Pyridine-substituted desoxyritonavir is a more potent inhibitor of cytochrome P450 3A4 than ritonavir

Article abstract of DOI:10.1021/jm400288z

Utilization of the cytochrome P450 3A4 (CYP3A4) inhibitor ritonavir as a pharmacoenhancer for anti-HIV drugs revolutionized the treatment of HIV infection. However, owing to ritonavir-related complications, there is a need for development of new CYP3A4 in

Full text of DOI:10.1021/jm400288z

Article
pubs.acs.org/jmc
Pyridine-Substituted Desoxyritonavir Is a More Potent Inhibitor of
Cytochrome P450 3A4 than Ritonavir
Irina F. Sevrioukova*,† and Thomas L. Poulos†,‡,§
‡
§
†Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine,
California 92697, United States
S
* Supporting Information
ABSTRACT: Utilization of the cytochrome P450 3A4 (CYP3A4) inhibitor ritonavir as a
pharmacoenhancer for anti-HIV drugs revolutionized the treatment of HIV infection.
However, owing to ritonavir-related complications, there is a need for development of new
CYP3A4 inhibitors with improved pharmacochemical properties, which requires a full
understanding of the CYP3A4 inactivation mechanisms and the unraveling of possible
inhibitor binding modes. We investigated the mechanism of CYP3A4 interaction with three
desoxyritonavir analogues, containing the heme-ligating imidazole, oxazole, or pyridine group
instead of the thiazole moiety (compounds 1, 2, and 3, respectively). Our data show that
compound 3 is superior to ritonavir in terms of binding affinity and inhibitory potency owing
to greater flexibility and the ability to adopt a conformation that minimizes steric clashing and
optimizes protein−ligand interactions. Additionally, Ser119 was identified as a key residue
assisting binding of ritonavir-like inhibitors, which emphasizes the importance of polar
interactions in the CYP3A4−ligand association.
and gastrointestinal disturbance. Finally, owing to poor
physicochemical properties and, in particular, low aqueous
solubility, ritonavir cannot be conveniently coformulated with
other antiretroviral agents. Hence, it is highly desirable to
develop boosters with more favorable pharmacological and
physicochemical properties and fewer off-target effects.
INTRODUCTION
■
Human cytochrome P450 3A4 (CYP3A4) is the major drug-
metabolizing enzyme that oxidizes a wide range of structurally
diverse xenobiotics and prescribed pharmaceuticals. Bioavail-
ability and therapeutic efficiency of these administered drugs,
therefore, depends on whether/how fast they are degraded by
CYP3A4. CYP3A4 inhibition, on the other hand, leads to
increased plasma levels of pharmaceuticals. CYP3A4-related
drug−drug interactions are usually undesirable, as they may
cause toxicity and other adverse complications. Carefully
controlled CYP3A4 inactivation, however, can be clinically
useful.
Treatment of HIV infection is one example of a beneficial
CYP3A4 inactivation. HIV protease inhibitors, an important
part of antiretroviral therapy, are rapidly metabolized primarily
by liver and intestinal CYP3A4. Co-administration of low doses
of ritonavir, an HIV protease inhibitor and a potent CYP3A4
inactivator (Figure 1), elevates plasma levels of HIV protease
inhibitors, which enables more effective dosing regiments.1
Although ritonavir has been successfully utilized as a
pharmacoenhancer (booster) for over a decade, there are
several important complications associated with this drug.
Because ritonavir was originally developed as an HIV protease
inhibitor, its administration in subtherapeutic doses can
promote the emergence of HIV drug resistance. Another
disadvantage is the ability of ritonavir to affect activities of other
CYPs, as well as the phase II conjugating enzymes and drug
transporters.2,3 Ritonavir also activates xenobiotic-sensing
receptors that regulate expression of CYP3A4,4 which can
decrease the overall inhibitory potency of ritonavir upon its
prolonged use. Additional side effects include lipid disorders
Several companies are currently involved in the development
of novel CYP3A4 inhibitors that could be used as
pharmacoenhancers.1 One such compound, cobicistat, has
been approved recently as part of a combination pill for treating
̈
HIV-1 infection in treatment-naive patients. Cobicistat (Figure
1) is a ritonavir analogue lacking the key hydroxyl group
required for the HIV protease binding and inhibition and is
devoid of anti-HIV activity.5,6 Functional assays on human liver
microsomes showed that cobicistat acts on the CYP3A enzymes
more selectively than ritonavir and via a similar mechanism,
although with somewhat lower effectiveness (kinact/Ki of 0.57 vs
0.90 min−1 μM−1 for ritonavir).6 Other advantages of the new
booster include higher aqueous solubility, improved tolerability,
and reduced off-target drug−drug interactions. No structural
and functional information on the CYP3A4−cobicistat complex
is currently available and, thus, the precise mechanism of
binding and inhibition remain unknown.
