Review CYP2D in the Brain

p337 p.1 [100%] Drug Metab. Pharmacokin. 18 (6): 337–349 (2003). Review CYP2D in the Brain Yoshihiko FUNAE, Wataru KISHIMOTO,ToshioCHO, Toshiro NIWA, and Toyoko HIROI Department of Chemical Biology, Osaka City University, Medical School, Osaka, Japan Summary: CYP2D1, 2D2, 2D3, and 2D4 are major CYP2D isoforms expressed in the rat. In humans, only CYP2D6 is expressed. In rat brain, the mRNA for CYP2D4 is most abundant in cerebellum, striatum, pons and medulla oblongata. In human brain, CYP2D6 mRNA expression was detected in all regions with highest levels observed in cerebellum. CYP2D isoforms are involved in the metabolism of not only xenobiotics such as antidepressants, b-adrenergic blockers, antiarrhysthmics, and antihypertensives, but also endogenous compounds such as trace amine and neurosteroids. Among 11 isoforms of human recombinant P450s, only CYP2D6 exhibit- ed an ability to e‹ciently convert tyramine which exists in the brain, to dopamine. CYP2D4 and CYP2D6 which are the predominant CYP2D isoforms in the rat and human brain, respectively, possess 21-hydroxylation activity for both progesterone and allopregnanolone. CYP2D4, not P450c21, works as a steroid 21-hydroxylase in the brain. These results suggested that CYP2D in the brain may be involved in the metabolism of neuronal amines and steroids and in the regulation of the central nervous system. Key words: CYP2D; CYP2D4; CYP2D6; brain; dopamine formation; steroid 21-hydroxylation system or on the heart. It is thus of immense interest Introduction whether CYP2D6 is not only localized in the liver,8) but Six genes, named CYP2D1 through CYP2D5 and also found at high levels in these two target tissues, the CYP2D18,werefoundintherat.1–3) CYP2D1 catalyzes brain9,10) and heart.11) a debrisoquine 4-hydroxylase and exhibits a closest The polymorphic hydroxylation of debrisoquine was enzymatic relationship to human CYP2D6. CYP2D7P ˆrst described in the 1970s.12) Since then, the clinical and CYP2D8P are human pseudogenes. No cDNA has pharmacological as well as molecular biological fea- been cloned that corresponds to these genes, and their tures13) of this polymorphism have been studied. Several expression has never been demonstrated. groups of important drugs, such as tricyclic antidepres- The levels and substrate speciˆcity of P450s from sants and neuroleptics,14) are metabolized in the liver by humans were systematically characterized.4,5) Antibod- CYP2D6, and this has important clinical implications ies raised against puriêd P450 which were expressed for the usage of such drugs. In healthy volunteers, a in Saccharomyces cerevisiae were used to measure the signiˆcant diŠerence in personality between extensive levels of hepatic P450s. The level of CYP3A4 was (EM) and poor metabolizers (PM) of debrisoquine highest in human hepatic microsomes, comprising living in Sweden was reported.15) That study compared 30¿40z of total P450. CYP2C9 comprised 10¿20z PM and EM of debrisoquine using the Karolinska of the total. The level of CYP2D6 was 14z.4) It Scales of Personality inventory.15) This indicated that has been estimated that CYP2D6 is responsible for debrisoquine hydroxylase also metabolizes endogenous 20¿30z of the oxidation of all pharmaceutics substrate(s) important for central nervous system used by humans.6,7) Of particular note are tricyclic function.16) Poor metabolizers were more anxiety- antidepressants, selective serotonin reuptake inhibitors, prone and less successfully socialized than extensive 5-hydroxytryptamine receptor antagonists, anti- metabolizers of debrisoquine.17) This and a previous psychotics, opiates, and amphetamines, together with study among subjects in Sweden suggest that there may b-adrenoreceptor antagonists and antidysrhythmic be a relationship between personality and activity of drugs,7,8) all agents that act either in the central nervous CYP2D6. This polymorphic enzyme may have an Received; September 22, 2003, Accepted; October 24, 2003 To whom correspondence should be addressed: Yoshihiko FUNAE, Department of Chemical Biology, Osaka City University, Medical School, Osaka, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585 Japan. Tel. ＋81-6-6645-3915, Fax. ＋81-6-6645-3917, E-mail: funae＠med.osaka-cu.ac.jp 337 p337 p.2 [100%] 338 Yoshihiko FUNAE, et al. Table 1. Sequence identities (z) of rat and human CYP2D Table 2. Active site residues of human and rat CYP2D isoforms29) isoforms. Both the overall sequence identity (upper triangle) and that of the active site residues listed in Table 2 (lower triangle) is given. CYP b Residue SRS a number 2D1 2D2 2D3 2D4 2D5 2D6 1 103ProLeuGluProProPro 105 Pro Pro Pro His Pro Pro 106 Ile Ile Ile Phe Ile Ile CYP2D1 CYP2D2 CYP2D3 CYP2D4 CYP2D5 CYP2D18 CYP2D6 112 Val Tyr Tyr Phe Val Phe CYP2D1 73 79 72 95 72 71 120 Ile Val Val Val Val Phe CYP2D2 45 78 73 73 73 71 121 Leu Leu Leu Leu Phe Leu CYP2D3 59 59 75 79 75 72 2 213 Leu Phe Met Leu Leu Leu CYP2D4 50 41 45 73 99 77 216 Val Asp Gln Glu Val Glu CYP2D582415550 73 71 217 Ser Thr Thr Ser Ser Ser CYP2D18 50 41 45 100 50 77 3 243 Gly Lys Gly Gly Gly Phe CYP2D6594155595059 244 Gln Leu Gln Lys Gln Gln 4 301AspAspAspAspAspAsp 304 Thr Met Gly Met Thr Ser 305AlaAlaAlaAlaAlaAla endogenous neuroactive substrate or product, such as a 308 Val Val Val Val Val Val biogenic neurotransmitter amine. There is now substan- 309ThrThrThrThrThrThr 5 369IleIleIleIleIleIle tial evidence that CYP2D6 is present and functions also 370 Ala Val Val Leu Ala Val 18–20) in the human brain, although its activity is lower 374 Leu Ile Leu Val Leu Val than in the liver. 