the hydroxylase and will also inhibit the C17,20-lyase reaction, as in the present study. In those cases, all four progestagens may increase. The relative inhibition between the 17a-hydroxylase and C17,20-lyase reactions is drug specific, and will determine the shape of the curve for the 17-hydroxysteroids. Comparison of the dose–response curves (cf. Fig. 4) showed that the hydrophilic compounds 5 and 6 were less potent, whereas compound 2 and the hydrophobic compounds 3 and 4 were more active by a factor of five in the cellular assay, and compound 6 appears to be the most potent inhibitor in the purified enzyme assay. The recombinant CYP17A1 assay confirms the very important observation that the C17,20-lyase reaction is more sensitive to all five compounds, compared to the 17a-hydroxylase reaction. This may provide a possibility of obtaining inhibitors that are more selective towards the C17,20-lyase than the 17a-hydroxylation reaction. This is important since the C17,20-lyase reaction is one of the last steps in the androgen formation while strong inhibition of the 17a-hydroxylation may lead to severe side-effects due to corticoid deficiency.23 Furthermore, the present study shows that it is possible using structure-based methods to identify inhibitors of CYP17A1, and that small variations in inhibitor structures have significant
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Fig. 4 Inhibition of steroidogenesis for compounds 2–6. Left panel: Steroid production in the H295R cell line. Middle panel: Combined d4 and d5 product/substrate ratios for the H295R CYP17A1 a-hydroxylations (full line, closed symbols) and 17-lyase reactions (dotted lines, open symbols). Right panel: Inhibition of the recombinant CYP17A1 a-hydroxylation (full line, closed circles) and the subsequent 17-lyase reaction using progesterone as the substrate (dotted lines, open triangles) and the 17-lyase reaction using 17OH-progesterone as the substrate (dotted lines, open circles). x-axis: concentration of compound in mM; y-axis: relative concentration of the steroids in percent of the solvent control; loq: limit of quantification; hsd: hydroxysteroid dehydrogenase. All values are mean values. For experimental details, see the ESI.† Note the exponential y-axis for progesterone.
effects on the steroidogenesis. We have based our strategy on the CYP17A1 structure in complex with abiraterone. A 3D structure of one of the above-mentioned compounds bound to CYP17A1 would likely pave the way for a further optimization of this class of non-steroidal CYP17A1 inhibitors.
Notes and references 1 2 3 4 5 6 7 8 9 10 11 12
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