Previously, we utilized spectroscopic, kinetic and structural
approaches to investigate the mechanism of CYP3A4 inhibition
by ritonavir and desthiazolylmethyloxycarbonyl ritonavir
(DTMCR), an analogue that lacks the heme-ligating thiazole
moiety.7,8 These in vitro studies demonstrated that heme
Received: February 24, 2013
© XXXX American Chemical Society
A
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from the equilibrium titrations (Insets (a) and (b) in Figure 2,
Table 1), 1 and 3 have a 2-fold higher affinity for CYP3A4 than
ritonavir, whereas the binding affinity of 2 is 10-fold lower.
Similar to the ritonavir-ligated CYP3A4,7 the protein bound
to compound 3 was fully converted to a 442 nm absorbing
species upon reduction with sodium dithionite (Figure 2C).
The conversion was partial for the CYP3A4−2 complex
(∼60%) and negligible for the 1-bound form (Figure 2A,B).
Such spectral differences allowed us to test whether 1 and 2 can
be displaced from the active site by stronger ligands.
Displacement titrations showed that both ritonavir and 3 can
substitute 1 and 2 and that the binding affinity of 3 for the 1-
and 2-bound CYP3A4 was several-fold higher (Supporting
Information Figure 1S). Compound 3 also has a higher affinity
for CYP3A4 complexed with bromoergocryptine, a type I
substrate (Ks of 5 vs 50 nM for ritonavir).7 Finally, 1 and 2 but
not 3 dissociated from CYP3A4 during repetitive concen-
tration/dilution cycles. Thus, the pyridine-containing 3 binds to
CYP3A4 irreversibly and stronger than ritonavir.
It should be noted that ligation of compound 1 to CYP3A4
significantly slows down/impedes heme reduction, which could
not be completed under the experimental conditions used. This
could be the consequence of a large negative shift in the heme
redox potential caused by the 1 ligation (≫70 mV, Table 1). A
decrease in Eo,7 induced by 2 and 3 is ∼50 and 70 mV,
respectively.
Kinetic analyses showed that the reaction of CYP3A4
binding to 1, 2, and 3 was biphasic, with the rate constants
for the fast and slow phases (kfast and kslow, respectively)
increasing with an increase in ligand concentration (Figure 3).
Although the kfast vs [ligand] dependence was hyperbolic for all
compounds, the fits to the plots for 1 and 2 did not pass
through the origin. This differs from the ritonavir binding
reaction, which is monophasic at subequimolar
inhibitor:CYP3A4 ratios and biphasic when the ligand is
present in an excess.8 Most notably, the kfast vs [ritonavir] plot
is V-shaped,8 with the breaking point at the inhibitor:CYP3A4
ratio reaching unity (Figure 3). Finally, ritonavir ligates to the
heme slower than the investigated compounds, especially 3, kfast
which is 5-fold higher (Table 1). For irreversible type II
inhibitors of CYP3A4, kfast would be proportional to the
inactivation rate constant (kinact) and, hence, can serve a
measure of the inhibitory potency.
Effect of Ritonavir-Like Molecules on CYP3A4
Stability. Tight-binding ligands often increase thermal stability
of target proteins. To investigate how type II inhibitors affect
stability of CYP3A4, we compared melting curves for different
ligand-bound forms. Association of all investigated compounds
led to an increase in the melting temperature (Tm) of CYP3A4,
but the most pronounced stabilizing effect was caused by
ligation of 3 (Figure 4A, Table 1). Thus, a simple and quick
measurement of ligand-mediated shifts in Tm can be used as an
additional tool for identification of high affinity CYP3A4
binders.
Figure 1. Chemical structures of ritonavir, cobicistat, and compounds
1−3.
ligation is essential for tight association of ritonavir-like
compounds, whereas nonbonding interactions provided by
the side groups define the binding mode. To further investigate
the mechanism of CYP3A4 inactivation and to test whether the
affinity and inhibitory potency of ritonavir can be further
improved, we compared the properties of three desoxyritonavir
analogues where the thiazole moiety was replaced with
imidazole, oxazole, or pyridine (compounds 1, 2, and 3,
respectively; Figure 1). On the basis of inhibitor binding affinity
and association kinetics, as well as thermal denaturation, activity
inhibition assays, mutagenesis, and X-ray data, we conclude that
the pyridine-containing compound 3 is a stronger ligand and a
more potent CYP3A4 inhibitor than ritonavir. In addition,
Ser119 was shown to be a key residue that assists association of
ritonavir-like inhibitors and stabilizes the ligand-bound form.
