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School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom (A.G., J.B.H.); and Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire, United Kingdom (S.E.C.)
(Received March 28, 2003; accepted May 28, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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factor (defining the alteration in
the binding affinity in the presence of a modifier) ranging from 0.04 to 2.3.
The conclusions from the multisite kinetic analysis performed support the
hypothesis of distinct binding domains for each substrate subgroup.
Furthermore, the analysis of intersubstrate interactions strongly indicates
the existence of a mutual binding domain common to each of the three CYP3A4
substrate subclasses.
; Stresser et al.,
2000; Wang et al.,
2000; Lu et al.,
2001), pathway-differential effects
(Shou et al., 2001a;
Galetin et al., 2002), and
heteroactivation (Ludwig et al.,
1999; Tang et al.,
1999; Ngui et al.,
2000; Kenworthy et al.,
2001) are all phenomena observed in in vitro drug-drug interaction
studies that are not commonly incorporated into in vitro-in vivo scaling and
prediction procedures. The above, and other non-Michaelis-Menten kinetic
properties, are frequently linked with CYP3A4
(Kenworthy et al., 2001;
Shou et al., 2001b;
Tang and Stearns, 2001;
Galetin et al., 2002), but
recent studies indicate atypical kinetics for some other human enzymes, namely
CYP2C9 (Hutzler et al.,
2001b), UDP-glucuronosyltransferase 1A1
(Williams et al., 2002), and
2B7 (Stone et al., 2003). The
complexity of effects seen in the homotropic situation when more than one
molecule of the same substrate is present at the active site
(Shou et al., 1999;
Lin et al., 2001) is increased
for heterotropic interactions involving two different substrates
(Ueng et al., 1997;
Korzekwa et al., 1998).
Heterotropic effects (either activation or inhibition) require a more
elaborate approach involving either the simultaneous binding of two different
substrates to the active site (Shou et al.,
1994,
2001b) or possibly an effector
site (Ueng et al., 1997;
Domanski et al., 2001;
Kenworthy et al., 2001;
Galetin et al., 2002), and
hence there is an increase in the number of enzyme complexes formed.
The significance of nonstandard Michaelis-Menten data in vitro, and their
correlation with the in vivo situation, remains ambiguous
(Atkins et al., 2002); to date,
there are few confirmatory studies in vivo
(Tang et al., 1999;
Egnell et al., 2003), and in
certain cases, their relevance could be questioned
(Hutzler et al., 2001a;
Ngui et al., 2001). The
introduction of maximum clearance as an alternative for intrinsic clearance
(Houston and Kenworthy, 2000)
represents one attempt to introduce autoactivation into the in vitro-in vivo
scaling strategy. The dependence of clearance on substrate concentrations
below the Ks is associated with positive cooperativity and
indicates the possibility of clearance underestimation in rapid in vitro
screening procedures based on only one substrate concentration
(Houston and Kenworthy, 2000).
In terms of predicting drug-drug interactions in vivo, the use of multiple
substrates in vitro at various substrate concentrations is recommended to
explore the range of possible consequences of a heterotropic interaction
(Kenworthy et al., 1999).
The selection of CYP3A4 substrates employed for in vitro testing is mainly
based on three distinct CYP3A4 subgroups, first identified via a number of
statistical tests, including cluster analysis, by Kenworthy et al.
(1999) and substantiated by
others (Stresser et al., 2000;
Lu et al., 2001). A recent
collation (Galetin et al.,
2002) of CYP3A4 interactions from our laboratory
(Houston and Kenworthy, 2000;
Kenworthy et al., 2001;
Galetin et al., 2002) and
elsewhere (Wang et al., 2000;
Ngui et al., 2001,
Shou et al., 2001a) has
highlighted the value of multisite interaction factors to rationalize the
range of atypical Michaelis-Menten kinetic effects observed. Here we expand
this approach by performing a multisite kinetic analysis of mutual
interactions of the three most commonly used CYP3A4 substrates
(Yuan et al., 2002): midazolam
(MDZ1), testosterone
(TST), and nifedipine (NIF). In addition to their role as representative
prototypes of the CYP3A4 substrate subgroups, MDZ, TST, and NIF show
distinctive kinetic properties namely, hyperbolic, sigmoidal, and substrate
inhibition, respectively. Felodipine (FEL), a structural analog to NIF, was
included in these studies for further evaluation and possible inclusion in the
NIF distinct CYP3A4 subgroup.
