Discovery of novel non-steroidal cytochrome p450 17a1 inhibitors as potential prostate cancer agents

Article abstract of DOI:10.3390/ijms21144868

The current study presents the design, synthesis, and evaluation of novel cytochrome P450 17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces androgens among other steroids, and it is implicated in prostate cancer. T

Full text of DOI:10.3390/ijms21144868

International Journal of
Molecular Sciences
Article
Discovery of Novel Non-Steroidal Cytochrome P450
17A1 Inhibitors as Potential Prostate Cancer Agents
Tomasz M. Wróbel 1,2,
*
, Oksana Rogova 1, Kasper L. Andersen 3 , Rahul Yadav 4,
Simone Brixius-Anderko 4, Emily E. Scott 4,5, Lars Olsen 1,6, Flemming Steen Jørgensen 1 and
Fredrik Björkling 1
1
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2,
DK-2100 Copenhagen, Denmark; oksana.rogova@chem.lu.se (O.R.); lo@sund.ku.dk (L.O.);
fsj@sund.ku.dk (F.S.J.); fb@sund.ku.dk (F.B.)
Department of Synthesis and Chemical Technology of Pharmaceutical Substances,
2
Medical University of Lublin, Chodz´ki 4a, 20093 Lublin, Poland
Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5,
3
DK-2200 Copenhagen, Denmark; kasper.andersen@bric.ku.dk
Department of Medicinal Chemistry, University of Michigan, 428 Church
4
Street, Ann Arbor, MI 48109-1065, USA; ry205@msstate.edu (R.Y.); sbrixius@med.umich.edu (S.B.-A.);
scottee@med.umich.edu (E.E.S.)
Department of Pharmacology, University of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA
5
6
Protein Engineering, Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsvaerd, Denmark
*
Correspondence: tomasz.wrobel@umlub.pl; Tel.: +48-814-487-273
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 June 2020; Accepted: 7 July 2020; Published: 9 July 2020
Abstract: The current study presents the design, synthesis, and evaluation of novel cytochrome P450
17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces
androgens among other steroids, and it is implicated in prostate cancer. The obtained compounds
are potent enzyme inhibitors (sub µM) with antiproliferative activity in prostate cancer cell lines.
The binding mode of these compounds is also discussed.
Keywords: cytochrome P450 17A1; CYP17A1; prostate cancer; enzyme inhibition
1. Introduction
Prostate cancer (PCa) is expected to be the most frequently diagnosed cancer in 2020 and also the
second deadliest in men [
been established since 1941 and treatments aimed at blocking the synthesis of these hormones, so called
androgen deprivation therapies (ADT), were subsequently developed for clinical use [ ]. A notable
example involves usage of gonadotropin releasing hormone (GnRH) agonists and antagonists to
reduce serum testosterone levels known as “chemical castration” [ ]. Unfortunately, resistance to
ADT occurs within a short period of time and PCa evolves into castration resistant prostate cancer
(CRPC), in which the cancer cells produce androgens via de novo and “back door” pathways [ ].
1]. The link between the disease and the production of male hormones has
2
3
4,5
Previous treatments developed against CRPC, such as chemotherapy with docetaxel in combination
with prednisone, only have limited therapeutic efficacy and their use was associated with severe side
effects including anaemia and hepatotoxicity [6]. Thus, suppression of hormone levels remains the key
objective in the treatment of advanced prostate cancer. The cytochrome P450 17A1 (CYP17A1) enzyme
is required for androgen production via both the de novo and “back-door” pathways and has been
identified as the most promising target to block androgen synthesis [2]. Accordingly, both academic
and industrial research groups have focused on the development of CYP17A1 inhibitors, with such
Int. J. Mol. Sci. 2020, 21, 4868; doi:10.3390/ijms21144868
www.mdpi.com/journal/ijms

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efforts leading to the approval of abiraterone acetate (Figure 1) for the treatment of CRPC by the Food
and Drug Administration (FDA) and the European Medicines Agency (EMA) in 2011.