375ProProProProProThr In this review, we describe the genetic and catalytic 6 483 Phe Leu Phe Ala Ile Phe properties of rat and human CYP2D isoforms. 483 Pro Pro Leu Leu Ser Leu CYP2D4 and 2D6, which are expressed in the brain, a Residue numbering is that of CYP2D6. catalyze endogenous substrates such as amines and b SRS: substrate recognition sites. steroids. The pharmacological and physiological sig- niˆcance of the presence of CYP2D in the brain is described. CYP2D6 and rat CYP2D1-4, as deduced from rigid docking studies are shown in Table 2. The analogous Isoforms of the CYP2D family in rat and human residues in CYP2D5 and 2D18 have also been included. Six gene homologues of CYP2D6 have been isolated CYP2D4 and 2D18 were found to diŠer in the identity from the rat,1,2,21–23) namely, CYP2D1, 2D2, 2D3, 2D4, of only four residues, none of which were located in the 2D5, and 2D18. Human CYP2D6 is one of the most CYP2D active site. When the active site residues of the important phase I enzymes involved in the metabolism human and rat CYP2D isoforms listed in Table 2 are of therapeutic drugs. Another feature that has sig- examined, it is striking how few residues in the models niˆcantly contributed to the attention CYP2D6 has are completely conserved. Only ˆve of the 22 residues obtained concerns its polymorphic nature.24,25) To date are invariant in all human and rat CYP2D isozymes, of more than 70 diŠerent alleles have been identiêd.26) which four are located in the I helix (SRS 4). The eŠects of these polymorphic genes range from a Correspondingly, sequence identities between CYP2D complete loss of functional protein to an increase in isoforms were much lower for the active site residues enzyme activity. DiŠerences in substrate speciˆcity may than for the overall sequences (Table 1) except between also arise. As a result, drug treatment in polymorphic CYP2D4 and 2D18. The most notable diŠerences in individuals may cause adverse eŠects or a lack of drug active site residues of the homology models between e‹cacy.27) human and rat isoforms involved residues 120 and 216. Immunoblotting studies have demonstrated hepatic As described above, residue 120 constitutes a phenylala- expression of CYP2D1, 2D2, 2D4, and 2D5, but not nine in CYP2D6 and provides p-stacking interactions CYP2D3, in various rat strains.28) CYP2D18 is believed with ligands. In all rat isoforms, residue 120 is nonaro- to be the rat brain variant of CYP2D4.23) The rat and matic and therefore only capable of contributing to human CYP2D isoforms share high sequence identity ligand binding a‹nities by van der Waals interactions. (À70z)29) (Table 1). Nevertheless, signiˆcant diŠer- Rigid docking studies with CYP2D6 identiêd Glu216 ences in binding and regioselectivity of metabolism have as a key ligand-binding residue forming hydrogen bonds been observed between these CYP2D isoforms.28–32) For with several substrates. In the cases of CYP2D1W5and example, R-mianserin was N-oxidated by only CYP2D1 2D3, a valine and glutamine, respectively, are located at while 8-hydroxylation was oxidated by all isoforms this position. These isoforms are unable to form a salt investigated.30) The active site residues of human bridge with the basic nitrogen of ligands. p337 p.3 [100%] CYP2D in the Brain 339 Although it is known that rat CYP2D2, similar to human CYP2D6, is capable of metabolizing debrisoquine, bufuralol, and other substrates,1,33,34) the catalytic characteristics of most rat CYP2D enzymes remain to be elucidated. Therefore, we expressed four rat P450 isoforms (CYP2D1, 2D2, 2D3, and 2D4) placed in the CYP2D subfamily in yeast and compared their biochemical properties and substrate speciˆcity simul- taneously.32) We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simultane- ously. Three are believed to be alleles of CYP2D1, 2D2, and2D3,respectively,basedonhighnucleotide sequence similarity, while CYP2D4 had sequences of both CYP2D4 and CYP2D18. Expression plasmids carrying CYP2D cDNAs were transformed into Saccharomyces cerevisiae. Typical P450 CO-diŠerence Fig. 1. mRNA expression of rat CYP2D isoforms in tissues.

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