Several distinctive types of CYP3A4 interactions are reported here for MDZ, TST, NIF, and FEL (e.g., cooperative and partial inhibition, pathway-differential effects, concentration-dependent positive and negative homotropy), and their association with specific multisite interaction factors is defined. Furthermore, additional kinetic evidence for the existence of mutual and distinct substrate-binding domains for particular substrate subgroups within the CYP3A4 active site is presented.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Incubation Conditions. Interaction studies were performed at
incubation times and protein concentrations within the linear range for each
individual substrate. Microsomes from human B-lymphoblastoid cells containing
recombinant human CYP3A4/OR were suspended in phosphate buffer (0.1 M, pH
7.4). The final incubation volume was 0.2 ml, containing 47 to 111 pmol of
P450/ml. Samples were preincubated for 5 min in a shaking water bath at
37°C, and each reaction was initiated with an NADPH-regenerating system (1
mM NADP+, 7.5 mM isocitric acid, 15 mM magnesium chloride, and 0.2
unit of isocitric dehydrogenase). The substrates (concentration ranged from at
least 
Km to 2Km) were added to
each incubation in either methanol or phosphate buffer depending on the
solubility. Neither of the substrates showed significant microsomal binding
(<10%). The final concentration of methanol in incubation media was
0.5% (v/v). The range of modifier concentrations applied was from 0.5 to
100 µM in most studies. The reaction was terminated by 0.1 ml of ice-cold
methanol. Samples were then centrifuged at 13,400g for 5 min and
analyzed by high-pressure liquid chromatography-UV or liquid
chromatography-tandem mass spectrometry as described earlier
(Galetin et al., 2002).
Data Analysis. The kinetic parameters for each substrate alone and
in the presence of an inhibitor were obtained from untransformed data by
nonlinear least-squares regression using GraFit 4 (Erithacus Software Ltd.,
Horley, Surrey, UK). In the case of FEL and MDZ, the Michaelis-Menten equation
with the weighting factor of 1/y was used for preliminary kinetic analysis.
Preliminary analysis of NIF kinetic data was carried out assuming single-site
Michaelis-Menten kinetics with substrate inhibition
(Houston and Kenworthy, 2000).
Kinetic parameters Vmax, S50, and Hill
coefficient (n) were calculated from untransformed data using the
Hill equation for initial analysis of TST kinetics. In addition to the Hill
equation, a two-site model (eq. 3;
Kenworthy et al., 2001) was
also used for the preliminary analysis of the TST data in the presence of
increasing concentrations of the modifiers. The changes in kinetic parameters
observed in the presence of various modifiers were significance tested using
analysis of variance.
Further data analysis to provide a more detailed model of the molecular events was based on the application of various steady-state and rapid equilibrium multisite kinetic approaches. Two- and three-site models and the corresponding equations derived assume the existence of particular substrate-binding domains within the active site. Various interaction factors are defined in order to characterize the effect of a certain modifier. There are more factors involved in a heterotropic than a homotropic interaction due to the increased number of enzyme complexes and binding sites involved and a possible overlap between the sites for substrate and modifier.
The kinetic models applied assume rapid equilibrium, i.e., the rate at
which ES/SE complex dissociates is much faster than the rate of product
formation (Segel, 1975). In
all the cases (apart from pathway-differential effects and substrate
inhibition kinetics), two substrate-binding sites were assumed to be
identical, with no distinguishable difference between ES and SE conformations.
Each complete data set (n = 20–30) in the presence and absence
of the modifier was fitted to the rate equations for various multisite kinetic
models using GraFit. The least number of parameters, lowest standard errors of
the parameter estimates, and consistency with kinetic properties of both the
substrate and modifier represent the principal criteria for the selection of a
certain model. Goodness of fit was determined by comparison of statistical
parameters (
2 and Akaike information criterion values) between
the models and a reduction in the standard errors of the parameter estimates.
Kinetic parameter estimates generated from different multisite kinetic models
were used to simulate velocity curves for metabolite formation. A major
advantage of this type of kinetic analysis is the ability to simultaneously
fit all the data covering the range of modifier concentrations, in contrast to
the preliminary analysis (e.g., Hill plots) in which individual fits are
obtained for each specific concentration of modifier. The enzyme complexes
that are involved in metabolite formation are in the numerator of all the
equations, whereas the denominator contains all the enzyme complexes present
(Segel, 1975).
