Mol Biol Rep DOI 10.1007/s11033-015-3863-0
CYP2D44 polymorphisms in cynomolgus and rhesus macaques Yasuhiro Uno • Shotaro Uehara • Sakae Kohara • Naoki Osada • Norie Murayama • Hiroshi Yamazaki
Received: 3 July 2014 / Accepted: 10 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Macaques, including cynomolgus and rhesus macaques, are important animal species used in drug metabolism studies. CYP2D44 is expressed in cynomolgus macaque liver and encodes a functional drug metabolizing enzyme, metabolizing typical human CYP2D substrates such as bufuralol and dextromethorphan. CYP2D44 is highly homologous to human CYP2D6 that is known to be polymorphic with a large inter-individual variation in metabolic activities, however, genetic polymorphisms have not been investigated in macaque CYP2D44. In the present study, screening of 78 cynomolgus and 40 rhesus macaques found a total of 67 variants, including 64 non-synonymous variants, 1 nonsense mutation, and 2 frameshift mutations, and 1 gene conversion, of which 14, 19, and 15 variants were unique to Indochinese cynomolgus macaques, Indonesian cynomolgus macaques, and Chinese rhesus macaques, respectively. Eleven of the 64 non-synonymous variants were located in substrate recognition sites, the regions important for protein function. By site-directed mutagenesis and
Y. Uno (&) S. Uehara S. Kohara Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minami Akasaka, Kainan, Wakayama 642-0017, Japan e-mail: [email protected] S. Uehara N. Murayama H. Yamazaki Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo, Japan N. Osada Department of Population Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan N. Osada Department of Genetics, Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan
metabolic assays, S175N, V185L, A235G, R242G, R245K, and N337D showed substantially decreased activity in bufuralol 10 -hydroxylation as compared with wild-type proteins. Moreover, two null alleles (c.128T[del and c.664G[T) were found in Indonesian cynomolgus macaques, but not in Indochinese cynomolgus macaques or Chinese rhesus macaques. These results suggest that genetic polymorphisms might account for the variability of CYP2D44-dependent metabolism in macaques. Keywords Cytochrome P450 Genetic polymorphism Metabolic activity Monkey
Introduction Animal studies are conducted in the preclinical drug development to predict metabolism of the newly developed drugs in humans; however, intra- and inter-species differences are occasionally seen in the studies, making it difficult to extrapolate the animal data to humans [1]. Such differences are partly accounted for by cytochrome P450 (P450), a family of important drug-metabolizing enzymes, which is involved in metabolism of a large number of drugs [2]. In the preclinical studies, rodent and non-rodent species are used, and the non-rodent species frequently used are dogs and monkeys. Therefore, it is important to understand the intra- and inter-species differences in a P450mediated drug metabolism in monkeys. CYP2D6, one of P450s, is involved in the metabolism of approximately 20–25 % of the prescribed drugs, including bufuralol and dextromethorphan [3]. CYP2D6 is highly polymorphic with a number of alleles (see http://www. cypalleles.ki.se). A large inter-individual variation that has been noted for CYP2D6-dependent metabolic activities is
123
Mol Biol Rep
partly explained by the genetic polymorphisms at CYP2D6 locus [3]. For example, CYP2D6*4 and CYP2D6*10 result in a defective or unstable enzyme, and the entire gene is deleted in some human genomes (CYP2D6*5). Moreover, these alleles are distributed differently in populations; CYP2D6*4 and CYP2D6*10 are mainly found in Caucasians and Asians, respectively, making these alleles important when prescribing drugs at hospital or in clinical studies during drug development. Cynomolgus macaques (Macaca fascicularis), and also rhesus macaques (Macaca mulatta), are frequently used in drug metabolism studies due to their evolutionary closeness to humans. In vivo analysis using CYP2D probe drug (dextromethorphan) revealed inter-animal variability in pharmacokinetics [4], which could be accounted for by genetic polymorphisms. In cynomolgus macaques, a number of genetic variants have been identified, including P450 genes [5–9]. However, investigation on involvement of genetic polymorphisms in the inter-animal variability of CYP2D-dependent drug metabolism has been hampered by the lack of information on the CYP2D genetic polymorphisms in this species. In cynomolgus and rhesus macaques, two CYP2D genes, CYP2D17 and CYP2D44, are expressed predominantly in liver and encode functional drug-metabolizing enzymes, metabolizing human CYP2D6 substrates, bufuralol and dextromethorphan [10]. In the present study, a screening was conducted using genome samples from 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia) and 40 Chinese rhesus macaques to identify genetic variants in CYP2D44. Distribution of the variants found was analyzed in Indochinese and Indonesian cynomolgus macaques, and Chinese rhesus macaques. For some of the variants, protein was expressed in Escherichia coli, with which metabolic assays were performed using CYP2D substrate (bufuralol).