Inhibitory Potency of Compounds 1−3. The inhibitory
potency of the ritonavir analogues toward CYP3A4 was
compared in vitro in a reconstituted system with the redox
partner cytochrome P450 reductase (CPR) and 7-benzyloxy-4-
(trifluoromethyl)coumarin (BFC) as a substrate. Formation of
the hydroxylated product was monitored fluoroscopically, and
the percentage of the activity remaining was plotted vs ligand
concentration to determine a concentration required for half-
maximal inactivation (IC50), a measure of the inhibitory
RESULTS
■
Binding Affinity and Association Kinetics of Com-
pounds 1−3. All three desoxyritonavir analogues induced type
II spectral changes in CYP3A4 (Figure 2), indicative of direct
nitrogen ligation to the heme iron. The largest red-shift in the
Soret band (416−424 nm) was caused by compound 1.
According to the spectral dissociation constants (Ks) calculated
B
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Figure 2. Spectral changes induced in CYP3A4 upon binding of 1 (A), 2 (B), and 3 (C). Absorbance spectra of ferric ligand-free (), ligand-bound
(---), ferrous ligand-bound (···), and ferrous CO complex (-·-·-·) were recorded at room temperature in 50 mM phosphate buffer, pH 7.4, containing
20% glycerol and 1 mM dithiothreitol. Insets (a) and (b) are absorbance changes observed during titration of CYP3A4 with 1−3 and plots of
absorbance change vs ligand concentration, respectively.
Table 1. Properties of Ritonavir and Compounds 1−3
a
b
c
d
e
442 nm band (Fe2+)
Ks (nM)
Eo,7 (mV)
kfast (s−1
)
Tm (°C)
IC50 (nM)
f
f
f
f
ritonavir
+
51 10
22
570 30
25
−350
≪−370
≈ −370
−350
5
1.4 0.3
1.9 0.3
2.2 0.4
7.0 0.5
54.8 0.1
56.5 0.2
54.8 0.1
57.2 0.2
550 50
280 40
3400 60
1
2
3
−
4
+
5
5
130 15
a
b
c
Spectral dissociation constant. Midpoint redox potential of the inhibitor-bound CYP3A4. Eo,7 of the ligand-free CYP3A4 is ∼−300 mV.7 Rate
d
constant of the fast phase of the ligand binding reaction. Melting temperature of the CYP3A4−inhibitor complex. Tm for the ligand-free CYP3A4 in
e
the absence and presence of 2% DMSO is 52.6 0.1 and 52.1 0.1 °C, respectively. Concentration required for half-maximal inactivation of
recombinant CYP3A4. Determined previously.7
f
C
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○
●
Figure 3. Plots of the observed rate constants for the fast ( ) and slow ( ) kinetic phases of the ritonavir and 1−3 binding to WT CYP3A4 vs
ligand concentration.
Figure 5. (A) The active site of CYP3A4 crystallized in the presence of
1. Compound 1 is not bound to the heme (shown in pink) or
anywhere else. Instead, there are three water molecules (cyan spheres),
one of which is directly coordinated to the heme iron. Residues
comprising the F−F′ loop are rendered in orange. The side chain of
Arg212 is disordered but can be modeled in an inward position as
observed in the 1TQN model. (B) 2 remains bound to the heme
during crystallization. However, only the heme-ligating oxazole moiety
is well-defined in the X-ray structure, with the rest of the molecule
being disordered. Compound 2 is modeled in an orientation similar to
that of 3 (see Figure 6). Parts of the I-helix in the ligand-free and 2-
bound CYP3A4 are displayed in gray and pink, respectively. A notable
displacement in the I-helix is caused by steric hindrance between the
Figure 4. Effect of ritonavir and compounds 1−3 on thermal stability
(A) and the BFC hydroxylation activity of CYP3A4 (B). The
denaturation and inhibitory assays were conducted as described in the
Experimental Section. The Tm and IC50 values derived from the plots
(A) and (B), respectively, are given in Table 1.
ligand and Phe304. The 369−370 peptide, however, remains in place.
2Fo − Fc and Fo − Fc electron density maps contoured at 1σ and 3σ
are colored in blue and green, respectively.
heme-ligating oxazole group (Figure 5B). Another indication of
2 present in the active site was a notable displacement of the I-
helix, likely caused by steric hindrance with Phe304 (explained
below).
effectiveness (Figure 4B). The derived IC50 values are in good
agreement with the binding affinity (Table 1): compounds 1
and 3 inhibited the BFC hydroxylation 2−4-fold more
effectively than ritonavir, whereas the inactivating potency of
2 was 6-fold lower. Further, the ratio between IC50s measured
for ritonavir and its analogues under conditions of co- and
preincubation with NADPH were close to unity (∼1.1−1.3).
This suggests that all compounds are very weak time-
dependent inhibitors, inactivating CYP3A4 mainly through
heme ligation rather than formation of a reactive metabolite(s).
Taken together, the experimental data indicate that 3 is a
stronger type II ligand and more potent inhibitor than ritonavir
acting via a similar mechanism.