Use of the Multisite Kinetic Approach. Many authors seem uncertain
about how to deal with cooperativity/unusual kinetics, and the literature
contains numerous examples of standard Michaelis-Menten hyperbolic curves
forced through data that clearly show atypical kinetic features. In some of
these cases, the insufficient number of data points rules out any meaningful
selection of an alternative model. We have found that multisite models provide
a valuable insight into complex CYP3A4 interactions
(Kenworthy et al., 2001;
Shou et al., 2001b;
Galetin et al., 2002), once
certain practical issues of dealing with the data that cannot be described by
the Michaelis-Menten model are addressed.
For the purposes of the mechanistic studies, recombinant systems have
proved to be a better defined and more controlled system in comparison to
human liver microsomes. However, concentrations of accessory proteins (e.g.,
OR and cytochrome b5) can differ considerably between
these two systems (Venkatakrishnan et al.,
2000), and their lack/addition, as well as membrane lipid
composition and ionic strength of the in vitro matrix, may also affect CYP3A4
catalytic activity.
According to the kinetic properties of the substrate, variations of the
generic two-site model (Galetin et al.,
2002) were applied to rationalize the inhibition profiles obtained
for NIF, FEL, and MDZ. These two-site kinetic models accommodated a range of
effects and varied in the number and type of corresponding interaction
factors, associated with either binding affinity (
, ) or rate of
product formation (ß, 
) (Fig.
1A). The assumption of a fast release of products
(Segel, 1975) was the
rationale for considering the metabolism of each substrate molecule
independently. However, the kinetic properties of the effector, alterations in
substrate and/or modifier binding affinity (, ), and catalytic
efficiency upon effector binding () were also considered.
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The initial step in the selection of a model and the relevant interaction
factors (, ß, , or ) is highly dependent on the
kinetics of the substrate (hyperbolic, substrate inhibition, or sigmoidal), as
illustrated in Scheme 1 by the
values of and ß. The net effect of the increased binding affinity
in the presence of another substrate ( < 1) can range from
heteroactivation to inhibition depending on the corresponding value
(changes in the rate of metabolite formation). In contrast, partial inhibition
is typically characterized by the decreased affinity of a second inhibitor
molecule for a binding site in the presence of another substrate.
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The possibility of enzyme-product complex formation, which would lead to
reduced enzyme availability for the substrate interaction and decreased rate
of the reaction (Narasimhulu et al.,
1998), could be an issue of concern. However, these complexes were
not included in the total sum of the metabolically productive complexes in the
model derivation, in order to keep the modeling procedure relatively
simple.
Positive Cooperative Inhibition. Cooperative inhibition profiles
result from binding of a second inhibitor molecule in a cooperative manner to
the enzyme active site, indicated by the steeper slope of IC50
plots (>1) compared with the standard one-site type of inhibition. This
phenomenon is analogous to the positive cooperativity observed for some CYP3A4
substrates (e.g., TST), as the binding affinity of the second inhibitor
molecule increases in the presence of the first. The enhanced extent of
inhibition with increasing inhibitor concentrations is characterized by the
changes in Ki value by the factor i,
where i < 1.
In addition to alterations in the binding affinity, cooperative inhibition
profiles can be attributed to changes in the product formation
(Kp) by the factor , with a decrease in the overall
rate of the reaction when < 1 (eq. 1). In cases where no changes to
the effective catalytic rate constant are observed ( = 1), the
corresponding equation can be simplified by eliminating .
 | (1) |
The opposite effect, decreased binding affinity of the second inhibitor
molecule in the presence of the first (negative cooperativity,
I > 1), is associated with partial inhibition, where full
inhibition is not achieved at high inhibitor concentrations.
Negative Cooperativity and Partial Inhibition. Partial inhibition is
characterized by incomplete inhibition, even at saturating concentrations.
Competitive and noncompetitive types could be distinguished, depending on
whether changes in either binding affinity (Ks) or product
formation (Kp) are observed in the presence of a modifier.
