The effects of type II binding on metabolic stability and binding affinity in cytochrome P450 CYP3A4

doi:10.1016/j.abb.2010.03.011

Archives of Biochemistry and Biophysics

Volume 497, Issues 1–2, May 2010, Pages 68-81

https://doi.org/10.1016/j.abb.2010.03.011 Get rights and content

Abstract

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound’s metabolic stability can be increased by incorporation of a sp² nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme-iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1200-fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding.

Introduction

Cytochrome P450s (P450s) are heme-containing monoxygenases that are responsible for a plethora of oxidation reactions important in plants, bacteria, and mammals [1]. In mammals, P450s carry out the biotransformation of several endogenous compounds in the body such as bile acids, biogenic amines, fatty acids, and steroids. This family of enzymes also plays a role in the metabolism of drugs, and more importantly can be a major factor in failures in drug development. Metabolic stability is an important factor in determining the amount of a potential drug that will reach the site of action. It has been shown that metabolic stability can be increased by the incorporation of a sp² nitrogen into an aromatic ring [2], [3]. Two potential (non-exclusive) mechanisms for nitrogen containing aromatic systems to slow metabolism are by (1) decreasing electron density on the aromatic carbons, reducing metabolic rate by the electrophilic active-oxygen species, and (2) nitrogen coordination with the heme-iron to form what is known as a type II binder (Fig. 1). The electronic mechanism will play a role anytime aromatic hydroxylation is the major pathway for metabolism, and both can contribute to altering metabolic rates.

With respect to type II binding, it is hypothesized that coordination to the iron slows down the catalytic cycle by trapping the ferric iron in low-spin state forming a dead-end complex which cannot be reduced by P450 reductase to initiate the catalytic cycle (Fig. 1) [2], [3]. Three questions remain unanswered with respect to type II binding: (1) How much is the affinity increased by nitrogen coordination to the heme-iron? (2) Does type II iron coordination slow or prevent metabolism? (3) How does a compound that coordinates to the heme-iron get metabolized? Our recent studies indicate that nitrogen coordination to the heme-iron of 2C9 leads to a very strong interaction, yielding up to a 4200-fold increase in binding affinity [4], [5], [6]. Given this strong affinity, it is difficult to envision how a type II binding substrate can be metabolized significantly, since the fraction of high-spin five-coordinate heme that could be reduced would be very small. In an attempt to answer these questions we use experimental and computational methods to study pyridinyl quinoline-4-carboxamide analogs (Fig. 2) which have been designed to change from type II, nitrogen coordination, to type I binding by altering the position of the nitrogen in the aromatic ring. Measurements of binding affinity, metabolic stability, and regioselectivity of metabolism in CYP3A4 are used to explore the affect of type II binding on reaction rates. The substrates are designed to modulate the amount of type II binding without altering other properties, such as solvation. The ortho substituted compound (Z = N, X = C, Y = C in Fig. 2) should not be able to coordinate to the heme-iron since coordination is precluded by steric interactions with the heme [5]. We chose to study CYP3A4 instead of CYP2C9 because CYP3A4 gives better type II UV/vis difference spectra [7], [8]. This enzyme also allows us to see if the increases in binding affinity observed for 2C9 can be extrapolated to other P450 enzymes. Furthermore, CYP3A4 is a major drug P450 enzyme that metabolizes more than 50% of clinically used drugs.

Section snippets

K_i Measurements

CYP3A4 Supersomes® were obtained from BD Biosciences (Gentest, Woburn, MA, USA) and contained human CYP3A4 at a concentration of 1 μM, with P450 reductase (3200 nmol/min × mg protein) and cytochrome b₅ (1300 pmol/mg protein). Testosterone was used as the CYP3A4 marker substrate and run at four concentrations (7.81, 15.6, 31.2, and 125 μM) against five different inhibitor concentrations. Incubations (200 μL) consisted of 0.5 pmol of 3A4 enzyme in 100 mM potassium phosphate buffer at pH 7.4 containing 1 mM

Results

The affinity for the active-site of CYP3A4 for each compound listed in Table 1 was measured by their ability to inhibit CYP3A4 dependent metabolism of testosterone. The compounds were synthesized as previously reported [5]. All of the compounds studied are competitive inhibitors versus testosterone and their K_i values are shown in Table 1. Within each series as shown in Table 1, the para nitrogen containing compounds bind more tightly than meta or ortho nitrogen containing compounds (a series

Discussion

Coordination to the heme-iron of P450 by sp² nitrogen containing compounds (type II binding) has been shown to affect the binding modes of the ligand and increase metabolic stability for some compounds [2], [3]. It is tempting to speculate that this is a general phenomenon that can be used to modulate affinity and metabolic stability for the P450 enzyme family. Given the potential for drug design it is important that we understand the underlying chemistry of this interaction.

Previously, we have

Acknowledgment

This work was supported by GM84546 for the National Institute of General Medical Sciences.

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Original paperThe effects of type II binding on metabolic stability and binding affinity in cytochrome P450 CYP3A4

Abstract

Introduction

Section snippets

Ki Measurements

Results

Discussion

Acknowledgment

Biochem. Pharmacol.

Bioorg. Med. Chem.

J. Biol. Chem.

J. Biol. Chem.

Drug Metab. Dispos.

J. Med. Chem.

J. Med. Chem.

Mol. Pharmacol.

Drug Metab. Dispos.

A statistical treatment of dose–response curves

Biochem. J.

Original paper
The effects of type II binding on metabolic stability and binding affinity in cytochrome P450 CYP3A4

K_i Measurements