Crystal Structures of CYP3A4 Bound to Compounds 2
and 3. Although compound 1 promoted CYP3A4 crystal-
lization, it did not remain in the active site of the crystalline
protein or bind somewhere else. Instead, three water molecules
were observed near the heme, one of which was directly
coordinated to the iron (Figure 5A). This and an inward
orientation of the F−F′ loop, characteristic for the ligand-free
1TQN and 1WOE structures,9,10 indicate that the protein is in
a six-coordinated resting state. Compound 2 did cocrystallize
with CYP3A4, but the electron density was seen only for the
In contrast, in the CYP3A4−3 complex structure, only the
isopropyl-thiazole of the ligand was undefined (Figure 6A).
Compound 3 orients in the active site similarly to ritonavir but
with a few notable differences. As observed for ritonavir,7 the
phenyl group closest to the heme-ligating moiety in 3 (Phenyl-
1) is imbedded into a hydrophobic pocket lined with the
phenylalanine and leucine residues and, owing to steric clashing
with Phe304, displaces the I-helix (Figure 6A). The second
phenyl (Phenyl-2), however, does not protrude into a cavity
above the heme plane as deeply as the respective group of
ritonavir does. This eliminates steric hindrance with the Ile369-
Ala370 peptide and prevents the heme shift observed in the
3NXU structure (Figure 6B). As a result, the Fe−N bond in the
CYP3A4−3 complex is slightly shorter (2.1 vs 2.2−2.3 in the
ritonavir-bound protein).
Another important difference is the relative orientation of
Phenyl-2. In compound 3, this moiety is placed closer and near
parallel to the heme-ligating pyridine and the guanidinium
group of Arg105 (Figure 6B,C), thereby promoting π-stacking
and cation−π interactions. The conformation of 3 is further
stabilized by the inhibitor amide nitrogen donating a hydrogen
D
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Figure 6. Crystal structure of the CYP3A4−3 complex. (A) Compound 3 (in magenta) binds to the heme via the pyridine nitrogen. Most of the
ligand−protein interactions are provided by the phenyl side groups, labeled as (1) and (2). Phenyl-1 is imbedded into a hydrophobic pocket lined
with Phe108, Leu210, Leu211, Phe241, and Phe304 (shown in yellow sticks). Steric clashing between Phenyl-1 and Phe304, which is also observed
in the CYP3A4−ritonair complex, leads to the I-helix displacement. Phenyl-2 of 3, however, imposes no steric hindrance on the 369−370 peptide
and, as a result, there is no heme shift. For comparison, the heme, 369−370 peptide, and I-helix of the superimposed ligand-free 1TQN structure are
shown in pink. 2Fo − Fc and Fo − Fc electron density maps contoured at 1σ and 3σ are displayed in blue and green, respectively. (B,C) Relative
orientation of 3 (magenta) and ritonavir (green). The heme, 369−370 peptide, and residues lining the hydrophobic pocket in the ritonavir-bound
3NXU structure are colored in cyan. Owing to higher flexibility, 3 binds in a conformation that eliminates clashing between Phenyl-2 and Ile369-
Ala370 (B). Furthermore, Phenyl-2 orients near parallel to the pyridine ring and the guanidinium group of Arg105. This promotes π-stacking and
cation−π interactions, stabilizing the complex. A peptide flip in 3 allows formation of a stronger hydrogen bond with Ser119 via the amide nitrogen
(depicted as a blue dashed line in (C)). In contrast, ritonavir is H-bonded to Ser119 via the carbonyl oxygen (red dashed line). Such conformational
differences could increase the binding affinity and inhibitory potency of compound 3. (D) A closer view at the Phe-2−Arg105 interaction site
demonstrates that only the phenyl ring of 3 (magenta) can establish cation−π interactions with the Arg105 guanidinium group.
atom to the Ser119 hydroxyl group. In contrast, the carbonyl
oxygen of ritonavir accepts an H-bond from Ser119 (Figure
6C). Because 3 is in a more elevated position due to a larger
pyridine ring, the peptide bond flip optimizes and strengthens
the H-bond, whose length is 2.5 vs 2.8 Å in the ritonavir-bound
structure. Altogether, such dissimilarities may increase the
binding affinity and, consequently, the inhibitory potency of 3.
Effect of the S119A Substitution on the CYP3A4−
Inhibitor Interaction. To elucidate the role of Ser119 in the
ligand binding process, we investigated properties of the S119A
mutant of CYP3A4. Elimination of the H-bond forming
hydroxyl group in Ser119 significantly perturbed association
of the ritonavir-like molecules. The mutation led to a several-
fold decrease in the binding affinity of all investigated
compounds, most notably of ritonavir (Table 2; Supporting
Information Figure 2S). The more drastic effect was on the
kinetics of inhibitor ligation. While all reactions remained
biphasic, the kfast values were decreased by 6−22-fold (Figure 7
and Supporting Information Figure 2S, Table 2). Most
strikingly, the kfast vs (compounds 1−3) plots were no longer
hyperbolic (compare respective panels in Figures 3 and 7), in
that the rate constants did not change or slightly decreased
until the inhibitor:CYP3A4 ratio reached unity and then
gradually increased. kfast for ritonavir, on the other hand,
remained virtually unchanged at supra-equimolar ligand
concentrations.
