Multistep Binding of the Non-Steroidal Inhibitors Orteronel and Seviteronel to Human Cytochrome P450 17A1 and Relevance to Inhibition of Enzyme Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b01849

Orteronel (TAK-700) is a substituted imidazole that was developed for the treatment of castration-resistant prostate cancer but was dropped in phase III clinical trials. Both enantiomers of this inhibitor of cytochrome P450 (P450) 17A1 show some selectivity in differentially blocking the 17α-hydroxylation and lyase activities of the enzyme. Although both enantiomers of this compound have sub-micromolar IC50 values and bind to the enzyme with a type II spectral change (indicative of nitrogen-iron bonding) and reported Kd values of 56 and 40 nM (R and S, respectively), the rates of binding to P450 17A1 were relatively slow. We considered the possibility that the drug is a slow, tight-binding inhibitor. Analysis of the kinetics of binding revealed rapid formation of an initial complex, presumably in the substrate binding site, followed by a slower change to the spectrum of a final iron complex. Similar kinetics were observed in the interaction of another inhibitor, the triazole (S)-seviteronel (VT-464), with P450 17A1. Kinetic tests and modeling indicate that the further change to the iron-complexed form of the orteronel- or seviteronel-P450 complex is not a prerequisite for enzyme inhibition. Accordingly, the inclusion of heme-binding heterocyclic nitrogen moieties in P450 17A1 inhibitors may not be necessary to achieve inhibition but may nevertheless augment the process.

Full text of DOI:10.1021/acs.jmedchem.9b01849

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Article
Multi-Step Binding of the Non-Steroidal Inhibitors Orteronel and Seviteronel to
Human Cytochrome P450 17A1 and Relevance to Inhibition of Enzyme Activity
Stella A. Child, and F. Peter Guengerich
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01849 • Publication Date (Web): 30 Mar 2020
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Multi-Step Binding of the Non-Steroidal Inhibitors Orteronel and Seviteronel to Human Cytochrome
P450 17A1 and Relevance to Inhibition of Enzyme Activity
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Stella A. Child and F. Peter Guengerich
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146,
United States
Address correspondence to:
Prof. F. Peter Guengerich
Department of Biochemistry
Vanderbilt University School of Medicine
638B Robinson Research Building
2200 Pierce Avenue
Nashville, Tennessee 37232-0146, U.S.A.
Telephone: (615) 322-2261
FAX: (615) 343-0704
E-mail: f.guengerich@vanderbilt.edu
Running title: Orteronel and seviteronel inhibition of P450 17A1
Keywords: Cytochrome P450, P450 17A1, CYP17A1, enzyme inhibition, orteronel, TAK-700, seviteronel,
VT-464, prostate cancer, androgen inhibition
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ABSTRACT
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Orteronel (TAK-700) is a substituted imidazole that was developed for the treatment of castration-resistant
prostate cancer but was dropped in Phase III clinical trials. Both enantiomers of this inhibitor of cytochrome
P450 (P450) 17A1 show some selectivity in differentially blocking the 17-hydroxylation and lyase
activities of the enzyme. Although both enantiomers of this compound have sub-µM IC50 values and bind
to the enzyme with a Type II spectral change (indicative of nitrogen-iron bonding) and reported Kd values
of 56 and 40 nM (R- and S-, respectively), the rates of binding to P450 17A1 were relatively slow. We
considered the possibility that the drug is a slow, tight-binding inhibitor. Analysis of the kinetics of binding
revealed rapid formation of an initial complex, presumably in the substrate binding site, followed by a
slower change to the spectrum of a final iron complex. Similar kinetics were observed in the interaction of
another inhibitor, the triazole (S)-seviteronel (VT-464), with P450 17A1. Kinetic tests and modeling
indicate that the further change to the iron-complexed form of the orteronel- or seviteronel-P450 complex
is not a pre-requisite for enzyme inhibition. Accordingly, the inclusion of heme-binding heterocyclic
nitrogen moieties in P450 17A1 inhibitors may not be necessary to achieve inhibition but may nevertheless
augment the process.
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INTRODUCTION
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Cytochrome P450 (P450, CYP) 17A1 is the enzyme that converts the 4 steroid progesterone to 17-
hydroxy (OH) progesterone and then to androstenedione (Scheme 1).1 It also converts the 5 steroid
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Scheme 1.
Reactions catalyzed by P450 17A1. DHEA, dehydroepiandrosterone.
pregnenolone to 17-OH pregnenolone and on to dehydroepiandrosterone, which can be oxidized to
androstenedione by 3-hydroxysteroid dehydrogenase. P450 17A1 is present in gonadal tissues, including
the adrenals and testes. The production of androgens is important for normal reproductive function, and
androgens are also necessary for synthesis of estrogens. The 17-hydroxy steroids are also used in the
biosynthesis of mineralocorticoids and glucocorticoids.