Materials and methods Preparation of genomic DNA Whole blood samples were collected from 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia, 4–5 years of age, weighing 3–5 kg). Blood samples were also obtained from 40 rhesus macaques (from China, 7 years of age, weighing 3–5 kg). From these samples, genomic DNA was extracted using the Puregene DNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd.
123
PCR amplification, sequencing, and sequence analysis To identify genetic variants, genomic DNA samples of 78 cynomolgus macaques and 40 rhesus macaques were subjected to direct sequencing of exons 1–7. PCR was carried out in a 20-lL reaction containing 1 ng of genomic DNA, 5 pmol of forward and reverse primers, and 1 unit of LATaq DNA polymerase (Takara, Ohtsu, Japan) for exons 1–3, AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) for exons 4–7, and Ex-Taq DNA polymerase (Takara, Ohtsu, Japan) for exons 8–9. The thermal cycler conditions included an initial denaturation at 95 °C for 2 min, and 35 cycles of 20 s at 95 °C, 30 s at 62 °C, and 2 min at 72 °C, followed by a final extension step of 10 min at 72 °C. For exons 4–7, an initial denaturation was conducted at 95 °C for 10 min. The PCR primers were mmCYP2Dv (5flk1a) 50 -GCAGCCCCTCAG TCAGCTA-30 and mfCYP2Dv (3int1c) 50 -CTTTGGAAA ATCTCATCTTTCATGC-30 for exons 1–2, mfCYP2Dv (5int2x1) 50 -GGGACAGACACCTCGTTTCC-30 and mfCYP 2Dv (3int4a) 50 -GGGTCTCCATCTCTCCTGTG-30 for exons 3–4, mfCYP2Dv (5int4a) 50 -GCATAGAAGGGTTTGGGA AA-30 and mfCYP2Dv (3int5a) 50 -AGAGGGTACTTGGG GACACA-30 for exon 5, mfCYP2Dv (5int5a) 50 -GTGAG GACCAAGGGTGGTG-30 and mfCYP2Dv (3int6a) 50 TCAGGGGACCAGCCCTGACCCTCTCCT-30 for exon 6, mfCYP2Dv (5int6a) 50 -CAGCACAGGCTTGACCAGTCT -30 and mfCYP2Dv (3int7a) 50 -CACACAGCTGGACTCTG TCAA-30 for exon 7, and mmCYP2D44 (5int7a) 50 -CCA GTCTAGTAGGGAAGACAGATCA-30 and mmCYP2D44 (3flk1a) 50 -GCCACTAGATTCATTTTCTGTACAAC-30 for exons 8 and 9. Sequencing was performed using an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequencing primers were mmCYP2Dv (5flk1b) 50 -CGCTATGCTCCCAGTACACA-30 or mfCYP 2Dv (3int1d) 50 -GGAAGTCCCTCAAATCTGCTTC-30 for exon 1, mfCYP2Dv (5ex1b) 50 -GGACTTCAAGAACA CACCATACTG-30 or mf2Dv (3int2xx2) 50 -TGTGGTTTTC CTGGTCTTCC-30 for exon 2, mf2Dv (3int3xx1) 50 -CCCC ACCTTCCCAGTTCC-30 for exon 3, mfCYP2Dv (5int3a) 50 AAGGCGGGAACTGGGAAG-30 for exon 4, mfCYP2Dv (5int4a) or mfCYP2Dv (3int5a) for exon 5, mfCYP2Dv (5int5a) for exon 6, mfCYP2Dv (5int6a) for exon 7, and mmCYP2D44 (5int7b) 50 -CTGTGTGCCAGGCA GTGT-30 and/or mmCYP2D44 (3flk1a) for exons 8 and 9. Sequence analysis was conducted using Sequencher (Gene Codes, Ann Arbor, MI, USA) and DNASIS Pro (Hitachi Software, Tokyo, Japan). To determine genetic variants, the sequence was compared to cynomolgus CYP2D44 cDNA sequence (GenBank accession DQ297684).