Abiraterone (ABT), an FDA approved drug, was designed based on the pregnenolone structure
with a pyridyl substituent in position 17 [
7]. Other discovery efforts include development of the
abiraterone analogue galeterone (discontinued after phase III clinical trials), and the nonsteroidal
compounds S-orteronel/TAK-700 [8] (terminated after phase III trials) and S-seviteronel/VT 464 [9]
(completed phase II trials).
Figure 1. Structures of the cytochrome P450 17A1 inhibitors.
Crystal structures of CYP17A1 complexed with the steroidal inhibitors reveal that in each case
an sp2 hybridized nitrogen atom on a substituent extending from C17 coordinates directly to the
heme iron [10,11]. In abiraterone and galeterone, the coordinating substituents are pyridine and
benzimidazole respectively. Their steroid cores form a 60◦ angle with the heme plane and both
substrates and inhibitors have the distal, C3 end of the steroid interacting with Asn202 [10].
In our efforts towards the discovery of novel non-steroidal ligands, we wanted to investigate
compounds that have the possibility of binding either with the pyridine or benzimidazole moiety
interacting with the heme group by combining both moieties into the same structure (Figure 2a).
Linking those two moieties with benzene ring provided the most versatile way to reveal structures
which would be similar in size and shape to abiraterone, so that they could be expected to occupy
the same binding pocket. These compounds were assessed for their binding affinity and inhibition of
purified CYP17A1 enzyme and their antiproliferative activity in selected prostate cancer cell lines.
2. Results and Discussion
2.1. Synthesis
Simple non-steroidal analogues were designed which would allow exploration of the effect of a
different substitution pattern of pyridine on enzymatic activity. Compounds where the benzimidazole
fragment was replaced with indole (1e) and the pyridyl group replaced with phenyl (1d) were also
synthesized to allow only one part of a molecule to be able to interact with the heme group. Thus, five
novel compounds (Figure 2b) were prepared where the aniline linker was selected to facilitate synthesis
and provide a symmetrical linker with two nitrogen atoms para to each other. The final compounds
were obtained via the Buchwald–Hartwig cross-coupling of heterocyclic bromides with corresponding
amines, providing a most versatile route to the target compounds [12] (Supplementary Materials S1).
Starting benzimidazole (3a) or indole (3b) was arylated by nucleophilic aromatic substitution with
4-bromofluorobenzene to provide N-substituted bromides (2a and 2b) (Figure 2c) [13]. The subsequent

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C-N bond formation in compounds 1a
,
1b
,
1d, and 1e was then performed with the target aminopyridine
or aniline amino groups. After evaluating several pre-catalysts, tBuXPhos Pd was selected for this
transformation. Compound 1c was approached differently taking advantage of the inhouse availability
of 3-bromopyridine. This coupling required reversing polarity during Buchwald-Hartwig reaction
which was enabled by employing aniline 2d. This aniline derivative was obtained from catalytic
hydrogenation of the corresponding nitro compound (2c), which was prepared similarly to the
bromo analogues.
Figure 2. Compounds in the present study (a) highlighting the pyridine and benzimidazole moieties
connected with the aniline linker (
b
) full structures and isolated yields (c) synthesis scheme with reagents
and conditions: (i) 1-bromo-4-fluorobenzene or 1-fluoro-4-nitrobenzene, K3PO4, DMF, 150 ◦C, (ii) tBuXPhos
Pd G1/G3, tBuXPhos, amine or 3-bromopyridine, tBuONa, tBuOH, RT to 70 ◦C, (iii) 10% Pd/C, MeOH, RT.