Thermal stability of CYP3A4 was also affected by the S119A
substitution in a ligand-dependent fashion. The melting
temperature for the ligand-free and ritonavir-bound mutant
was increased by 1.4 and 0.4 °C, respectively, indicative of some
stabilization of the protein fold. In contrast, Tm for the 1-, 2-,
and 3-bound CYP3A4 was 1−1.6 °C lower than the respective
values derived for the wild type (WT; Tables 2 and 3). It can be
concluded, therefore, that formation of the Ser119-mediated H-
bond not only assists association of compounds 1−3 but also
stabilizes the resulting complexes. The lower flexibility of
ritonavir and its inability to optimally orient in the active site
are the likely reason for the lack of the stabilizing effect of the
Ser119-mediated interaction.
Table 2. Effect of the S119A Mutation on the CYP3A4−
Inhibitor Interaction
a
Ks (nM)
kfast (s−1
)
Tm (°C)
ritonavir
240 20
0.25 0.03
0.17 0.02
0.20 0.03
0.32 0.04
55.2 0.2
55.5 0.2
53.2 0.1
55.6 0.2
1
2
3
67
970 80
65
5
9
a
Melting temperatures for the ligand-free CYP3A4 S119A in the
absence and presence of 2% DMSO are 54.0 0.1 and 53.5 0.1 °C,
respectively.
○
●
Figure 7. Plots of the observed rate constants for the fast ( ) and slow ( ) kinetic phases of the ritonavir and compounds 1−3 binding to CYP3A4
S119A vs ligand concentration.
E
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Table 3. X-ray Data Collection and Refinement Statistics
ligand
water
2
3
Data Statistics
space group
I222
I222
I222
unit cell parameters
resolution range
total reflections
unique reflections
redundancy
a = 76 Å, b = 99 Å, c = 125 Å; α, β, γ = 90° a = 76 Å, b = 99 Å, c = 126 Å; α, β, γ = 90° a = 78 Å, b = 100 Å, c = 131 Å; α, β, γ = 90°
a
65.3−2.60 (2.67−2.60)
115923
49.8−2.72 (2.79−2.72)
114890
65.7−2.90 (2.98−2.90)
50075
16260
12236
11329
7.1 (4.5)
9.4 (5.4)
4.4 (4.5)
completeness
average I/σI
98.4 (97.5)
10.3 (2.3)
99.9 (98.9)
18.9 (3.5)
97.2 (99.0)
10.9 (2.5)
Rmerge
0.082 (0.516)
0.072 (0.505)
0.068 (0.548)
Refinement Statistics
b
R/Rfree
22.0/29.8
20.7/26.9
19.5/26.9
rmsd
bond lengths
(Å)
0.011
2.0
0.009
1.89
0.011
1.98
bond angles
(deg)
a
b
Values in brackets are for the highest resolution shell. Rfree was calculated from a subset of 5% of the data that were excluded during refinement.
In contrast, here we show that the pyridine-substituted
desoxyritonavir (compound 3) is superior to ritonavir in terms
of binding affinity and inhibitory potency for CYP3A4 (Table
1). The extremely tight binding of 3 to CYP3A4 is likely due in
part to a favorable interaction of the heme iron with the
pyridine ring in the ligand, involving both σ-donation by the
pyridine nitrogen lone pair and back-bonding of iron d-orbitals
to antibonding orbitals of the aromatic π-system. An analogous
mode of binding is possible with an sp2 lone pair of the
imidazole-containing analogue, compound 1, which on the
basis of σ-donating ability alone would be expected to form the
tighter complex because of its greater intrinsic basicity. The
reduced effectiveness of back-bonding from iron to imidazole,
however, results in a lower overall binding affinity of 1. In
addition, the imidazole ring has two tautomeric forms each with
an sp2 lone pair that could in principle complex with the iron,
and neither of which appears to map closely onto the
corresponding nitrogen of the pyridine analogue. Thus, it is
also possible that the stereoelectronic requirements for
favorable binding play a significant role in controlling the
binding affinities due to the constraints imposed by the
tethered heteroatoms in each ligand. The magnitude of the 442
nm absorption of the ferrous species (negligible for the
CYP3A4−1 complex; Figure 1) may reflect the Fe−N bonding
strength and, hence, this parameter should be taken into
consideration together with the Ks values during spectral
evaluation of potential CYP3A4 inhibitors. The fact that
compound 1 potently inhibits the BFC hydroxylating activity of
CYP3A4 (Table 1) can be explained by a large negative shift in
the heme redox potential, which may completely block an
electron flow from CPR. Most likely, 1 gets expelled from the
active site during crystallization because of the less favorable
stereoelectronic properties of the imidazole nitrogen, leading to
a weaker, dissociable Fe−N bond.