Partial competitive inhibition (regardless of the substrate kinetic
properties) is characterized by decreased binding affinity of a second
I molecule for the binding site in comparison with the first
(Ki > Ki), analogous to the
negative cooperativity. Simultaneous presence of both S and
I at the active site and access to active oxygen enables the
complexes with I to be productive, leading to unchanged
Kp and Vmax for the reaction (
= 1, and it can be eliminated from the equation). In cases when partial
inhibition occurs via competition at only one binding site, the application of
a simpler model is possible, as in the case of NIF-FEL interaction (eq. 2).
 | (2) |
Inhibition of a Substrate Showing Substrate Inhibition Kinetic
Properties (Loss of Negative Homotropy). A two-site model, with only one
catalytically active site, has been applied for all NIF interactions, as
described previously (Galetin et al.,
2002). The "substrate inhibition" site cannot be
occupied until the active site is filled (sequential binding of substrate
molecules). The presence of a substrate in the second binding site causes a
decrease in product formation from SES, defined by the factor ß (<1).
Similar to all cases described by the generic two-site model, the interaction
factor is associated with the alterations in the product formation due
to the presence of an inhibitor molecule at the active site.
When is comparable to ß, the effect of a modifier is analogous
to the binding of a second substrate molecule, and the substrate inhibition
phenomenon remains. However, at high concentrations of substrate and
inhibitor, the profile changes to a hyperbolic curve due to dominance of the
nonproductive S(EI) complex.
 | (3) |
Heterotropic Inhibition of a Substrate Showing Sigmoidal Kinetics (Loss
of Positive Homotropy). Derived from a three-site model described
previously for the interactions of quinidine (QUI) and TST
(Galetin et al., 2002), the
model presented in Fig. 1B describes the inhibition of substrates showing sigmoidal kinetics, in which
the inhibitor eliminates substrate cooperativity. In the absence of the
inhibitor, the substrate binds cooperatively with an interaction factor
(<1). However, the interaction between two substrate-binding sites
resulting in an increase in the affinity of the vacant substrate sites is
prevented in the presence of the inhibitor. The increased affinity of SE/ES
and SES complexes for the inhibitor molecule is defined by an alteration in
the Ki value by the factor (<1) (eq. 4). At the
same time, the enzyme complexes containing both substrate and inhibitor
molecules are not productive; hence, the Vmax could be
driven to zero values at very high inhibitor concentrations.
 | (4) |
Pathway-Differential Effects. In addition to describing interactions
for substrates with sigmoidal kinetic properties, the three-site kinetic model
approach is more appropriate for elucidating the phenomenon of
pathway-differential effects. Equation 5 is derived for 1'-OH MDZ
formation using a three-site model with two distinct substrate-binding sites;
ES1 is preferable for 1'-OH MDZ (defined by
Ks1 and Kp1) and S2E for
4-OH MDZ formation (Ks2 and Kp2)
(Fig. 1C). In contrast to a
similar model applied for the effect of QUI on MDZ
(Galetin et al., 2002), no
interaction between the two occupied MDZ binding sites is assumed. Competition
between TST and MDZ for the mutual site is characterized by the inhibition of
1'-OH MDZ formation. At the same time, binding of TST molecules to a
distinct effector site alters the kinetic properties of the substrate site
preferential for the 4-OH formation, stimulating the metabolite formation
(
Kp2 > Kp2).
 | (5) |
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). A range of
substrate-dependent differences is observed in the inhibitory potency that
prevents any consistent rank ordering. The lack of relationship between the
Ks for the particular probes and their respective
Ki values is shown in
Fig. 2.
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TST activated NIF oxidation in lymphoblast-expressed CYP3A4 (36% of control value 2.59 pmol/min/pmol of P450 at a 10 µM substrate concentration). Similarly, in the TST-FEL interaction, slight activation of FEL metabolism occurred at low substrate concentrations, changing to inhibition at higher concentrations of both substrate and modifier; the effects did not exceed 20% of the control value. No further modeling of the TST effect on NIF/FEL metabolism was performed.
Inhibition was observed for the 10 other interactions and was characterized by changes either in the binding affinity or in the product formation or a combined effect, defined by following multisite interaction factors (see Tables 1, 2, 3):
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< 1, increased binding affinity
for the formation of ISE/ESI/ISES complexes (as seen for the NIF/FEL/MDZ
effect on TST, the FEL effect on NIF, and the MDZ effect on FEL); or b)
> 1, nonfavorable formation of ISE/SEI/ISES resulting in partial
inhibition (TST effect on MDZ and NIF effect on FEL). < 1, less productive complexes with
I (NIF/FEL effect on MDZ resulting in cooperative inhibition and
MDZ/FEL effect on NIF), or b) = 0, nonproductive complexes
(interactions with TST as a substrate).
To systemically approach the various kinetic phenomena observed and link the observations with particular interaction factors, four distinct types of inhibition (A-D) and a pathway-differential effect (E) have been identified.