2.2. Enzyme Inhibition
The target compounds were initially tested for binding mode and affinity for CYP17A1 using a
spectral binding assay. This assay can distinguish the mode of interaction, with a blue shift of the Soret
absorbance occurring when ligand displaces water from the heme iron to generate a five-coordinate iron
without direct coordination to the heme iron (type I, similar to most substrates) or a red shift of the Soret
peak when the ligand nitrogen replaces the water to directly interact with the six-coordinate iron (type II,
similar to many inhibitors) [14]. All compounds herein displayed type II binding, consistent with direct
binding between the ligand nitrogen and heme iron (Supplementary Materials S2). Comparison of
compounds 1a–1c indicated that the position of the pyridine nitrogen atom is important, with 1c
containing 3-pyridyl (Kd 96 nM) having a 2–3-fold higher affinity than the 2-pyridyl (Kd 420 nM)
or 4-pyridyl (Kd 290 nM) analogues (Table 1). This observation suggests that 3-pyridyl fragment
is important for optimal ligand alignment. This 3-pyridyl fragment is also found in abiraterone.
Interestingly, only somewhat reduced affinity for CYP17A1 was observed for compound 1d, in which
the pyridine is replaced by a phenyl ring and compound 1e, in which the benzimidazole moiety was
replaced with indole (Table 1). This indicates that either end of the molecule appears to be able to
coordinate the iron atom.
Regardless of their binding affinity, all compounds were tested for their ability to inhibit progesterone
17
the low affinity of compounds 1a and 1b, they had high IC50 values, 8
The higher affinity compounds 1c 1d, and 1e were more potent with IC50 values of 0.83
and 0.56 µM, respectively.
α
-hydroxylation by recombinant purified CYP17A1 (Supplementary Materials S2). Consistent with
M and >10 M, respectively.
M, 1.76 M,
µ
µ
,
µ
µ

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Table 1. Binding and enzyme inhibition; ND—not determined; *—data from Ref. [10], **—data from
Ref. [11].
CYP17A1
Kd (nM)
CYP17A1
IC50 (µM)
CYP21A2
IC50 (µM)
CYP3A4
IC50 (µM)
CYP2D6
IC50 (µM)
Compound
1a
290 ± 55
420 ± 70
96 ± 22
150 ± 37
120 ± 34
<100 *
8.06 ± 3.9
>10
ND
ND
ND
>10
ND
>10
-
ND
1b
1c
ND
ND
0.83 ± 0.19
1.76 ± 0.19
0.56 ± 0.10
0.08 **
1.5 ± 0.67
>10
1d
ND
ND
1e
0.19 ± 0.03
2.5 ± 0.60
Abiraterone
Galeterone
Orteronel
-
-
-
-
-
-
<100 *
0.13 **
-
<40 **
0.95 **
-
Since iron-binding heterocycles have the potential to bind and inhibit other human cytochrome
P450 enzymes with similar active site topology, we tested the two most potent CYP17A1 inhibitors,
1c and 1e, against the important drug-metabolizing enzymes CYP3A4 and CYP2D6. In human drug
metabolism, CYP3A4 and CYP2D6 are dominant enzymes and undesired inhibition could potentially
alter the pharmacokinetics and reduce the clearance of co-administered drugs. CYP3A4 is particularly
important in this respect, with a large and flexible active site able to accommodate many different
molecular scaffolds including steroids. At concentrations up to 10
CYP3A4 nifedipine metabolism by 50% (IC50 > 10 M). Similarly, compound 1c poorly inhibited CYP2D6
dextromethorphan metabolism (IC50 > 10 M). Compound 1e did demonstrate moderate inhibition of
CYP2D6 (IC50 = 2.5 M), but this was 4.5-fold weaker than inhibition of CYP17A1. The most similar
µM, neither compound inhibited
µ
µ
µ
steroidogenic human enzyme to CYP17A1 is CYP21A2, which also uses progesterone as a substrate,
in this case to form 21-hydroxyprogesterone. Undesired inhibition of this enzyme could adversely impact
the biosynthesis of the corticosteroids cortisol and aldosterone which control stress/immune responses
and blood pressure, respectively [15]. Abiraterone itself has this liability [16] and is thus co-administered
with the synthetic corticosteroid prednisone [15]. Compound 1e potently inhibited CYP21A2 with a sub
micromolar IC50 (IC50 = 0.19