DISCUSSION
■
Inhibition of the drug-metabolizing CYP3A4 is usually
undesired and considered as a liability.11 A successful utilization
of CYP3A4 inhibitors in treatment of HIV infection,1 however,
suggests that such compounds could help fighting other chronic
conditions requiring administration of costly drugs that are
predominantly cleared by CYP3A4. Therefore, there is a need
for development of new boosters with improved pharmaco-
logical and physicochemical properties, which is not possible
without a full understanding of the CYP3A4 inhibitory
mechanism.
In contrast to the prevalent notion that ritonavir is a
mechanism-based inactivator of the CYP3A family of
enzymes,12−16 our studies on highly purified recombinant
CYP3A4 demonstrated that the key contribution to CYP3A4
inhibition is the ability of ritonavir to bind tightly and
irreversibly, causing a negative shift in the heme redox potential
which precludes an electron flux from CPR.7 Biochemical and
structural comparisons of ritonavir and DTMCR binding, in
turn, showed that tight association of ritonavir-like molecules to
CYP3A4 requires heme coordination, while hydrophobic
interactions provided by the side groups define the overall
binding mode.8 The atypical (nonhyperbolic, V-shaped)
ritonavir association kinetics was proposed to arise from the
existence of a peripheral site, to which the drug may dock prior
to moving into the active site cavity.8
In this work, we investigated how elimination of the ritonavir
hydroxyl group, strictly required for the HIV protease
inactivation, and substitution of the heme-ligating moiety
influence the ability to bind and inhibit CYP3A4. The effect of
thiazole-to-pyridine replacement in ritonavir has been pre-
viously investigated by assessing CYP-related spectral changes
and IC50 for the CYP3A terfenadine hydroxylase activity in
human liver microsomes.17 The magnitude of type II spectral
changes induced by ritonavir and its pyridine-substituted
derivative (Abbott laboratories compound A-83962) in micro-
somal CYPs was found to be similar, but the inhibitory potency
of A-83962 was 4-fold lower. Such a big difference in IC50 was
explained by a greater liability of the pyridine moiety to
chemical oxidation.
Our study also emphasizes the importance of two other
factors that could control the CYP3A4−ligand interaction and
define ligand binding mode. The first one is a structural
flexibility of the ritonavir-like molecules, which increases upon
elimination of the hydroxyl group. This allows 3 to adopt a
conformation that minimizes steric hindrance imposed by
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Phenyl-2 (Figure 6B). Most importantly, Phenyl-2 can now
orient parallel to the pyridine ring and the guanidinium group
of Arg105 (Figure 6C,D) to form π-stacking and cation−π
interactions, respectively, that stabilize the inhibitor-bound
complex. Clashing between Phenyl-1 and Phe304 and the
resulting displacement of the I-helix, however, remain
significant (Figure 6A). This unfavorable interaction can be
prevented if Phenyl-1 is substituted with a smaller hydrophobic
moiety.
Asp61, Asp76, Arg106, Arg372, and Glu374), analogous to that
observed in the ritonavir-bound structure,7 can be easily
established. In either case, the data presented here is sufficient
to conclude that compound 3 is a higher affinity ligand and a
more potent CYP3A4 inhibitor than ritonavir and, hence, it can
serve as a better template for developing novel pharmacoen-
hancers.
CONCLUSIONS
■
The second factor that can modulate both the rate of
formation and stability of the CYP3A4−inhibitor complexes is
the H-bonding interaction with Ser119 (Figure 6C). The active
site Ser119 was reported to regulate substrate specificity and
steroid hydroxylation activity of CYP3A4,18 as well as the
binding/egress of type II inhibitors metyrapone and
ketoconazole, and association of 4-aminopiperidine
drugs.19−22 Here we show that elimination of the Ser119
hydroxyl group significantly lowers the binding affinity of
ritonavir and compounds 1−3 and perturbs their ligation
kinetics (Tables 1 and 2; Supporting Information Figure 2S).
Furthermore, the S119A mutation transforms the hyperbolic
character of the kfast vs [compound 1−3] dependence into V-
shaped (Figures 3 and 7), and affects thermal stability of
CYP3A4 in a ligand-dependent manner (Figure 4A, Tables 1
and 2). On the basis of these findings, we suggest that (i)
Ser119 interacts with the inhibitor molecules at earlier stages of
the binding process, (ii) deviations from the hyperbolic binding
kinetics may, in part, be caused by conformational restraints
affecting the Ser119-mediated protein−ligand interaction, and
(iii) formation of the H-bond with Ser119 increases stability of
the CYP3A4−inhibitor complex when conformational re-
straints in the ligand are minimized or eliminated. The notably
decreased Tm and kfast and higher Ks values for the CYP3A4
S119A−3 complex (Tables 1 and 2) imply that the peptide flip,
bringing the carbonyl oxygen of 3 closer to the Ser119 hydroxyl
group, stabilizes the overall conformation and strengthens
inhibitor binding.