A. Cooperative Competitive Inhibition—Exemplified by the NIF-MDZ and FEL-MDZ Interactions. The enhanced inhibition of MDZ 1'-hydroxylation observed with increasing inhibitor (FEL, NIF) concentrations and the steeper slopes of the IC50 plots (>1) at high S and I concentrations indicate the cooperative binding of the inhibitor (Fig. 3, A and B, respectively). Preliminary kinetic analysis, applying a one-site model, suggested a competitive nature of the inhibition, demonstrated by a 4- and 17-fold (p < 0.05) increase in the Km value for 1'-OH MDZ metabolic pathway in the presence of NIF and FEL, respectively. Although the simple one-site competitive inhibition model generated a satisfactory fit for the range of low I concentrations, it did not predict the enhancement of the inhibition observed with increasing concentrations of I.
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Ki values obtained from the generic two-site model are
similar for FEL and NIF (Table
1). A correlation plot of the inhibitory effects of NIF and FEL at
various MDZ concentrations (Fig.
3C) is consistent with these findings. The binding affinity of the
second FEL molecule is higher in the presence of the first ( = 0.24).
However, this cooperativity in binding of FEL molecules contrasts with the
Michaelis-Menten kinetics displayed by this substrate
(Eriksson et al., 1991;
Galetin et al., 2002). In
addition, the enhanced inhibition can be attributed to a significant decrease
in product formation in the presence of a modifier, defined by a low
value (0.4). The formation of the IES complex is less favorable, described by
altered values for the MDZ binding constant (Ks
> Ks).
NIF substrate inhibition kinetic properties and sequential binding to the
active site are incorporated in the generic two-site model. The enhancement of
1'-OH MDZ inhibition at higher NIF concentrations, also manifested by
changes to Vmax, is analogous to the binding of the second
NIF molecule to the active site causing substrate inhibition. Minimal
interaction between NIF molecules is observed ( = 0.9), but the
substantial effect on product formation ( = 0.2) correlates well with
the observed cooperativity in IC50 plots
(Fig. 3B).
B. Partial Inhibition—Exemplified by the TST-MDZ and NIF-FEL
Interactions. Similar to previously reported studies with other
substrates, erythromycin (Wang et al.,
1997), triazolam (Schrag and
Wienkers, 2001), and terfenadine
(Wang et al., 2000), TST also
partially inhibited MDZ 1'-OH pathway
(Fig. 4A), while activating
4-hydroxylation (described below). The IC50 plots obtained over the
range of substrate (MDZ) concentrations (5–20 µM) and the 5-fold
increase in IC50 values observed (114–537 µM) are
consistent with a competitive type of inhibition. However, a slope <1, the
high plateau of uninhibited activity at high I concentrations, and a
interaction factor of 2.2 (Table
1) are associated with partial inhibition. The competitive nature
of the observed interaction is confirmed in a 3-fold increase in MDZ
Km value (4.2–11.4 µM, p < 0.05) and
no alterations in Vmax values, even at high TST
concentrations (100 µM).
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Partial inhibition may be a result of either partial interactions at both
sites or via competition at only one binding site. An example of the latter
type is the effect of NIF on FEL pyridine formation, over the 5 to 100 µM
FEL concentration range. In this case, NIF partially shields only one of the
FEL sites from the active oxygen, allowing the application of a simpler
kinetic model (eq. 2) to generate a Ki value of 33 µM
and a factor of 2.1 (Table
2). Figure 4B
illustrates another way of distinguishing partial from pure competitive
inhibition by plotting the rates of metabolism at fixed substrate
concentrations (e.g., 100 µM FEL) in the presence of increasing inhibitor
concentrations (0.5–500 µM NIF). Unlike the competitive inhibition
situation (velocity of the reaction minimized at high concentrations of an
I), partial or negative cooperative inhibition results in a limiting
plateau (ES and ESI are still productive), the level determined by the value
of the interaction factor .
C. Inhibition of a Substrate with Substrate Inhibition Kinetics (Loss of
Negative Homotropy)—Exemplified by the Effect of MDZ/FEL on NIF.
Reduced product formation at high NIF concentrations, associated with
substrate inhibition, is defined by the interaction factor ß < 1
(Fig. 1A). In the presence of
low MDZ concentrations, this phenomenon is still observed, as the rate of NIF
product formation from IES complex is analogous to that from SES, due to
comparable values of interaction factors and ß (0.44 and 0.41,
respectively). However, at higher S and I concentrations,
the nonproductive I complex (S(EI)) dominates, changing the shape of
the profile into a hyperbolic type (Fig.