that compound 1e has the potential to yield significant off-target effects. While compound 1c did not
demonstrate significant inhibition of CYP3A4 or CYP2D6, it inhibited CYP21A2 with an IC50 (1.5 M)
only two-folder higher than CYP17A1 (0.83 M). This finding suggests that retaining the benzimidazole
µM). Combined with moderate inhibition of CYP2D6, these results suggest
µ
µ
moiety would be at least partly beneficial for the compound selectivity. However, at the same time
it might be possible to achieve similar results by altering the nitrogen atom position in the pyridine
fragment in compound 1e. Further optimization of 1c is required to eliminate off-target effects on the
steroidogenic human CYP21A2 enzyme to prevent disruption of glucocorticoid and mineralocorticoid
biosynthesis in vivo. This might be achieved by adding substituents compatible with the CYP17A1 active
site topography forming interactions with the bordering Arg239 and/or Asp298 residues, but incompatible
with the more spatially constrained CYP21A2 active site, as has been accomplished for abiraterone [17].
2.3. Antiproliferative Activity
Next, we tested the effect of compounds 1a
The anti-proliferative activity was determined in vitro by adding 10
–
1e on prostate cancer derived cell lines.
M compound to each of three
µ
prostate cancer cell lines (LNCaP, DU145, and PC3). As an additional comparison, immortalized
non-cancerous fibroblasts (BJ hTERT) were used. These cancer cell lines are considered the classic
standard of PCa cell culture [18]. Since these cell lines grew at very different rates, inhibition
of growth rate (GR) was used as a metric [19]. All compounds except 1e were non-toxic to
non-cancerous cells. Compounds 1a and 1c were both cytotoxic in the PC3 cell line (Figure 3).

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The other compounds caused >50% growth inhibition in this cell line. DU145 cells were weakly
affected by only compound 1e with growth rate inhibition more than 50% (GR < 0.5). A similar level
of growth inhibition was observed for compound 1d
The reference compound abiraterone, a potent CYP17A1 inhibitor, showed low antiproliferative
activity in androgen independent PC3 cells at 10 M. These findings suggest the involvement of
, 1e in LNCaP cells, and 1b and 1d in PC3 cells.
µ
additional mechanisms of action. We observed substantial susceptibility of PC3 cells in comparison
to DU145 (see Supplementary Materials S3 for GR50 in PC3 cells). These two cell lines are similar in
many respects. They are both androgen independent, express lower levels of androgen receptor
(AR) protein compared to LNCaP [20], do not express prostate specific antigen (PSA) or human
glandular kallikrein 1 (hK1) and they have similar doubling times [21]. However, they differ in
their origins, PC3 being derived from a lumbar vertebral metastatic prostate tumour and DU145
from a brain metastatic prostate tumour [21]. Notably, PC3 cells have higher metastatic potential
than DU145 and LNCaP cells [22]. Autophagy is also differently induced among them with DU145
cells being particularly impaired [23]. By observing a uniform response of all cell lines used in the
present study to 5-fluorouracil (5-FU), we were able to rule out any general cell dependent variability
in chemosensitivity towards anti-metabolite treatment. Still, chemoresistance in PC3 and DU145
may be mediated by other mechanisms [24,25]. While AR also functions as a tumour suppressor
inhibiting cell proliferation, it is possible in AR deprived cells to observe growth inhibition when
cells retain necessary coregulators. Indeed, it was shown that PC3, but not DU145 retained these
coregulators to achieve AR tumour suppressor function [26]. It is conceivable that our compounds
can potentially influence this pathway, however no clear mechanism can be derived from this
observation and further research is therefore warranted.