One approach to treating prostate cancer is by suppression of androgen synthesis, i.e. inhibiting
P450 17A1 (Scheme 1). A number of inhibitors have been considered, and one (abiraterone acetate, a
prodrug) is currently on the market.2, 3 One goal in P450 17A1 inhibition is to preferentially inhibit the lyase
step compared to the initial 17-hydroxylation step (Scheme 1), because the 17-OH steroids are needed
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to produce cortisol. In clinikcal practice, administration of abiraterone currently involves supplementation
with prednisone. Some discernment of inhibition of the two reactions has been reported with drug
candidates, one of which is orteronel (TAK-700) (Scheme 2).4 Both enantiomers are inhibitory, with the
(S)-enantiomer
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Scheme 2.
(R)- and (S)- enantiomers of orteronel and (S)-seviteronel.
being more potent. Another naphthalene-containing non-steroidal drug candidate is seviteronel (VT-464),
and the (S)-enantiomer is more inhibitory (Scheme 2).6 Although orteronel was dropped in Phase III clinical
studies, seviteronel is apparently still under development at this time. We5 and others6 have considered the
selectivity of orteronel for the lyase reaction. However, as pointed out by Petrunak et al.,6 the apparent
potency of inhibition and the selectivity are dependent upon the experimental design.
Crystal structures of P450 17A1 bound to both the (R)- and (S)-enantiomers of orteronel and to (S)-
seviteronel have been reported by the Scott laboratory.6 The protein structures differ for the two
enantiomers, suggesting that conformational changes are involved in achieving these differences. Structures
of unliganded P450 17A1 are not available for comparison. As we noted previously,7 selective inhibition
of the two P450 17A1 reactions is not possible using a single binding site unless conformational changes
are involved.
The crystal structures of human P450 17A1 bound to abiraterone, galeterone (TOK-001), (S)-
seviteronel, and (R)- and (S)-orteronel all show the interaction of a heterocyclic ring nitrogen atom with the
heme iron, i.e. a classic Type II complex with an associated low-spin spectrum and a UV-visible Soret max
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near 430 nm.6, 8 Such binding is also seen in the abiraterone complexes of both zebrafish P450 17A1 and
17A2.9, 10 Consideration of the known inhibitors of P450 17A16, 11, 12 might suggest that the presence of
iron-heterocyclic nitrogen bonding is critical for P450 17A1 inhibitors.
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In the course of our studies on the mechanisms and kinetics of binding of steroids to P450 17A1,7
we identified conformational selection as a mechanism in substrate selection, i.e. multiple conformations
of P450 17A1 existing in solution prior to binding and selecting for substrate recognition. This mechanism
was also implicated for abiraterone,7 which is also a steroid. Surprisingly the kinetics of orteronel binding
were very different than observed for the steroids, and we further investigated these. The results indicate
rapid binding followed by a change to the final spectrally-observed complex with orteronel, presumably
the one characterized by its crystal structure(s).6 Similar behavior was observed with (S)-seviteronel. Some
enzyme inhibitors, including drugs, act through slow, tight-binding inhibition, characterized by initial loose
fitting to an enzyme followed by a slower transition to a more tightly bound form.13-17 We considered such
a mechanism but have ruled it out in favor of one in which the initial binding of the enzyme is tight and
leads directly to inhibition.
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RESULTS
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Kinetics of orteronel binding. Previous work on the binding of several steroids (including
substrates, products, and the inhibitor abiraterone) showed multiphasic kinetics, which could be described
in terms of a conformational selection mechanism, as opposed to induced fit.7 Binding of the substituted
imidazole inhibitor orteronel yields a classic Type II Soret difference spectrum, with a max at 430 nm and
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
min at 410 nm.5, 6
Mixing (final concentrations of) 2 µM P450 17A1 and 2 µM (S)-orteronel yielded a plot (A430-
A410) that was first-order with regard to each component (Figure 1A), but the rate was slower than might be
Fig. 1
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Figure 1.
Kinetics of spectral changes observed in binding of (S)-orteronel with P450 17A1. The
final concentrations of the compound and P450 were both 2 µM. (A) Plot of A430-A410 (wavelengths for
maximum difference) vs. time. (B) Plot of reciprocal of residual unbound concentration vs. time (residual
unbound concentrations determined by subtraction from 2 µM, in an Excel file). With equimolar starting
concentrations, the rate constant for a reaction A + B  AB, if beginning with equimolar concentrations
of A and B, is mathematically equivalent to 2A  AB and can be estimated, using the expression kt =
1/[A]t – 1/[A]0,18 to be 7.5  105 M-1 s-1 in this case (using the first 400 ms of data). (A: P450 17A1; B:
orteronel; C: P450-orteronel complex)
expected for a strong inhibitor (IC50 0.27 µM, Kd 40 nM).6 Similar kinetics were observed with (R)-
orteronel. Fitting the data to a plot of 1/[unbound P450 17A1] vs. time (Figure 1B) yielded an estimated
on-rate constant of 7.5  105 M-1 s-1 for the initial portion of the reaction, which is less than expected for
diffusion-limited binding.19, 20
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Spectra of P450 17A1 and orteronel complexes
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The slow kinetics suggested that the binding of orteronel might be more complex than a simple 2-
component system. Analysis of the reactions presented in Figures 1 and 2 by singular value decomposition
(SVD) suggested the possibility of an intermediate species (Figure 3A). Species 2 resembles the unbound
enzyme (Species 1), but is not identical, and is converted to the (final) Species 3.
