CYP450 Enzymes in Drug Discovery

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Transcript of CYP450 Enzymes in Drug Discovery

CYP450 Enzymes in Drug Discovery and Development: An OverviewLIN XU, BIPLAB DAS, and CHANDRA PRAKASHDepartment of Drug Metabolism and Pharmacokinetics, Biogen Idec, Cambridge, MA

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Introduction Nomenclature and classication of human CYP enzymes Catalytic activity of CYP enzymes Common CYP-mediated biotransformation reactions Species variation in the expression and activity of CYP enzymes Ethnic variability in expression and activity of cytochrome P 450 enzymes Tools for in vitroin vivo extrapolation Summary and future perspectives Acknowledgment References

1 2 2 4 9 24 25 27 28 28

2.1

INTRODUCTION

The CYP450 (P 450) is a collective name for a very large group of enzymes found in all domains of life and are responsible for the metabolism of a vast array of xenobiotic chemicals, including drugs, carcinogens, pesticides, pollutants, and food toxicants as well as endogenous compounds, such as steroids, prostaglandins, and bile acids [1,2]. The origin of the cytochrome P 450 name, rst coined in 1962, was from the fact that these enzymes are cellular (cyto) colored (chrome) proteins, which contain heme pigments (P) that absorb light at a wavelength of 450 nm when exposed to carbon monoxide [3,4]. P 450 enzymes are predominantly expressed in the liver as well as in extrahepatic tissues such as lungs, kidneys, intestine, brain, and skin. Since their discovery at the end of 1950, P 450 research has grown and the multiplicity and complexity of the P 450 system has been evident for more than ve decades [5]. Over 11,500 members or distinct P 450s genes are currently known that are present in the majority of species from all biological kingdoms [6,7]. The P 450 enzymes catalyze oxidative as well as some reductive (phase I) reactions. These reactions introduce or unmask a functional group (e.g., OH, CO2 H, NH2 , or SH) within a molecule to enhance its hydrophilicity. It can occur through direct introduction of the functional group (e.g., aromatic and aliphatic hydroxylation) or byEncyclopedia of Drug Metabolism and Interactions, 6-Volume Set, First Edition. Edited by Alexander V. Lyubimov. 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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2

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

modifying existing functionalities (e.g., oxidative hydrolysis of the esters and amides, oxidative N, O, and S-dealkylation, and reduction of aldehydes and ketones) [8]. As a result, more hydrophilic (water soluble) and polar entities are formed, which are eliminated from the body. In general, metabolism leads to compounds that are generally pharmacologically inactive and relatively nontoxic. However, metabolic biotransformation of drugs at times can lead to the formation of metabolites with pharmacological activity [9] or toxicity [10]. P 450 enzymes have long been of interest in the metabolism of pharmaceuticals and other xenobiotics, since these enzymes are responsible for the elimination of majority of the marketed drugs. These reactions account for 95% of the drug metabolism. In addition, there are a number of endogenous and exogenous factors, such as genetic variation, age differences, hormone levels, diet, and exposure to a variety of drugs, that can inuence the expression and catalytic properties of P 450 enzymes. Tremendous progress has been made in the last six decades in the characterization, expression, function, and regulation of P 450 enzymes in animals and humans [2,11]. In this chapter, we summarize the most recent advances in our knowledge and application of P 450 enzymes in drug discovery and development with particular emphasis on their involvement in the metabolism of drugs. In addition, we describe the species and ethnic variation in the expression of P 450 enzymes and the tools used to extrapolate metabolism and toxicity in animals to humans.

2.2 NOMENCLATURE AND CLASSIFICATION OF HUMAN CYP ENZYMES P 450 enzymes are categorized into families, subfamilies, and specic enzymes according to their amino acid sequence similarity. P 450s that share at least 40% sequence identity are placed within the same family, designated by an Arabic numeral, while those with greater than 55% homology are placed in the same subfamily, designated by a capital letter and those with 97% homology represent individual enzymes, designated again by a number. Individual alleles are designated by appending a star and a number (human cytochrome P 450 allele nomenclature committee, http//drnelson.utmem.edu/Cytochrome450.html) (Fig. 2.1).

2.3

CATALYTIC ACTIVITY OF CYP ENZYMES

The P 450 enzymes are referred to as hydroxylases, monooxygenases, or mixed function oxidases and possess three known types of activities. P 450s, acting as hydroxylases, activate molecular oxygen and insert one atom of molecular oxygen into the substrate (S or X) while reducing the other atom of oxygen to water (Eqs. 2.1 and 2.2). As a result, the xenobiotics can undergo hydroxylation, epoxidation, heteroatom (N, S) oxygenation, heteroatom (N, S, O) dealkylation, ester cleavage, isomerization, dehydrogenation, and oxidative dehalogenation. SH + O2 + NADPH + H+ SOH + H2 O + NAD(P)+ X + O2 + NADPH + H+ XO + H2 O + NAD(P) (2.1) (2.2)

CATALYTIC ACTIVITY OF CYP ENZYMESCYP Superfamily

3

CYP1

CYP2

CYP3

Family

CYP2A

CYP2B

CYP2C

CYP2D

CYP2E

CYP2J

Subfamily

CYP2C8

CYP2C9

CYP2C18 CYP2C19

Individual Enzyme

CYP2C9*2

CYP2C9*3

Allele

Figure 2.1

Nomenclature of CYP450 enzymes.