Figure 3. Growth rate inhibition (GR) of 5-fluorouracil (5-FU), Abiraterone (ABT) and compounds 1a
All compounds were tested at 10 M, with DMSO as a control; The sign of the GR value relates directly to
response phenotype: Values between 0 and 1 show partial growth inhibition, a value of 0 equals cytostasis,
and values between 0 and 1 show compounds are cytotoxic. Dunnett’s test used for multiple comparison
to a single control. The stars signify an adjusted p value: ** p < 0.01; *** p < 0.001; **** p < 0.0001.
-e.
µ
−

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2.4. Molecular Modelling
In order to probe possible additional details in how these compounds bind to CYP17A1, all five
compounds 1a 1e were subjected to docking studies. For 1a and 1c, we observed poses with either
–
the pyridine or benzimidazole moiety coordinating to the iron atom in the heme group. The other
compounds showed predominantly only one binding pose. Compounds 1d and 1e with only
one nitrogen atom available for coordination were positioned accordingly and compound 1b was
coordinating with benzimidazole moiety (Supplementary Materials S4).
We submitted each complex for 100 ns molecular dynamics (MD) simulations without any
constraints with different initial velocities to explore if the docking poses represented the optimal
binding mode. The focus of our analyses of the MD trajectories was coordination to the iron atom in
the heme group and the possibility for the interactions with Arg239 and/or Asp298. Simulations of
compounds 1a and 1c, which displayed mixed binding modes, were most revealing and generally
showed a preference for pyridine-iron coordination. This is in agreement with previous DFT calculations
showing stronger heme affinity towards pyridine than benzimidazole [27]. A comparison of the
average free energies of binding calculated by the MM/GBSA method (Table 2) shows that compounds
1a, 1c and 1e should bind better to the CYP17A1 enzymes than 1b and 1d. However compound 1a
suffers a penalty due to unfavourable nitrogen atom position in the pyridine ring which agrees with
the experimentally determined binding (Table 1). It is also noteworthy to add that, although binding
through pyridine nitrogen atom seems to be preferred, the relatively low binding energy of compound
1d together with high affinity demonstrates that binding through benzimidazole cannot be ruled out.
However, in the case of compound 1d, there is additional interaction where NH between the benzene
linker and pyridine ring forms a hydrogen bond to Asp298 and pi-cation interaction between benzene
ring and the charged sidechain of Arg239.
Table 2. Average binding free energy of compounds 1a
n/a—non applicable.
-1e with two possible binding modes.
Compound
Kcal/mol
Kcal/mol
1a
1b
1c
–18.7
–20.8
–18.5
–19.9
n/a
–24.6
n/a
–26.7
n/a
1d
1e
–23.0
3. Materials and Methods
3.1. Synthetic Protocols
All reagents and solvents were used as purchased from commercial sources and reactions were
carried out under anhydrous and air-free conditions under inert atmosphere unless stated otherwise.
Reaction conditions and yields were not optimized. Dry column vacuum chromatography (DCVC)
was performed with silica gel 60 (15–40 µm, Merck KGaA, Darmstadt, Germany). Ion exchange
chromatography was performed on ISOLUTE® MP-TsOH columns (sulfonated macroporous
polystyrene resin, 500 mg, 6 mL, Biotage, Uppsala, Sweden). Ion exchange column was first conditioned
with MeOH (4 mL), then the compound dissolved in MeOH was loaded onto the column, and the
column was washed with MeOH (10 mL) to remove non-basic impurities. Then, 2M solution of
ammonia in MeOH (4 mL) was added to elute the compound from the column and washed with
MeOH (8 mL) for complete compound recovery upon solvent removal.

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3.2. NMR
1H and 13C spectra were recorded on 600 MHz Bruker Avance III HD or 400 MHz WB Bruker
Avance spectrometers (Bruker, Billerica, MA, USA). Coupling constants (J) are reported in Hertz (Hz).