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Fig. 2
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Figure 2.
Raw spectra recorded after mixing 2 µM P450 17A1 with 20 µM (S)-orteronel (final
concentrations after mixing), spaced over 4 s.
The difference spectrum calculated between Species 3 and Species 1 has a max of 430 nm and min of 410.
SVD analysis of the (S)-orteronel data yielded a rate of conversion of Species 1 to Species 2 of 37 s-1 and
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from Species 2 to Species 3 at a rate of 0.93 s-1 , and the results with the (R)-enantiomer were similar (Figure
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Species 2
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Figure 3.
Global fitting of kinetic data from Figure 2. Parts A-C: (R)-Orteronel; Parts D-F: (S)-
Orteronel. (A) Fitting of total data (4,000 spectra collected over 4 s) using OLIS GlobalWorks singular
value decomposition (SVD) with a 3-component system. (B) SVD fitting of the three species from Part A.
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Species 1 converted to Species 2 at a rate of 73 ± 6 s-1 and species 2 converted to Species 3 at 0.55 ± 0.10
s-1. (C) Residuals analysis from Part B. (D) Fitting of total data (4,000 spectra collected over 4 s) using
OLIS GlobalWorks SVD with a 3-component system. (E) SVD fitting of the three species from Part D.
Species 1 converted to Species 2 at a rate of 37 ± 2 s-1 and species 2 converted to Species 3 at 0.93 ± 0.02
s-1. (F) Residuals analysis from Part E.
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Some of the spectra were selected from individual experiments. Reaction of 2 µM P450 17A1 with
20 µM (S)-orteronel (final concentrations) is shown in Figures 2 and 4A, with the actual spectral traces at
35 ms and 3.85 s shown. These
Fig. 4
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Figure 4.
Spectral changes observed in reaction of (S)-orteronel with P450 17A1. The P450
concentration was 2 µM and the (S)-orteronel concentration was 20 µM (final concentrations in observation
cell). A total of 4,000 spectra was collected over a period of 4 s. (A) The spectra are shown at times 0, 35
ms, and 3.85 s. Compare with Figure 3A. (B) Kinetic traces are shown for different wavelengths. Note the
opposite directions of the changes following mixing and the gradual changes following the initial response.
(C) Plot of A390 changes. The rate of the initial increase was 60 ± 7 s-1 and the second (decrease) was 0.88
± 0.04 s-1. (D) Residuals trace for plot and rate estimates in Part D.
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resemble those predicted by SVD analysis (Figure 3A, 3D). Traces at 360, 390, 413, and 430 nm are shown
in Figure 4B, documenting the varying changes in the absorbance at these wavelengths and validating the
SVD modeling. The A390 data were fit to a biphasic plot of k1 at 60 ± 7 s-1 (increase) and k2 of 0.88 ± 0.04
s-1 (Figure 4C, 4D), similar to Figure 3B, 3E.
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Traces of A430-A410 were obtained with a lower concentration of P450 17A1 (1 µM, final
concentration) and varying concentrations of (S)-orteronel (Figure 5A). The binding was slow and not
complete even after 8 seconds
Fig. 5
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and the orteronel concentrations are indicated. (A) A430-A410 traces. (B) Magnitude of spectral change
observed after 4 s. (C) Rates of changes (Part A) as a function of orteronel concentration (estimated from
fits to single exponentials). (D) Rates of A392-A416 changes plotted versus orteronel concentration.
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(8 second data not shown). A plot of the absorbance changes at 4 seconds vs. the orteronel concentration
was consistent with tight binding (i.e., Kd 40 nM) (Figure 5B).5, 6 A plot of the single-exponential rates of
binding vs. orteronel concentration suggested a hyperbolic fit (Figure 5C). The first portion of the plot
yields a possible rate constant of ~ 5  105 M-1 s-1 (compare with Figure 1B). The rates of the initial change
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(A392-A416) were linear with respect to the orteronel concentration (Figure 5D), yielding an apparent on-
rate constant of ~ 2.5  107 M-1 s-1.