That preincubation of 3 and other ritonavir analogues with
NADPH does not significantly affect IC50 suggests an inhibitory
mechanism that does not involve formation of a reactive
metabolite. This agrees with our previous conclusions7 and a
recent study on human microsomal CYP3A4.23 Although
formation of reactive metabolites of ritonavir was proposed
over a decade ago,12,13,15 their identity remains unknown.
Comparative metabolomic screening of WT and Cyp3a-null
mice led to a conclusion that CYP3A enzymes can bioactivate
ritonavir via glycine conjugation and thiazole ring-opening
pathways.24 Metabolism of compound 3 has not been
investigated in this study, but the primary oxidation site is
expected to be the terminal isopropyl group. This follows from
the structural analogy of 3 to ritonavir12,24 and cobicistat, as
well as biphasic binding kinetics (Figure 3), indicative of
multiple orientation modes for ritonavir-like molecules within
the active site of CYP3A4.
The CYP3A4 inhibitor ritonavir has been successfully used as a
booster for anti-HIV drugs for over a decade. However, there is
presently a search for new CYP3A4 inhibitors that have
improved pharmacochemical properties and cause fewer side
effects. In this study, we investigated whether the CYP3A4
inhibitory potency of ritonavir can be further improved by
comparing the properties of three desoxyritonavir analogues
that contain the imidazole, oxazole, or pyridine group instead of
the heme-ligating thiazole (compounds 1, 2, and 3,
respectively). On the basis of the binding affinity, association
and inhibitory kinetics, stabilization effect, and the binding
mode of the investigated compounds, we conclude that 3 is a
stronger ligand and a more potent CYP3A4 inhibitor than
ritonavir. This, in part, is due to the favorable stereoelectronic
properties of the pyridine nitrogen and increased structural
flexibility of the desoxyritonavir backbone, which allows 3 to
establish a stronger Fe−N bond and adopt a conformation that
minimizes steric clashing and optimizes protein−ligand
interactions, in particular, with Ser119. Our mutagenesis data
show that the active site Ser119 is a key residue that not only
assists binding of ritonavir-like inhibitors but also stabilizes the
ligand-bound form. Thus, polar interactions are important in
the CYP3A4−ligand association and need to be taken into
consideration during the drug design process. Compound 3
may serve as a starting template for the development of a new
generation of potent CYP3A4 inhibitors. One way for its
structural optimization could be replacement of the Phenyl-1
side group with a smaller hydrophobic moiety in order to
prevent steric clashing with Phe304 and the I-helix displace-
ment.
EXPERIMENTAL SECTION
■
Synthesis of compounds 1−3 is described in the Supporting
Information. The purity of 1−3 was >95% as determined by HPLC
analysis.
Protein Expression and Purification. C-terminal His-tagged WT
and S119A CYP3A4Δ3-22 were expressed in Escherichia coli, purified,
and quantified as reported previously.7
Spectral Binding Titrations. Ligand binding to WT and S119A
CYP3A4 was monitored spectrophotometrically. Proteins were titrated
with small aliquots of dimethyl sulfoxide (DMSO) solutions of
ritonavir (Toronto Research Chemicals), 1, 2, or 3 in 50 mM
phosphate, pH 7.4, 20% glycerol, and 1 mM dithiothreitol (buffer A).
The final solvent concentration did not exceed 2%. Spectral
dissociation constants (Ks) were determined as described in detail
elsewhere.7
Kinetics of Ligand Binding. Kinetics of ligand binding to
CYP3A4 was monitored at room temperature in a SX.18MV stopped
flow apparatus (Applied Photophysics, UK). Protein solutions (2 μM)
were mixed with various concentrations of inhibitors in 50 mM
phosphate, pH 7.4, and absorbance changes were followed at 426 nm
for ritonavir and 2, and at 430 and 425 nm for 1 and 3, respectively.
Owing to low solubility, the maximal concentration of compounds 1−
3 used in the stopped flow experiments was 36 μM. Kinetic data were
analyzed using the program IgorPro (WaveMetrics, Inc.).
Owing to insufficient resolution or/and high thermal motion
of the 3 isopropyl-thiazole, the role of this terminal group in the
interaction with CYP3A4 could not be determined. It should be
noted though that the CYP3A4 active site region is similar in
both 3- and ritonavir-bound structures (the rms deviation for
the 457 Cα atoms is only 0.53 Å), and the isopropyl-thiazole of
3 can be modeled in a nearly identical conformation (Figure
6B). This means that a water-mediated bridge between the
isopropyl-thiazole nitrogen of 3 and “polar umbrella” (residues
G
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Redox Potential Measurement. Redox potentials of the
inhibitor-bound CYP3A4 were estimated spectroscopically as
previously described7 using benzyl viologen (−359 mV) as a redox
dye.