Chemical shifts are reported in parts per million (ppm,
(TMS) or residual solvent peak.
δ scale) relative either to internal standard
3.3. MS
High resolution mass spectroscopy (HRMS) was carried out on a Bruker Solarix XR 7T
ESI/MALDI-FT-ICR mass spectrometer (Bruker, Billerica, MA, USA) with positive MALDI ionization
mode using NaTFA cluster-ions for external calibration. Data obtained were processed in Bruker
DataAnalysis Software version 4.4.
3.4. HPLC
Analytical HPLC was carried out on Dionex UltiMate HPLC system (Thermo-Fisher, Waltham,
MA, USA) consisting of LPG-3400A pump, WPS-3000SL autosampler, and DAD-3000D diode array
detector using Gemini-NX C18 column (4.6 × 250 mm, 3 µm, 110 Å).
Preparative HPLC was carried out on an Dionex UltiMate HPLC system consisting of HPG-3200BX
pump, Rheodyne 9725i injector, 10 mL loop, MWD-3000 detector, and FCA_Multi automated fraction
collector using Gemini-NX C18 (21.2 × 250 mm, 5 µm, 110 Å).
Both analytical (1 mL/min) and preparative (21.2 mL/min) HPLC were performed with gradient
elution, 0–100% solvent B (ACN-H2O-TFA 90:10:0.1) in solvent A (H2O-TFA 100:0.1) over 15 min.
Data were acquired and processed using the Chromeleon Software version 6.80.
3.5. Spectral Binding Assay
Initial ligand binding was evaluated by measuring absorbance changes in CYP17A1 upon addition of
ligands. Purified recombinant human cytochrome P450 17A1 enzyme (1 µM) in buffer (25 mM potassium
phosphate, pH 7.4, 200 mM NaCl, 20% glycerol) was titrated with ligands. Ligands were dissolved in
DMSO and added to three concentrations (500, 1000, and 2000 nM). The spectral intensity difference
(∆A) was measured as difference between minimal absorption (Amax) and maximal absorption (Amin).
The Amin was typically at ~410 nm and Amax at ~440 nm for the type II changes observed for these ligands.
3.6. Cytochrome P450 17A1 17α-Hydroxylation Inhibition Assay
Purified recombinant human CYP17A1 (20 pmol) and human full-length cytochrome P450
reductase (80 pmol) were used to convert progesterone (purchased from Acros Organics, Fair Lawn, NJ,
USA) (6
µ
M) to 17
α
-hydroxyprogesterone. Test inhibitors were dissolved in DMSO and initially tested
M. In subsequent assays the range of inhibitor concentrations
in concentrations ranging from 1.3 to 41
µ
was optimized to best define the IC50 of individual compounds. Reactions were run and the 17
α
-hydroxyprogesterone product measured by LC-MS as previously published [17].
3.7. Inhibition Assays for CYP3A4, CYP2D6, and CYP21A2
Selected inhibitors were tested for off-target inhibition of purified recombinant human CYP3A4
nifedipine metabolism and CYP2D6 dextromethorphan metabolism as described with minor
modifications [28]. CYP3A4 nifedipine (purchased from Sigma-Aldrich, St. Louis, MO, USA) activity
assays were carried out with 200
from Sigma-Aldrich, St. Louis, MO, USA) assays were carried out with 100
steroidogenic cytochrome P450 21A2 progesterone 21-hydroxylation was evaluated as reported [17].
µM nifedipine for 30 min. The CYP2D6 dextrometorphan (purchased
µM DXM. Inhibition of

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3.8. Prostate Cancer Cell Line Proliferation Screening
LNCaP, PC-3, and DU-145 prostate cancer cells were propagated in RPMI-1640, GlutaMAX + 25 mM
HEPES (Gibco) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (P/S) (Gibco) and
10% (LNCaP) or 6% (PC-3 and DU-145) fetal bovine serum (FBS) (HyClone). Non-cancerous BJ fibroblast
cells immortalized with expression of hTERT cells were propagated in DMEM GlutaMAX + 4.5 g/L
D-glucose and pyruvate supplemented with 10% FBS and P/S.