Spectra of P450 17A1 and (S)-seviteronel complexes
Seviteronel (VT-464) is another non-steroidal, naphthalene-based drug candidate, which is still in
clinical trials. It is also a strong inhibitor of P450 17A1 and was of interest in terms of the unexpected
kinetic results obtained with orteronel (Figures 2-4).
Steady-state spectroscopy showed a classic Type II binding spectrum with P450 17A1 (Figure 6),
due to the triazole moiety. Mixing P450 17A1 and (S)-seviteronel yielded a series of spectra, and SVD
Fig. 6
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Figure 6. Difference spectra of P450 17A1 with methanol and (S)-seviteronel. The concentration of P450
17A1 was 1 µM (in 50 mM potassium phosphate buffer, pH 7.4). Methanol was added to 2% (v/v) and the
spectrum was recorded, subtracting the original spectrum. The decrease in absorbance at 420 nm (~ 0.005
compared to 0.14) is approximately what would be expected for the dilution effect. Note that the absorbance
changes at several wavelengths (e.g. 390 nm, 430 nm) are not in the same direction as the initial spectral
complex formed with orteronel or seviteronel and the methanol effect cannot explain the observed changes
(Figures 4,B, 8B). (S)-Seviteronel (50 µM) was then added (along with another 0.5% methanol, v/v) and
the 2% methanol spectrum was subtracted from this.
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global fitting indicated similarity to those observed with (R)- and (S)-orteronel (Figure 3), although possibly
with different rate constants (Figure 7).
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Fig. 7
Species A
0.5
0.4
0.3
0.2
0.1
0.0
A
Species B
Species C
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Wavelength, nm
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Time, s
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Time, s
Figure 7. Global fitting of mixing of 2 µM P450 17A1 and 20 µM (S)-seviteronel (final concentrations, in
50 mM potassium phosphate buffer, pH 7.4). (A) Global fitting to a 3-species system using OLIS
GlobalWorks SVD. (B) Time courses predicted from Part A. (C) Residuals traced for kinetic fits in Part B.
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Actual analysis of the spectra recorded at different times did show three spectra (at times of 115
ms (mixing, corrected t= 0), 180 ms (actually 65 ms after mixing occurred), and 4 s (Figure 8A). Although
(as in Figure 4), the spectra are very similar,
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Fig. 8
0.4
A
t = 1 ms
t = 115 ms
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Wavelength, nm
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B
412 n
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430 n
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Time, s
Figure 8. Spectra and kinetics of reaction of P450 17A1 with (S)-seviteronel. (A) Spectra of P450 17A1
species obtained upon mixing 2 µM P450 17A1 and 20 µM (S)-seviteronel (final concentrations after
mixing, methanol concentration 0.1%, v/v) in 50 mM potassium phosphate buffer (pH 7.4). The spectra
were collected at the indicated times. The 115 ms spectrum (red) is immediately prior to the mixing step
and in of unliganded P450 17A1. The peak shifted to the 180 ms spectrum (elapsed time 65 ms) at its
maximum intensity and then changed to the t = 4 s spectrum. (B) Traces of absorbance at 390, 412, 418,
430, and 450 nm. Note opposite directions for absorbance changes after mixing. The 450 nm trace is
included to show the lack of absorbance drift, aside from the initial mixing artifact.
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Analysis of kinetic traces at the critical wavelengths (Figure 6) showed (i) the existence of an intermediate
species at 180 ms and (ii) absorbance changes in opposite directions at these wavelengths (Figure 8B).
Further analysis of the spectral changes (Figure 9) showed, as in the case of orteronel (Figure 4C),
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-0.19
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Time, s
Figure 9. Kinetic analysis of spectral changes observed upon mixing P450 17A1 and (S)-seviteronel
(Figures, 7, 8). (A) Plot of A390-A418 vs. time. The two changes were separated (115-180 ms and 180 ms -
4 s) and fit to first-order rates of 44 ± 3 s-1 and 1.9 ± 0.1 s-1, using the OLIS GlobalWorks software and
single-exponential fitting. (B) Plot of A430-A412 vs. time, which was fit in the same way to first-order rates
of 45 ± 2 s-1 and 1.9 ± 0.1 s-1.
biphasic changes with rather similar rates. In this case the rate of the first step was 44-45 s-1 and the rate of
the second reaction was 1.9 s-1 (compare with 60 and 0.88 s-1 for orteronel, Figure 4C).