5A). An analogous phenomenon occurs with NIF in the presence of
FEL (ß and of 0.44 and 0.56, respectively). Comparable inhibitory
potency of FEL and MDZ based on Ki comparison (see
Table 2) was not expected from
the Ks values for these substrates
(Fig. 2). This discrepancy and
the higher inhibitory potency of FEL are more evident when the alterations in
binding affinities for FEL/MDZ in the presence of NIF are considered (lower
value of = 0.62 for FEL in comparison to MDZ).
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D. Inhibition of a Substrate with Sigmoidal Kinetics (Loss of Positive Homotropy)—Exemplified by the NIF/FEL/MDZ Interactions with TST. The effect of NIF/FEL on TST 6ß-hydroxylation was substrate/modifier concentration-dependent with no effect at a low TST (10 µM) concentration and 1 µM FEL, achieving full inhibition in the 50 to 200 µM TST concentration range. In the case of MDZ, similar inhibitory potency was observed (IC50 = 4.4 µM) regardless of the TST concentration studied.
The initial analysis by a two-site model (eq. 3;
Kenworthy et al., 2001) shows
a decrease in Vmax values to 14, 21, and 28% of control
values in the presence of 50 µM FEL, NIF, and MDZ, respectively. Positive
cooperativity in TST binding is defined by the interaction factor <
1 (Kenworthy et al., 2001;
Galetin et al., 2002).
Depending on the affinity of a modifier for the active site, the possible
overlap with TST binding sites, or the binding to a separate effector site,
interactions can affect TST cooperativity. The extent of the decrease in
Ks values (2.5- to 4.8-fold) and the increase in
Ks values (9- to 38-fold) are consistent for these
three modifiers. At concentrations above 5 µM NIF, FEL, and MDZ appear to
prevent the interaction between two TST substrate-binding sites and reduce the
cooperativity, as seen in the linear shape of Eadie-Hofstee plots
(Fig. 5B).
Table 3 shows the kinetic
parameters for the effect of NIF, FEL, and MDZ on TST, generated from the
multisite model by a simultaneous fit to eq. 4. The increased affinity of
ES/SE/SES complexes for the I is defined by low values of
(0.04, 0.08, and 0.14 for FEL, MDZ, and NIF, respectively). In all three
studies, Vmax values were minimized at high I
concentrations, as a result of the formation of the metabolically
nonproductive ESI, SEI, and SESI complexes. The binding of an effector (NIF,
FEL, or MDZ) at two sites is consistent with the previously described effects
of QUI and haloperidol (HAL) on these compounds when investigated as CYP3A4
substrates (Galetin et al.,
2002).
E. Pathway-Differential Effects for MDZ. In addition to partially
inhibiting MDZ 1'-hydroxylation, TST activates the minor 4-OH pathway up
to 50% at 50 µM MDZ, in a manner similar to QUI
(Galetin et al., 2002). In
contrast, neither the NIF nor the FEL interaction results in a
pathway-differential effect. A three-site kinetic model, with two distinct
substrate-binding sites (Ks1 = 5.1 ± 0.3 and
Ks2 = 8.6 ± 0.3 µM; each preferable to one
particular MDZ pathway), best accommodated this differential effect of TST on
MDZ pathways. Competition for the mutual binding site for MDZ and TST causes
the partial competitive inhibition of 1'-hydroxylation (described
earlier), as a result of a decreased binding affinity of a second TST molecule
(Ki - 306 µM > Ki). TST
binding to the distinct effector site influences the substrate site
preferential for 4-OH MDZ, stimulating this pathway by increasing the product
formation (Kp2 > Kp2),
without affecting the binding (Ks2).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Whenever possible, the simplest kinetic inhibition model should be employed
to obtain kinetic parameters, and often the inclusion of all possible enzyme
complexes in the data analysis is not necessary (e.g., steric restrictions
resulting in the interaction at only one site). However, adoption of one-site
models for the analysis of enzymes known to exhibit atypical interactions has
severe limitations. For example, a competitive inhibition model may generate a
satisfactory fit for the range of low inhibitor concentrations, but the
cooperativity of the inhibition at higher inhibitor concentrations cannot be
predicted (e.g., the effect of FEL on 1'-OH MDZ formation).