Cells were grown to approximately 80% confluence in 150 mm culture dishes and harvested by
0.25% Trypsin/EDTA (Gibco) treatment. Released cells were counted and seeded in 384 well plates
(Falcon, ref. 353962) in 30 µl media with a cell count of 1500 (BJ cells, PC-3) or and 3500 (LNCaP
(Lund)) cells per well. Liquid handling steps involving cell seeding and staining were performed on a
MicroLab STARlet liquid handling workstation with a CO-RE 384 probe head (Hamilton Company).
The cells incubated for 24 h, after which cell counts in wells of one 384-well plate were determined
as described below (time zero). In parallel, the indicated CYP compounds (10 mM stock in DMSO)
or DMSO alone were delivered to wells in the remaining plates (30 nL) by acoustic droplet ejection
using an Echo 550 Liquid Handler (Labcyte) for a final concentration of 10 µM compound and 0.1%
DMSO. Alternatively, compound and DMSO were added in combination to achieve dilution series all
with a final 0.1% DMSO concentration. Cells were then incubated for an additional 48 h. Cells were
live-stained for 30 min. using 1:1200 dilutions of Höechst (Thermo Fisher Scientific 33342; 10 mg/mL)
and propidium iodide (PI; 2 mg/mL) for assessment of total and dead cell numbers, respectively. Image
acquisition was done using the automated microscope InCell Analyzer 2200 (GE Healthcare) with four
fields (10 objective) acquired per well. Image analysis was performed using the InCell Analyzer
×
2200 Workstation 3.7.3 software (GE Healthcare) where nuclei were segmented by the Höechst signal
(total number of cells) and the mean PI intensity in the nuclei was used to gate live and dead cells
(PI-negative cells: live cells). The number of live cells per well from three independent plates were
determined (compounds n = 3; DMSO wells n = 15–30) and normalized growth rate inhibition (GR)
metrics were calculated according to Hafner et al. 2016 [19]. GR50 curves were visualized using the
GR-calculator webserver (www.grcalculator.org) [29].
3.9. Molecular Modelling
The Protein Preparation Wizard in Maestro Software version 11.1 was used to prepare the proteins
structures [30]. The cytochrome P450 17A1 structures were obtained from Protein Data Bank [31
]
(PDB protein codes 3SWZ [10] and 5IRQ [11]). Bond orders were assigned, hydrogens were added,
and zero-order bonds to metals were created. For protein structures the A chains were selected, and all
water molecules were removed. The formal charge on heme iron was set to +3 and non-protonated
ligand state was used. The hydrogen bonding network was optimized at pH 7.0. A restrained protein
minimization was performed using OPLS3 [32] force field with convergence of heavy atoms to RMSD
0.30 Å. Ligands preparation was performed with LigPrep in Maestro [30]. Possible tautomers and
protonation states were generated at pH 7.0
±
2.0. The Epik program was used to predict pKa values of
ligands [33]. Docking was performed with GOLD (Genetic Optimisation for Ligand Docking) program
version 5.6 [34]. Proteins prepared by Protein Preparation Wizard were used without additional
modifications in GOLD. The co-crystalized ligand was extracted, and the binding site was defined
around the center of the mass of the co-crystalized ligand within 15 Å. Ligands prepared by LigPrep
were exported from Maestro. Ligands were docked 10 times with slow genetic algorithm and with
ChemScore as the scoring function [35]. For constrained docking the distance between the heme iron
and the atom expected to be coordinated to Fe was constrained between 1.5 and 3.5 Å.