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Kinetic analysis of inhibition mechanism
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The multi-step binding of both (R)- and (S)-enantiomers of orteronel to P450 17A1 (Figures 1-5)
raised the possibility that the inhibition could be the result of a slow, tight-binding mechanism, which has
been demonstrated for several enzyme inhibitors21-24 but to our knowledge has not been documented with
a P450. We considered two alternative mechanisms (Table 1): (i) a slow, tight-binding mechanism in which
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Table 1. Alternate Kinetic Models for Orteronel Bindinga
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A. Slow, tight-binding inhibition
B. Direct inhibition
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k1
k1
E + S
ES
EP
k1 106 M-1 s-1, k-1 0.45 s-1
E + S
ES
EP
k1 106 M-1 s-1, k-1 0.45 s-1
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k-1
k2
k-1
k2
ES
EP
k2 0.06 s-1
ES
EP
k2 0.06 s-1
k3
k3
E + P k3 0.8 s-1, k-3 106 M-1 s-1
E + P k3 0.8 s-1, k-3 106 M-1 s-1
k-3
k4
k-3
k4
k4 15  106 M-1 s-1, k-4 1.5 s-1
E + I
EI
k4 15  106 M-1 s-1, k-4 150 s-1
E + I
EI
k-4
k5
k-4
k5
EI
FI
k5 0, k-5 0
EI
FI
k5 1 s-1, k-5 0.001 s-1
k-5
k-5
a System used in Kintek Explorer software. E: P450 17A1, F: conformationally distinct form of E (E´in Scheme 3), S: progesterone, P: 17-OH
progesterone, I: orteronel.
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the initial binding of the inhibitor (I) is loose (Kd ~ 10 µM) and then conversion to a more tightly bound
form (E´I) occurs at a rate of ~ 1 s-1 (Figures 3, 4; Table 1A) with a forward to reverse equilibrium of 1000.
The value k5 = 1 s-1 is drawn from the spectral work (Figures 3, 4) and k-5 =0.001 s-1 is arbitrary (but will
influence some of the fitting). The value 0.001 s-1 corresponds to a t1/2 of 13 min, which seems reasonable
in light of the literature on slow, tight-binding inhibitor drugs.13, 15, 16 (ii) The alternative mechanism has
tight binding of the initial complex (Kd ~ 100 nM), and the complex is inhibitory itself. It does rearrange to
form the final stable complex but that step is mathematically silent in the inhibition model, in that both EI
and FI are equally inhibitory (Table 1B).
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The two mechanisms can be tested with classic approaches. In the first approach (Table 2), varying
Table 2. Experiment Used to Test Orteronel Inhibition Kinetic Mechanism (Table 1, Figure 10). a
(add equal volume)
T1 period (90 s)
[E]: 0.08 µM
[S]: 20 µM
T2 period (300 s)
[I]: 0, 2, or 6 µM
a E: P450 17A1, S: progesterone, I: orteronel.
concentrations of orteronel (I) were added to a reaction (P450 17A1 plus progesterone) after 90 s and
product formation (17-OH progesterone) was measured as a function of time (Figure 6). A slow, tight-
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Figure 10.
Orteronel inhibition data and fits to two alternate models (Tables 1, 2). The assay was
initiated with the concentrations described in Table 1 (T1 period), and the formation of 17-OH
progesterone was measured at the indicated time points. After 90 s (beginning of T2 period, Table 2), (R)-
orteronel was added to final concentrations of either 0 µM (red), 1 µM (green), or 3 µM (blue) and the
reactions continued. The same data points were used in both parts. The lines shown were calculated in
KinTek Explorer for using the parameters shown in Tables 1 and 2. (A) Slow, tight-binding model (Table
1A). (B) Direct inhibition model (Table 1B). Note curvature in Part A.
binding mechanism will show curvature as the reaction proceeds (Figure 6A), but a direct inhibition model
will show immediate inhibition (Figure 6B). However, the fit of the data did not allow discrimination
between the two models in this case (Figure 6).
One of the problems encountered with using some classic approaches to analyzing the kinetics of
orteronel inhibition is that we observed a slow loss of P450 17A1 activity following pre-incubation in the
absence of substrate or inhibitor, which we had mentioned previously regarding binding titration analysis.5
Accordingly, the preincubation step was performed on ice. An experiment was designed in which a low
concentration of orteronel (final concentration 0.5 µM) was incubated with P450 17A1 (0.2 µM) for varying
periods of time before a large excess of the substrate progesterone (100 µM) (and NADPH) was added to
initiate the 17-hydroxylation reaction (Figure 7).
Table 3. Experiment Used to Test Orteronel Inhibition Kinetic Mechanism (Table 1, Figure 11). a
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(add equal volume)
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T1 period (variable time: 0-1200 s)
[E]: 0.4 µM
T2 period (300 s)
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[S]: 200 µM
[I]: 0 or 1 µM
a E: P450 17A1, S: progesterone, I: orteronel.
Application of the model and rate constants in Tables 1
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Progesterone only
(S)-orteronel 0.5 µM
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Direct inhibition
No inhibition
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Preincubation time, min
Figure 11.
