In Vitro Enzyme Inhibition 541 Reversible Inhibition

The four types of reversible inhibition are competitive, noncompetitive, uncompetitive, and linear mixed.

5.4.1.1 Competitive Inhibition

In competitive inhibition, the inhibitor and the substrate both bind to the active site of the enzyme in a reversible manner.

(Km x a) + [S] Ki vj = velocity of the reaction in the presence of an inhibitor Kj = inhibitory constant

The presence of the inhibitor does not change Vmax, but Km changes by a factor of a.

IC50 is the inhibitor concentration that results in 50% inhibition.

Based on Cheng and Prusoff (1973) calculations, IC50 for a competitive inhibitor is as follows:

5.4.1.2 Noncompetitive Inhibition

In noncompetitive inhibition, the inhibitor and the substrate bind to the enzyme at different sites. As a result, Km is unchanged and Vmax is changed by a factor of 1/a.

5.4.1.3 Uncompetitive Inhibition

In uncompetitive inhibition, the inhibitor binds to the ES complex. Both Vmax and Km are changed by a factor of 1/a.

If [S] = Km then Ki = IC50/2. Note that compared to the IC50 equation for competitive inhibition, the ratio of [S] to Km is inverted.

5.4.1.4 Linear Mixed Inhibition

In linear mixed inhibition, the inhibitor binds to both the E and ES with inhibitory constants of Kj and dKj, respectively. Vmax and Km are changed by factors of 1/b and a/ft, respectively (Table 5.3).

vi = -n,\ r'l, where a = 1 + ^- and b = 1 + ^- (5.8)

Table 5.3. Effect on Vmax and Km under different reversible inhibition conditions

Competitive Noncompetitive Uncompetitive Linear mixed

Data analysis is conducted using a nonlinear regression program. The general rule is to input data into the different models and see which fits the data best. The simplest model that explains the data should be used. In addition, using a graphical representation is important for visual inspection of the data.

See Table 5.4.

Table 5.4. Preferred in vitro inhibitors of P450 isoforms

P450 isoform

Inhibitor

Kia (mM)

IC50b (mM)

CYP1A2

Furafylline

0.6-0.73

1.8

CYP2A6

Tranylcypromine

0.02-0.2

0.45

Methoxsalen

0.01-0.2

CYP2B6

2-Phenyl-2-(1-piperdinyl)

7.7

propane

CYP2C8

Montelukast, Quercetin

1.1

3.1

CYP2C9

Sulfaphenazole

0.3

0.27

CYP2C19

(+)-N-3-Benzylnirvanol

0.41

CYP2D6

Quinidine

0.027-0.4

0.058

CYP2E1

Tranylcypromine

8.9

CYP3A4/5

Ketoconazole

0.0037-0.18

0.016-0.026

aFrom the preferred list of P450 inhibitors in the FDA's Draft Guidance for Industry on Drug Interaction Studies (2006)

bIC50 values in human liver microsomes reported by Walsky and Obach (2004)

aFrom the preferred list of P450 inhibitors in the FDA's Draft Guidance for Industry on Drug Interaction Studies (2006)

bIC50 values in human liver microsomes reported by Walsky and Obach (2004)

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