The Desmond system builder was used to create the molecular dynamics (MD) systems with the
protein-ligand complex embedded in a SPC water model yielding an orthorhombic box with a buffer
size of 10 Å between the protein and the box boundary. The final system comprised close to 70,000 atoms
including approximately 7500 atoms for the protein including the heme group, 36 atoms for the ligand
(in the case of 1a), one chloride ion to neutralize the system, and approximately 21,000 water molecules.

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The MD simulations were performed with the Desmond program (version 3.6) using the OPLS-2005
force field [36]. For equilibration of the system prior to the production runs, the Desmond default
equilibration protocol was used. Subsequently, the systems were simulated for 100 ns and 1000 frames
collected. A total of 36 simulations were performed based on the 3SWZ and 5IRQ protein structures
combined with the different poses from the GOLD dockings. For each of the MD simulations the
free energy of binding were determined by the Molecular Mechanics/Generalized Born Surface Area
(MM/GBSA) method [37] using the thermal_mmgbsa.py script provided as part of the Schrodinger
software system [38]. The values listed in Table 2 are averages of 4–6 determinations each based on a
100 ns MD simulation.
4. Conclusions
In summary, a set of early hit molecules based on a benzimidazole/indole scaffold were found
to have sub-micromolar inhibitory activity for CYP17A1 with two most potent molecules being 1e
IC50 = 0.56 µM and 1c IC50 = 0.83 µM. Our compounds incorporate a novel, non-steroidal scaffold and,
due to a simple chemistry, allow for the rapid generation of a library of easily accessible analogues.
Compound 1c displayed favourable inhibitory selectivity against other drug-metabolizing CYP
family enzymes, however it demonstrated only two-fold selectivity for CYP17A1 over CYP21A2
inhibition. Furthermore, compound 1c displayed high cytotoxicity towards PC3 cancer cells while
maintaining remarkably non-toxicity towards fibroblasts. This constitutes a starting point for further
optimization towards small molecule therapeutics for the treatment of CRPC.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/14/
4868/s1.
Author Contributions: Conceptualization, T.M.W.; Formal analysis, T.M.W., K.L.A., R.Y., S.B.-A., E.E.S. and
F.S.J.; Funding acquisition, T.M.W. and E.E.S.; Investigation, T.M.W., O.R., K.L.A., R.Y., S.B.-A., F.S.J. and F.B.;
Methodology, T.M.W., O.R., K.L.A., R.Y., S.B.-A., L.O. and F.S.J.; Supervision, F.B. and E.E.S.; Writing—original
draft, T.M.W.; Writing—review & editing, T.M.W., K.L.A., R.Y., S.B.-A., E.E.S., F.S.J. and F.B. All authors have read
and agree to the published version of the manuscript.
Funding: This research was funded by the Narodowe Centrum Nauki grant Miniatura 3—2019/03/X/NZ7/00900
(T.M.W.). Production of the CYP17A1 and CYP21A2 enzymes and development of the corresponding assays
was supported by the NIH grant R01 GM102505 (E.E.S.). Production of the CYP3A4 and CYP2D6 enzymes and
development of the corresponding assays was supported by NIH grant R37 GM076343 (E.E.S.). Biochemical
evaluation of the compounds herein was supported by startup funds from the University of Michigan (E.E.S.).
Acknowledgments: Malika Godamudunage and Aaron Bart generously provided the CYP3A4 and CYP2D6
enzymes, respectively. We would like to thank Henning Gram Hansen, Linda Elin Pålsson, and Cornelia Steinhauer
from the HT Cell Based Screens core facility, BRIC, University of Copenhagen, for assistance with automated liquid
handling and image-based cell quantification. Many thanks go to Agnieszka Kaczor for her ongoing support.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
PCa
prostate cancer
ADT
CRPC
AR
androgen deprivation therapy
castration resistant prostate cancer
androgen receptor
PSA
hK1
MD
prostate specific antigen
human glandular kallikrein 1
molecular dynamics
ABT
5-FU
GnRH
abiraterone
5-fluorouracil
gonadotropin releasing hormone

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