Inhibition data and fits to two alternate models (Tables 1, 3). The rate constant k2 (Table 1)
was adjusted to fit the uninhibited data points (changed from Figure 10) resulting from the use of a different
enzyme preparation. P450 17A1 (final concentration of 0.2 µM) and orteronel (0.5 µM) were preincubated
for the times indicated on the x-axis. Reactions were then initiated by the addition of both 100 µM
progesterone and an NADPH-generating system (begin T2 period, Table 3), proceeding for 300 s and
followed by product analysis. The solid lines show the profiles calculated for a slow, tight-binding model
(blue line) and a direct inhibition model (red line), in KinTek Explorer, using the parameters shown in
Tables 1 and 2. The black line shows the expected uninhibited rates. The data points show the rates of
progesterone 17-hydroxylation measured in the presence and absence of orteronel.
and 3 yields two predictions (Figure 11):
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(i) in the slow, tight-binding model an exponential loss of activity is seen, resulting in an asymptote with
~ 70% inhibition. (ii) In the direct inhibition mode, very little inhibition is seen, and the extent of inhibition
is observed and does not change with time. As shown with both the (R)- and (S)-enantiomers, the direct
inhibition model fits the data points best for both enantiomers, i.e. only very limited inhibition (Figure 11).
Experiments done with (S)-seviteronel gave similar results (Figure 12). We conclude that its
behavior is similar to that of orteronel, as seen in the case of the spectral assays (Figures 6-9).
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Seviteronel (0.5 µM)
Direct inhibition
No inhibition
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Preincubation time, min
Figure 12.
Inhibition data and fits to two alternate models (Tables 1, 3). The rate constant k2 (Table 1)
was adjusted to fit the uninhibited data points (changed from Figure 10) resulting from the use of a different
enzyme preparation. P450 17A1 (final concentration of 0.2 µM) and seviteronel (0.5 µM) were
preincubated for the times indicated on the x-axis. Reactions were then initiated by the addition of both
100 µM progesterone and an NADPH-generating system (begin T2 period, Table 3), proceeding for 300 s
and followed by product analysis. The solid lines show the profiles calculated for a slow, tight-binding
model (blue line) and a direct inhibition model (red line), in KinTek Explorer, using the parameters shown
in Tables 1 and 2. The black line shows the expected uninhibited rates. The data points show the rates of
progesterone 17-hydroxylation measured in the presence () and absence () of seviteronel.
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DISCUSSION AND CONCLUSIONS
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The binding of both enantiomers (R-, S-) of orteronel to the enzyme target P450 17A1 was analyzed and
found to be slower than what would be expected for a diffusion-limited reaction19, 20 (Figure 1). Examination
of the spectral changes with SVD analysis over time (Figure 2) suggested the existence of multiple species
of orteronel-P450 17A1 complexes after mixing (Figure 3), and the phenomenon was established using
direct analysis of the spectra (Figure 4A). The opposite directions of absorbance changes at different
wavelengths (Figure 4B) validated this view, and the data could be fit to fast, diffusion-limited binding of
orteronel followed by a slower conversion of the complex to an Fe-N complex. However, the kinetic
modeling indicates that there is no need to invoke a slow, tight-binding mechanism for enzyme inhibition
(Figure 11).
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The point should be made that the initial spectral changes were not atypical for P450 reactions and
could be used to calculate an on-rate constant of ~ 2  107 M-1 s-1 (Figure 5D, Table 1), which might be
regarded as a simple bimolecular reaction. Visual inspection of the spectral traces (Figure 2) indicated a
lack of isosbestic points, even though the spectral changes were small, and SVD analysis was also more
consistent with a three-species system than two (Figure 3). The SVD predictions could be confirmed when
the individual spectra were isolated (Figure 5A) and the traces at different wavelengths examined in more
detail (Figure 5B).
Narasimhulu25 had earlier postulated that rabbit P450 2B4 binds norbenzphetamine in this manner.
That is, an initial high-spin complex was formed, which then changed to a Type II (low-spin) complex,25
similar to the mechanism we propose (Scheme 3).
Scheme 3.
Interactions of P450 17A1 (E) with orteronel or (S)-seviteronel (I).
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A number of drugs exert their activities via slow, tight-binding inhibition.13, 21-24 To our knowledge,
none have ever been reported for P450 enzymes, with the possible exception of cilengitide.26 However,
there are many instances of mechanism-based inactivation of P450s,27 which differs only from the slow,
tight-binding model of Table 1 in that k-5 is zero. The kinetic course of the inactivation is similar to slow,
tight-binding but the inhibition is not reversed by substrate (as in an experiment such as that of Figure 7).