Thermal Denaturation. CYP3A4 (2 μM) in the absence and
presence of 10 μM inhibitors or 2% DMSO was heated in 100 mM
phosphate buffer, pH 7.4, in a controlled way using a Cary 3
spectrophotometer, with a ramp rate of 0.5 °C/min and the 37−65 °C
temperature range. Protein denaturation was monitored at 280 nm. A
denaturation midpoint (melting temperature, Tm) was determined as a
temperature at which the folded and unfolded states are equally
populated.
Inhibitory Potency Assays. The inhibitory potency of ritonavir
and compounds 1−3 on the BFC hydroxylase activity of CYP3A4 was
evaluated fluorometrically in a reconstituted system with CPR. The
reaction was carried out at room temperature in 25 mM phosphate
buffer, pH 7.4, containing 3 mM MgCl2. CYP3A4 (0.3 μM) and rat
CPR (0.6 μM) were incubated for 5 min with various concentrations
of inhibitors in the absence and presence of 100 μM NADPH and then
mixed with 50 μM BFC. Formation of 7-hydroxy-4-trifluoromethyl-
coumarin was followed in a Hitachi F100 fluorimeter with λex = 430
nm and λem = 500 nm. A concentration required for half-maximal
inactivation (IC50) was derived from the [% activity] vs [inhibitor]
plots.
Crystallization and Determination of the X-ray Structures of
the CYP3A4−Inhibitor Complexes. CYP3A4 was crystallized in the
presence of inhibitors by a microbatch method under oil. Ligand-
bound CYP3A4 (0.6 μL, 54−56 mg/mL, with a 2−3-fold ligand
excess) in buffer A was mixed with 0.6 μL of 10−11% polyethylene
glycol 3350 and 90−100 mM sodium malonate, pH 5.9−6.0, and
covered with paraffin oil. Crystals grew within several days at room
temperature and belonged to the I222 space group, with one molecule
per asymmetric unit. X-ray diffraction data were collected at the
Stanford Synchrotron Radiation Laboratory beamline 7−1, using
Paratone-N as a cryoprotectant. Crystal structures were solved by
molecular replacement with PHASER25 and the 1TQN or 3NXU
structure as a search model. Only water molecules were present in the
active site of CYP3A4 crystallized in the presence of compound 1. The
water-, 2-, and 3-bound models were refined to 2.60, 2.72, and 2.9 Å
resolution with COOT26 and REFMAC,25 with the final R/Rfree values
of 22.0/29.8, 20.7/26.9, and 19.5/26.9, respectively. Data collection
and refinement statistics are summarized in Table 3.
the Stanford Synchrotron Radiation Laboratory, a national user
facility operated by Stanford University on behalf of the U.S.
Department of Energy, Office of Basic Energy Sciences. The
SSRL Structural Molecular Biology Program is supported by
the Department of Energy, Office of Biological and Environ-
mental Research, and by the National Institutes of Health,
National Center for Research Resources, Biomedical Technol-
ogy Program, and the National Institute of General Medical
Sciences. We thank Dr. R. Chamberlin for helpful discussions
and critical reading of the manuscript and Lianhong Xu and
Hong Ye of Gilead Sciences for synthesizing and providing the
compounds used in this study.
ABBREVIATIONS USED
■
CYP, cytochrome P450; CYP3A4, 3A4 isoform of cytochrome
P450; CPR, cytochrome P450 reductase; DMSO, dimethyl
sulfoxide; DTMCR, desthiazolylmethyloxycarbonyl ritonavir;
WT, wild type
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of compounds 1−3, experimental evidence for
reversibility of the 1 and 2 binding to CYP3A4, and spectral
and kinetic data on association of ritonavir, 1, 2, and 3 to
CYP3A4 S119A. This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
Atomic coordinates and structure factors for the water-, 2-, and
3-bound CYP3A4 have been deposited to the Protein Data
Bank with the ID codes 4I3Q, 4I4G, and 4I4H, respectively.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 1 949 8241953. Fax: 1 949 8243280. E-mail:
sevrioui@uci.edu.
Notes
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Huang, M.; McLeod, H. L. Mechanism-based inhibition of cytochrome
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(12) Koudriakova, T.; Iatsimirskaia, E.; Utkin, I.; Gangl, E.; Vouros,
P.; Storozhuk, E.; Orza, D.; Marinina, J.; Gerber, N. Metabolism of the
human immunodeficiency virus protease inhibitors indinavir and
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health grant
GM33688, Gilead Sciences, Inc., and the California Center for
Antiviral Drug Discovery and involves research carried out at
H
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