Additionally, certain data sets may show some of the features of competitive
inhibition but not consistently (e.g., the effect of QUI on TST;
Galetin et al., 2002). The
application of simple models for interactions involving substrates with
positive (testosterone, diazepam) or negative (terfenadine, nifedipine)
homotropic kinetic properties is particularly problematic, and the misuse of a
one-site model may lead to inaccurate estimation of kinetic parameters and
failure to identify important drug-drug interactions.
Interactions at the TST Site(s). TST slightly activated the
metabolism of NIF and FEL, whereas both dihydropyridines inhibited TST
6ß-hydroxylation. These diametrical drug-drug interaction patterns are in
agreement with other findings reported for TST
(Wang et al., 2000;
Kenworthy et al., 2001;
Lu et al., 2001). The fact
that this substrate represents the most used and recommended in vitro probe
for CYP3A4 (Yuan et al., 2002)
is, therefore, of concern.
Ki values for the inhibition of 6ß-HTS formation
by HAL, QUI (Galetin et al.,
2002), NIF, FEL, and MDZ, generated by applying various multisite
kinetic models, extend over a 10-fold range from 9.5 µM (NIF) to 99 µM
(QUI). In most cases, the IC50 values are not in good agreement
with the Ki values obtained from the multisite kinetic
models, indicating the need for caution over rapid screening protocols based
on IC50 plots for substrates with sigmoidal kinetic properties. In
the presence of NIF, FEL, and MDZ, the binding affinity of the first TST
molecule was increased, reflecting a difference between the effector site for
these modifiers and one of the TST binding sites. In contrast, the interaction
at the second site (defined by Ks) is of a
competitive nature (increase in Ks up to 40-fold)
and results in the elimination of cooperativity between the two TST binding
sites. This effect suggests that the second site is a mutual binding domain
for both TST and modifiers belonging to other CYP3A4 subgroups. Other
modifiers like diazepam (Kenworthy et al.,
2001), HAL, QUI (Galetin et
al., 2002), and progesterone (A. Galetin, unpublished data) while
decreasing the binding affinity for a second TST molecule do not affect the
cooperativity of TST binding, indicating the occupancy of a different effector
site from that of MDZ, NIF, and FEL as modifiers.
Interactions at the MDZ Site(s). The pathway-differential effect on
MDZ observed with TST in the current study is not exclusive as similar effects
have been reported previously for MDZ in the presence of various modifiers
(Ghosal et al., 1996;
Wang et al., 2000;
Galetin et al., 2002). These
findings and the difference in the Km values obtained for
1'- and 4-OH MDZ hydroxylation
(Gorski et al., 1994;
Mäenpää et al.,
1998) indicate the possible existence of two distinct
substrate-binding sites for MDZ.
To incorporate these differential effects for the two pathways, we have
applied a three-site kinetic model with a distinct effector site; the latter
is usually associated with substrates showing positive homotropic kinetic
properties. In this case, two substrate-binding sites are assumed to be
distinct, each generating one particular metabolite of MDZ, defined by their
respective Ks and Kp values. The
existence of two separate sites for MDZ was recently also indicated by
site-directed mutagenesis studies (Khan et
al., 2002). These authors revealed the significance of various
active site residues for the regioselectivity of MDZ and indicated the partial
overlap of the two MDZ binding sites.
Spectral titration analysis (Hosea et
al., 2000) has suggested the existence of a higher-affinity site
that overlaps with TST and MDZ and a lower-affinity binding site that overlaps
with -naphthoflavone (NF). Distinct Km and
Ki values observed for 1'- and 4-OH MDZ, and the
differential effects observed in the presence of NF, indicate the
possibility of a third site, distinct from both TST and NF. These
assumptions are supported by several findings from the current multisite
kinetic analysis. Interaction at the mutual site for TST and MDZ
1'-hydroxylation results in the mutual inhibition and a corresponding
increase in Ks and Ks values
for MDZ and TST, respectively, in the presence of each other. Partial
inhibition by TST is a consequence of the higher binding affinity of MDZ than
TST (Ks TST > Ks1 MDZ). The
increased formation of 4-OH MDZ in the presence of higher TST concentrations
and a switch from the major 1'-OH to the minor 4-OH pathway are not
phenomena unique to the TST-MDZ interaction
(Schrag and Wienkers, 2001;
Galetin et al., 2002). No
change in the MDZ binding affinity at the site preferential for
4-hydroxylation (Ks2 remains constant over the range of
TST concentrations) is consistent with the hypothesis of a second distinct
site.