Many amines are oxidized to form nitroso derivatives that interact very tightly with the heme iron and,
although they can be reversed, are functionally irreversible for a long period.27-29 We have not re-
investigated our work on the inhibition of P450 3A4 by the peptide cilengitide,26 which is complex but
appears to contain a component of this mechanism. It is also possible that some of the polycyclic
hydrocarbons, flavonoids, and other inhibitors that we have studied might fit into this group if examined in
more detail using the newer modeling approaches.30-34
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Orteronel was discontinued for development in the course of Phase III clinical trials. Although it
had shown promise in discovery and development,4, 35-37 it apparently did not exceed the efficacy of
abiraterone. Another P450 17A1 inhibitor understood to still be under development is (S)-seviteronel (VT-
464),6 currently in clinical trials and the subject of part of this report. Galeterone (TOK-001, VN/124-1)
was dropped in July 2016. These compounds contain, respectively, a benzimidazole and a 2H-triazole ring,
while abiraterone contains a pyridine. The two enantiomers of seviteronel have been reported to bind less
tightly to P450 17A1 than does orteronel.6 Spectral assays indicated very similar behavior (of the (S)-
enantiomer) of seviteronel to orteronel (Figures 6-9).
Overall, the mechanism of binding of orteronel to P450 17A1 would seem to resemble an induced
fit model more than conformational selection,38, 39 in that a discrete initial binding step can be identified
before a final change (Figure 4, Scheme 3). However, our earlier evidence for conformational selection
with P450 17A1 shows that multiple conformations of P450 17A1 exist in solution in the absence of ligand
(Scheme 3). One (or more) of those can bind orteronel or seviteronel. The initial EI complex is tight and
inhibitory, and then changes to a final E´I complex, the low-spin Fe-N entity seen in the spectra (Figures
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2-4) and the crystal structures.6 Thus, a combination of phenomena is involved in the interactions of P450
17A1 with the inhibitor orteronel and seviteronel. All of our evidence (i.e., Figures 1-5, 7-9) is consistent
with similar events occurring with both the R- and S-enantiomers, although the final structures differ.6
Alternative mechanisms to the one presented in Scheme 3 were also considered, using SVD
analysis in the OLIS GlobalWorks program. None of the 2-species options would fit the observed spectral
data (Figures 2, 3A, 3D, 7A). Other 3-species models were considered, particularly one involving a
conformational selection process (Scheme 4) in which there are two conformational states in slow
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Scheme 4. Alternate model with two conformational states in slow equilibrium.
equilibrium (step 1), and one of these binds orteronel to yield the low spin 430 (Type II) complex (step 2).
However, none of the models in the software provided satisfactory fits to the observed spectral traces
(Figures 2, 4A, 8A). Further, 3-species modeling with a slow conformational step (step 1, ~ 1 s-1) followed
by rapid reaction with orteronel did not predict the observed spectral changes, in contrast to Figures 3A,
3D, 7A). In Scheme 4, we know that all of the final complex is low spin (E´I) from the spectrum, so the
equilibration of E´I and EI (step 4) is necessary. (It should also be pointed out that we have shown
previously that the starting P450 17A1 enzyme (spectra shown in Figures 3, 5A, 8A) is > 95% low spin as
judged by second-derivative spectroscopy.7) Attempts to begin with E and E´ present, in slow equilibrium
(1 s-1), yield distinctly biphasic kinetic plots (A430-A410), which were not seen in the experiments (Figure
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3). Combining steps 2 and 4 in Scheme 4 yields the same mechanism we have already provided evidence
for and is described in Scheme 3.
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9
As mentioned earlier, orteronel is not being considered for development any longer but seviteronel
is. The conclusions about the behavior of (S)-seviteronel (Figures 6-9, 11) were the same as for orteronel.
All of the inhibition work presented here was done with the progesterone 17-hydroxylation
reaction, but the lyase reaction as also at least as important, in that a goal in development of inhibitors to
use in treating prostate cancer is to selectively inhibit the lyase activity compared to 17-hydroxylation.
This is not a trivial goal, in light of the evidence for a single active site in the the enzyme. Previous modeling
efforts were unsuccessful in the absence of invoking a different enzyme conformation at some point in the
two reactions.7 Exactly what the nature of such a conformational change is remains unclear. It might
be related to interaction with cytochrome b5. Further considerations of the details
of inhibition of the lyase reaction need to involve pre-steady-state measurements of products (by quench
flow kinetics), as indicated by modeling exercises.
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In summary, our spectral and kinetic results reveal a multi-step interaction of both enantiomers of
the drug candidates orteronel and (S)-seviteronel with the target, P450 17A1. The initial complex is tight
and capable of explaining the observed inhibition without invoking slow, tight-binding inhibition. The
initial complex goes on to yield a final Fe-N complex with a classic Type II low-spin character. However,
we cannot rule out the existence of further changes in the bound Fe-N complex that would be invisible in
the spectra.
EXPERIMENTAL SECTION
Chemicals. Progesterone was purchased from SigmaAldrich. Racemic orteronel (TAK-700) was
obtained from ApexBio (Houston, TX) (catalog no. A4236). The (R-) and (S)-enantiomers were separated
using chiral HPLC and their purity (>95%) and spectral properties have been characterized previously.5
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(S)-Seviteronel was purchased from Advanced ChemBlocks (Burlingame, CA) and its purity was evaluated
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by HPLC/UV and LC-MS (Supporting Fig. S2).
