P450 interaction with farnesyl-protein transferase inhibitors metabolic stability, inhibitory potency, and P450 binding spectra in human liver microsomes

doi:10.1016/S0006-2952(01)00724-9

Biochemical Pharmacology

Volume 62, Issue 6, 15 September 2001, Pages 773-776

https://doi.org/10.1016/S0006-2952(01)00724-9 Get rights and content

Abstract

Methyl substitution at the 2-position of the imidazole ring greatly improved drug metabolism profiles, in human liver microsomes, of ras farnesyl-protein transferase inhibitor (FTI) candidates for drug development. Methyl substitution markedly reduced the P450 inhibitory potency of non-substituted FTIs for CYP3A4 (by a factor of 12-403) and 2C9 (by a factor of 4.2-28), while it had little effect on the CYP2D6 enzyme. An immunochemical inhibition study demonstrated that CYP3A4 plays a predominant role in the metabolism of both non-substituted and 2-methyl-substituted imidazole-containing FTI candidates. Very strong type II binding spectra with human liver microsomes were observed for all non-substituted FTIs, while methyl substitution markedly weakened type II spectra or shifted the type of spectra from II to I. This indicated that methyl substitution on the imidazole moiety interfered with the substrate-P450 heme interaction, likely due to a steric effect caused by the methyl group. A kinetics study revealed that the methyl substitution increased V_max and K_m values to the same extent. These studies suggested that the 2-methyl substitution on the imidazole ring improved its drug metabolism profile by reducing the potential to inhibit CYP3A4-mediated metabolism without affecting intrinsic metabolic clearance (V_max/K_m).

Introduction

Mutational activation of the ras gene has been found in many types of human cancers including more than 90% of pancreatic, 50% of colon, and 30% of lung cancers [1], [2]. Ras proteins are synthesized as cytosolic precursor molecules and biologically activated after a series of posttranslational modifications including the prenylation reaction, which attaches the farnesyl group to the protein [3]. Therefore, inhibitors of the first step of the farnesylation of a cysteine residue, catalyzed by the enzyme FTase, have been considered as potential targets for the development of antitumor agents [4], [5], [6]. Although a number of thiol-containing compounds have been synthesized based on the sequence motif CAAX and have been proven to be effective for the inhibition of ras FTase activity [7], [8], [9], [10], the presence of oxidizable thiol functionality conferred disadvantages on their development as therapeutic agents. Therefore, discovery efforts were directed more toward the identification of non-thiol-containing FTIs. Among the non-thiol-containing FTIs, a series of imidazole-containing agents were effective ligands of the active site (zinc) of FTase [6], [11], [12], [13] and demonstrated inhibitory potency.

Imidazole-containing agents have generally been well known for their potent inhibition of P450 [14], [15]. A previous study on rats demonstrated that a 2-methyl substitution on the imidazole ring of one of the typical FTIs greatly increased the metabolic rate (V_max) and reduced the potency of the compound to inhibit CYP3A [16]. The purpose of the present study was to examine the effect of 2-methyl substitution of the imidazole ring on metabolic kinetics, P450 inhibitory potency, and the type and/or extent of substrate-induced binding spectra using a ferric form of P450 from human liver microsomes. Spectral analysis suggested that the 2-methyl substitution interferes with the stable coordination between the heterocyclic nitrogen of FTIs and P450 heme.

Section snippets

Materials and methods

Imidazole-containing FTIs [compounds A–H in Table 1; suffixed numbers (1 and 2) denote imidazole- and 2-methylimidazole-containing FTIs, respectively] were synthesized in the Department of Medicinal Chemistry at Merck Research Laboratories. Pooled human liver microsomes were obtained from the Keystone Skin Bank. Monoclonal anti-CYP3A4 antibody was prepared at Merck Research Laboratories. All other reagents were of analytical grade.

For inhibition studies, the FTI candidate was added at various

Results and discussion

Immunoinhibition studies with monoclonal anti-CYP3A4 antibody indicated that the metabolism of the FTI candidates tested in this study was inhibited > 90% by the antibody regardless of the 2-methyl substitution, suggesting that CYP3A4 plays a predominant role in the metabolism of both imidazole- and 2-methylimidazole-containing FTI candidates. The methyl substitution on the imidazole ring dramatically reduced the inhibitory potency of all non-substituted FTI candidates for the marker metabolism

Acknowledgments

The authors acknowledge the support and thoughtful discussion of Dr. Thomas A. Baillie during the course of this study. We also thank Ms. Joy A. Nishime for her technical support and Ms. A. Gibson for the preparation of this manuscript.

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¹: Abbreviations: FTI, farnesyl-protein transferase inhibitor; FTase, farnesyl-protein transferase; and LC-MS, liquid chromatography-mass spectrometry.

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Biochemical Pharmacology

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgments

References (21)

Nonfarnesylated tetrapeptide inhibitors of protein farnesyltransferase

J Biol Chem

Synthesis and biological activity of ras farnesyl protein transferase inhibitors. Tetrapeptide analogs with amino methyl and carbon linkages

Bioorg Med Chem

Peptide based p21^RAS farnesyl transferase inhibitorssystematic modification of the tetrapeptide CA₁A₂X motif

Bioorg Med Chem Lett

Imidazole-containing diarylether and diarylsulfone inhibitors of farnesyl-protein transferase

Bioorg Med Chem Lett

Structure-activity relationships in the effects of 1-alkylimidazoles on microsomal oxidation in vitro and in vivo.

Biochem Pharmacol

Measurement of substrate and inhibitor binding to microsomal cytochrome P450 by optical-difference spectroscopy

Methods Enzymol

Substrate interactions with cytochrome P450

Pharmacol Ther

ras Oncogenes in human cancera review

Cancer Res

ras Genes

Annu Rev Biochem

Protein isoprenylation and methylation at carboxy-terminal cysteine residues

Annu Rev Biochem

Cited by (18)

CW EPR parameters reveal cytochrome P450 ligand binding modes

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The effects of nitrogen-heme-iron coordination on substrate affinities for cytochrome P450 2E1

The kinetic mechanism for cytochrome P450 metabolism of Type II binding compounds: Evidence supporting direct reduction

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

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine

Short communicationP450 interaction with farnesyl-protein transferase inhibitors metabolic stability, inhibitory potency, and P450 binding spectra in human liver microsomes1

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgments

J Biol Chem

Bioorg Med Chem

Bioorg Med Chem Lett

Bioorg Med Chem Lett

Biochem Pharmacol

Methods Enzymol

Pharmacol Ther

ras Oncogenes in human cancera review

Cancer Res

ras Genes

Annu Rev Biochem

Protein isoprenylation and methylation at carboxy-terminal cysteine residues

Annu Rev Biochem

Short communication
P450 interaction with farnesyl-protein transferase inhibitors metabolic stability, inhibitory potency, and P450 binding spectra in human liver microsomes¹