Article Figures & Data
Figures
- Fig. 1.
Multisite kinetic equilibria models.
A, a generic two-site model for CYP3A4 interactions and the corresponding equation. Interaction factors associated with the changes in binding affinity (Ks/Ki) are as follows: α-homotropic substrate cooperativity, δ-heterotropic cooperativity, and αI-inhibitor cooperative binding. Interaction factors associated with the changes in catalytic rate constant (Kp) are as follows: β (SES) and γ (MES). B, a kinetic model for an enzyme that binds substrate cooperatively and where the inhibitor eliminates the sigmoidicity. C, three-site kinetic model with two distinct substrate-binding sites (S1 and S2) and an effector site (pathway-differential effect of a modifier).
- Scheme 1.
Summary of interaction factors from multisite kinetic models.
- Fig. 2.
Binding affinity constants for MDZ, TST, NIF, and FEL in lymphoblast-expressed CYP3A4 when incubated alone and in various combinations.
The Ks values represent the mean values of the Ks derived for a particular substrate for all the interactions studied. Multisite kinetic models applied for their derivation are stated in the data analysis for each interaction.
- Fig. 3.
Examples of cooperative competitive inhibition.
A and B, 1′-OH MDZ formation in the presence of increasing concentrations of FEL and NIF, respectively. Slopes increasing from 1.3 to 5.2 (FEL) and 0.6 to 1.4 (NIF) over the 5 to 50 μM substrate concentration range, consistent with cooperative binding of a modifier. C, correlation of FEL and NIF inhibitory effects on MDZ 1′-hydroxylation at various substrate concentrations. The dashed line represents the line of unity. Data points represent the mean of duplicate determinations.
- Fig. 4.
Examples of partial inhibition.
A, inhibition of MDZ 1′-hydroxylation by TST. B, inhibition of FEL oxidation by NIF, comparison of partial versus pure competitive inhibition at high S concentration (100 μM). In contrast to competitive inhibition, partial or negative cooperative inhibition shows a limiting plateau at high concentrations of an I, the level determined by the value of the interaction factor δ.
- Fig. 5.
Eadie-Hofstee plots for OX NIF and 6β-HTS formation in the presence of FEL and NIF, respectively.
A, loss of negative homotropy due to dominance of the nonproductive S(EI) complex. B, loss of positive homotropy [e.g., effect of NIF on TST at concentrations of 1 μM (•), 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.
Tables
- TABLE 1
Kinetic parameters for the in vitro effect of various CYP3A4 substrates on midazolam in human lymphoblast-expressed CYP3A4, generated by multisite kinetic model approach (mean ± S.E.) The kinetic parameters were determined using multisite kinetic models (n = 20-30) defined in footnotes a and b.
CYP3A4 Modifier
Vmax
Ks
Ki
γ
δpmol/min/pmol P450 μM μM NIFa 2.8±0.1 4.1±0.7 13.4±3.2 0.2±0.1 1.4±0.3 FELa 4.2±0.1 3.1±0.3 10.1±1.9 0.4±0.1 3.6±1.1 TSTb 5.3±0.1 5.1±0.3 139±26
2.2±0.6 -
↵ a Equation 1, α1 (cooperative binding of the modifier) = 0.24 and 0.9 for FEL and NIF, respectively
-
↵ b Equation 5, three-site model with distinct substrate-binding sites, Ks2 (4-OH MDZ) = 8.56 ± 0.25 μM. Ks in the Table refers to the Ks1 (1′-OH MDZ), α defining cooperative binding of TST = 0.25±0.04
-
- TABLE 2
Kinetic parameters for the in vitro effect of various CYP3A4 substrates on NIF/FEL in human lymphoblast-expressed CYP3A4, generated by multisite kinetic model approach (mean ± S.E.) The kinetic parameters were determined using generic two-site model (n = 20-30) defined in footnotes a, b, and c.
Substrate
CYP3A4 Modifier
Vmax
Ks
Ki
γ
δpmol/min/pmol P450 μM μM Nifedipinea MDZ 6.5±0.1 19.4±1.5 20.8±4.1 0.45±0.10 0.95±0.11 FEL 10.1±0.2 36.3±1.7 16.6±3.7 0.56±0.09 0.62±0.09 Felodipine MDZb 13.8±0.5 46.2±4.3 198±41 0.81±0.11 0.22±0.04
NIFc 16.5±0.4 34.8±2.0 33.1±8.0
2.1±0.1 -
↵ a Equation 3, β (Kp from SES) = 0.41±0.10 (MDZ) and 0.44±0.09 (NIF)
-
↵ b Equation 2 (Galetin et al., 2002), β = 2 as Vmax is equivalent to 2Kp[E]t, where [E]t is the total enzyme concentration (Segel, 1975)
-
↵ c Partial inhibition model defined by eq. 2 was applied
-
- TABLE 3
Kinetic parameters for the in vitro effect of various CYP3A4 substrates on testosterone in human lymphoblast-expressed CYP3A4, generated by multisite kinetic model approach (mean ± S.E.) The kinetic parameters were determined using kinetic model (n = 25-30) defined by equation 4, β (Kp from SES) = 2. Vmax is equivalent to 2Kp[E]t, in which [E]t is the total enzyme concentration (Segel, 1975).
CYP3A4 Modifier
Vmax
Ks
Ki
α
δpmol/min/pmol of P450 μM μM NIF 5.5±0.3 150±35 9.5±3.5 0.07±0.02 0.14±0.06 FEL 10.1±0.5 173±22 37.1±7.4 0.14±0.04 0.04±0.02 MDZ 10.3±0.5 128±19 23.1±5.4 0.15±0.05 0.08±0.03