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Enzymes. Human P450 17A1, with a C-terminal (His)6 tag, was expressed in Escherichia coli and
purified to near electrophoretic homogeneity as described elsewhere.40 Recombinant rat NADPH-P450
reductase41 and human cytochrome b542 were expressed in E. coli and purified as described.
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Catalytic Assays. Steady-state catalytic assays were done based on a modification of previous
methods.5, 7, 40 For the direct inhibition assay, CYP17A1 (0.04 µM) was incubated with NADPH-P450
reductase (2 µM), cytochrome b5 (0.5 µM), progesterone (10 µM), and L--1,2-dilauroyl-sn-glycero-3-
phosphocholine (16 µM) in 50 mM potassium phosphate buffer (pH 7.4) in a final volume of 0.5 mL. The
samples were preincubated for 5 min at 37 °C and the reaction was initiated by the addition of an NADPH
generating system (10 mM glucose 6-phosphate, 0.5 mM NADP+, and 2 μg/mL yeast glucose-6-phosphate
dehydrogenase). Orteronel and sevriteronel were dissolved in CH3OH and added to incubations (1 or 3 µM)
at 90 seconds, with the final CH3OH being ≤ 2% (v/v). The reactions were run for varying times and
quenched by addition of 2 mL of ethyl acetate. For the preincubation assay, the reaction mixture was as
follows: CYP17A1 (0.2 µM), NADPH-P450 reductase (2 µM), cytochrome b5 (0.5 µM), and L--1,2-
dilauroyl-sn-glycero-3-phosphocholine (16 µM) in 50 mM potassium phosphate buffer (pH 7.4) in a final
volume of 0.5 mL. Orteronel ((R)- or (S)-, 0.5 µM), seviteronel (0.5 µM), or the same volume of CH3OH
was added to the samples at differing pre-incubation times, and the mixture was left on ice. The samples
were then incubated at 37 °C and the NADPH-generating system (as above) was added 1 min before
progesterone (100 µM) was used to initiate the reaction. The reaction time was 5 min, after which the
reactions were quenched with 2 mL of ethyl acetate. Extraction and analysis of samples for both assays
were performed as reported previously, utilizing HPLC and UV detection.40
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Spectroscopy. Steady-state spectra were recorded using either a Cary 14-OLIS or an Aminco
DW2a-OLIS spectrophotometer (On-Line Instrument Systems (OLIS), Bogart, GA). Pre-steady-state
measurements were made with an OLIS RSM-1000 instrument operating in the rapid-scanning mode (4000
spectra/second) using a 4 mm  20 mm observation cell, 1.24 mm slits, a 16  0.2 mm ScanDisk, and 400
lines/mm, > 500 nm gratings at 23 °C in 100 mM potassium phosphate buffer (pH 7.4). At least four shots
were done under each experimental condition (e.g., orteronel or (S)-seviteronel concentration) and the OLIS
GlobalWorks software was used to extract A430-A410, A430-A410, or A390-A418 data, which were averaged
and fitted to single exponentials (e.g., Figure 5) or to other equations (e.g., Figure 1B). In some cases (e.g.,
Figures 3, 7), SVD analysis was done in the OLIS GlobalWorks program.
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Kinetic Analysis and Simulation. KinTek Explorer (v. 8.0) was used (KinTek, Snow Shoe, PA)
with an Apple iMac OXC version 10.13.6 system. Data files were imported as Excel files (saved and
transferred as .txt files), and the input parameters are cited in Tables 1-3. In some cases (e.g., Figure 11),
the Explorer output was imported into Prism software (GraphPad, San Diego, CA).
ANCILLARY INFORMATION
Supporting Information available: Analytical data for (R)- and (S)-orteronel,
analytical data for (S)-seviteronel. Molecular Formula Strings
available for (R)- and (S)-orteronel, (S)-seviteronel, and
progesterone.
Address correspondence to:
F. Peter Guengerich
Department of Biochemistry
Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 U. S. A. e-mail:
f.guengerich@vanderbilt.edu
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Author Contributions
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The manuscript was written through contributions of both authors.
Both authors have given approval to the final version of the
manuscript.
Funding Sources
This work was supported by National Institutes of Health Grant R01 GM118122 (F. P. G.). The content is
solely the responsibility of the authors and does not necessarily represent the official views of the National
Institutes of Health.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
We thank C. J. Wilkey for preparing P450 17A1 and E. Gonzalez for separating the enantiomers
of orteronel. Thanks are also extended to K. Trisler for assistance in preparation of the manuscript.
ABBREVIATIONS
P450 (or CYP), cytochrome P450; OH, hydroxy; SVD, singular value decomposition.
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