Interactions at the NIF/FEL Site(s). The similarities in NIF and FEL
metabolic pathways and the high correlation between their in vivo clearances
(Soons et al., 1993) suggest
that these two dihydropyridines belong to the same CYP3A4 substrate subgroup.
However, a substrate-dependent effect was observed previously with HAL and QUI
as modifiers (Galetin et al.,
2002). NIF was more susceptible to inhibition in comparison to FEL
(inhibition by both HAL and MDZ shows a difference of one order of magnitude).
Differential effects of QUI (activating FEL in contrast to inhibiting NIF
metabolism) indicated the possibility of different binding domains on CYP3A4
for NIF and FEL despite similarities in their chemical structures. However,
these sites must be in close proximity or even overlap to a certain degree to
allow simultaneous binding and access to the active oxygen on the heme. This
is also indicated by their mutual inhibition and a Ki
value for FEL inhibition of OX NIF formation (16.6 µM), in good agreement
with the affinity of FEL for CYP3A4 (26.4 µM;
Galetin et al., 2002). The
presence of NIF in the active site may partially shield one of the FEL sites
from the active oxygen, resulting in elimination of catalytic activity
associated with that site and partial inhibition.
How Many Sites? The existence of three binding sites, one for a
substrate, one for an effector, and one mutual for both, has been indicated by
various approaches to the analysis of CYP3A4 atypical kinetics
(Hosea et al., 2000;
Kenworthy et al., 2001,
He et al., 2003). The reduced
cooperativity in the binding of TST molecules in the presence of NIF/FEL/MDZ,
the inability of TST to inhibit the metabolism of NIF and FEL, and the ability
of TST to only partially inhibit metabolism of MDZ and terfenadine
(Wang et al., 2000) support
the hypothesis of distinct and preferential binding domains for each substrate
subgroup. However, interaction profiles observed between TST, MDZ, NIF, and
FEL also indicate the existence of a mutual site for all subclasses of CYP3A4
substrates.
Site-directed mutagenesis studies have indicated that CYP3A4 substrate- and
effector-binding sites are separate, but closely linked, and the residues
involved in the binding of either substrate and/or effector depend on the
molecules present (Domanski et al.,
2001; He et al.,
2003). This partial overlap of binding sites may explain certain
differences in the effects of substrates apparently belonging to the same
CYP3A4 subgroup [e.g., NIF and FEL interactions; the differential effect of
diazepam (Kenworthy et al.,
2001) and MDZ on the TST positive homotropy]. Sites for
"metabolism" and "regulation" may differ for the same
compound as proposed earlier for the CYP3A4 modifiers NF
(Shou et al., 2001a) and QUI
(Galetin et al., 2002). The net
effect depends on the particular substrate present at the active site, the
possible overlap of binding domains for the substrates involved in the
interaction, and the relative concentrations of both.
In conclusion, the multisite analysis presented here strongly supports the existence of one preferential binding domain for each of the three CYP3A4 substrate subgroups, indicating that extrapolation from one CYP3A4 substrate to another is only realistic within the same prototypical subgroup. Certain competitive features are apparent in the atypical interaction data set (but not consistently), and these can be attributed to the interactions at a mutual site for all CYP3A4 substrate subclasses.
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Footnotes |
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1 Abbreviations used are: MDZ, midazolam; TST, testosterone; NIF, nifedipine;
FEL, felodipine; 6ß-HTS, 6ß-hydroxytestosterone; OX NIF, oxidized
nifedipine; QUI, quinidine; , interaction factor for the change in
binding affinity (homotropic cooperativity); ß, interaction
factors for the change in catalytic rate constant; , interaction factor
for the change in binding affinity (heterotropic cooperativity); HAL,
haloperidol; NF, -naphthoflavone; OR, NADPH-cytochrome P450
reductase; P450, cytochrome P450. 
Address correspondence to: Dr. A. Galetin, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, UK. E-mail: Aleksandra.Galetin{at}man.ac.uk
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-naphthoflavone, terfenadine and testosterone.
Pharmacogenetics 8:
137-155.[Medline]
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), 5 µM (
), 10 µM (
), and 50
µM (
) of the modifier and control (
)]. The interaction between
TST substrate-binding sites is prevented at NIF concentrations >5 µM.
Data points represent the mean of duplicate determinations.