The oxidase activity of P 450s involves one electron transfer from reduced P450 to molecular oxygen with the formation of superoxide anion radical and H2 O2 (Eq. 2.3a,b). NADPH + O2 O2 + NAD(P)+ 2NADPH + 2H+ + O2 H2 O2 + NAD(P)+ (2.3a) (2.3b)

The reductase activity of P 450s involves direct electron transfer to reducible substrates such as quinones and proceeds readily under anaerobic conditions. The catalytic cycle of P 450 oxidation is a complex multistep processes as follows: 1. P 450 enzyme (Fe3+ ) rst binds to a substrate XH to form Fe3+ -XH. This results in lowering the redox potential, which makes the transfer of an electron favorable from its redox partner, NADH or NADPH. This is accompanied by a change in the spin state of the haem iron at the active site. 2. The next step in the cycle is the rst reduction of the Fe3+ -XH to Fe2+ -XH by an electron transferred from NAD(P)H via an electron-transfer chain. 3. In the third step, an O2 molecule binds rapidly to the Fe2+ -XH to form Fe2+ O -XH, which then undergoes a slow conversion to a more stable complex 2 Fe3+ -O -XH. 2 4. The next step in the cycle is a second reduction of Fe3+ -O -XH to Fe3+ -O2 2 2 XH via the electron donors either NADPH or cytochrome b5. This has been determined to be the rate-determining step of the reaction.

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CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

5. The Fe3+ -O2 2 -XH reacts with two protons from the surrounding solvent, breaking the OO bond, forming water and leaving an (Fe-O)3+ -XH complex. 6. The Fe-ligated O atom is transferred to the substrate forming a hydroxylated form of the substrate (Fe3+ -XOH). 7. The last step involves the release of product from the active site of the enzyme, which returns to its initial state.

2.4

COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS

P 450 catalyzed reactions can be classied into four broad categories: (i) hydroxylation reactions where a hydroxyl group replaces a hydrogen atom; (ii) epoxidation reactions where an oxygen atom is introduced into carboncarbon double or triple bond; (iii) heteroatom oxidation where an oxygen atom is added to a nitrogen or sulfur, (iv) dehydrogenation reactions where two hydrogen atoms are replaced by a double bond [12]. 2.4.1 Hydroxylation Reaction

Hydroxylation of an aliphatic carbon or an aromatic ring is one of the most common drug metabolism reactions. The other common biotransformation reaction is the hydroxylation at the carbon to a hetero atom, which resulted in oxidative cleavage of the molecule. 2.4.1.1 Aliphatic Hydroxylation. For aliphatic hydroxylation, one proposed mechanism is an abstraction of a hydrogen atom by (Fe-O)3+ to form a radical intermediate that reacts with the oxygen on the P 450 (Fe-OH)3+ to yield the alcohol and (Fe)3+ (Fig. 2.2). Drug molecules possess many alkane carbons with abstractable hydrogen atoms and therefore, hydroxylation can possibly occur at any one site that can result in more than one hydroxylated product, as shown for ezlopitant (Fig. 2.3) [13]. However, a product forms preferentially from the most stable radical (resonance stabilized such as benzylic or allylic). 2.4.1.2 Aromatic Hydroxylation. The aromatic hydroxylation occurs by epoxidation of the aromatic ring to form of an arene oxide, which undergoes a 1,2

(FeOH)3+ (Fe-O)3+ +H C CH3

C CH3

HO

CH3

+

Fe3+

(FeOH)3+ H2C C H

H

CH2OH

+

Fe3+

Figure 2.2

Proposed mechanism of aliphatic hydroxylation.

COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONSCH3O H NOH

5

N CH3O H N N Secondary alcohol

Ezlopitant N

CH3O H N

OH

Primary alcohol

Figure 2.3

Isomeric hydroxylated human metabolites of ezlopitant.

hydrogen shift (NIH shift) and subsequent tautomerization to yield a stable phenol product.H[FeO]3+

H

O

O H NIH Shift H H

OH

As a result, CYP-mediated aromatic hydroxylation often results in the formation of isomeric hydroxylated products. Owing to resonance stabilization, for monosubstituted phenyl groups,