Mol Biol Rep
Preparation of CYP2D44 proteins CYP2D44 protein variants were prepared as previously described [10], including the construction of pCW expression plasmids, protein expression, and membrane preparation, except that the pCW vector did not contain the reductase cDNA. The content of CYP2D44 protein variants in the membrane preparation was determined by Fe2?CO versus Fe2? difference spectra according to the method previously described [11]. QuikChange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used to introduce each mutation into the expression plasmid of cynomolgus CYP2D44 according to the manufacturer’s instructions. The primers used were: mfCYP2DvSNP_c128del (5qc1) 50 -CTGCCACTGC CGGGGCGGGCAACCTGCTGC-30 and mfCYP2DvSNP_ c128del (3qc1) 50 -GCAGCAGGTTGCCCGCCCCGGCAG TGGCAG-30 for c.128T[del, mfCYP2DvSNP_c524A[G (5qc1) 50 -CCCCTTTCGCCCCAaTGGCCTCCTGGACAA30 and mfCYP2DvSNP_c524A[G (3qc1) 50 -TTGTCCAGGAGGCCAtTGGGGCGAAAGGGG-30 for c.524G[A, mf CYP2DvSNP_c553G[T (5qc1) 50 -AGCGGCGAGCAACtTAATTGCCTCCCTCAC-30 mfCYP2DvSNP_c553G[T 0 (3qc1) 5 -GTGAGGGAGGCAATTAtGTTGCTCGCCGC T-30 for c.553G[T, mfCYP2DvSNP_c704G[C (5qc1) 50 CTGCGCATCCCAGgGCTGGCTGGCAAGGTC-30 mfCYP 2DvSNP_c704G[C (3qc1) 50 -GACCTTGCCAGCCAGCcC TGGGATGCGCAG-30 for c.704G[C, mfCYP2DvSNP_ c724C[G (5qc1) 50 -CTGGCAAGGTCCTAgGTTCCCAAA GGGCTT-30 mfCYP2DvSNP_c724C[G (3qc1) 50 -AAGCCC TTTGGGAACcTAGGACCTTGCCAG-30 for c.724C[G, mfCYP2DvSNP_c734A[G (5qc1) 50 -CTACGTTCCCAAA aGGCTTTCTTGACCCAG-30 mfCYP2DvSNP_c734A[G (3qc1) 50 -CTGGGTCAAGAAAGCCtTTTGGGAACGTAG30 for c.734A[G, mfCYP2DvSNP_c964G[A (5qc1) 50 GCCTCCTGCTCATGaTCCTGCACCCGGATG-30 mfCYP 2DvSNP_c964G[A (3qc1) 50 -CATCCGGGTGCAGGAtCA TGAGCAGGAGGC-30 for c.964G[A, and mfCYP2DvSNP_ c1009G[A (5qc1) 50 -CAACAGGAGATCGACgACGT GATAGGGCAG-30 mfCYP2DvSNP_c1009G[A (3qc1) 50 CTGCCCTATCACGTcGTCGATCTCCTGTTG-30 for c.1009A[G. Small letters indicate the nucleotide to be changed. The sequence of the insert was verified by sequencing. Drug-metabolizing assays Drug-metabolizing capabilities of the CYP2D44 protein variants heterologously expressed in E. coli were evaluated using a CYP2D substrate (bufuralol) as described previously [10, 12]. In brief, a typical mixture (0.25 mL) contained recombinant CYP2D44 protein (5 pmol), an NADPH-generating system (0.25 mM NADP?, 2.5 mM
glucose 6-phosphate, and 0.25 unit/mL glucose-6-phosphate dehydrogenase), and bufuralol (10 or 100 lM) in 50 mM potassium phosphate buffer (pH 7.4). The membrane preparation was fortified with NADPH-P450 reductase (0.10 nM). Reactions were incubated at 37 °C for 10 min, and subsequently terminated by adding 0.025 mL of 17 % perchloric acid. The supernatant obtained by centrifugation (900 g, 5 min) was analyzed by high-performance liquid chromatography with a fluorescence detector. CYP2D44 3D structure To establish a model of a three-dimensional structure, cynomolgus CYP2D44 amino acid sequence (wild type) was aligned with a crystal structure of human CYP2D6 (Protein Data Bank code 3TDA) using the MOE software (version 2013.10; Chemical Computing Group, Montreal, Canada) according to the manufacturer’s instructions. The energy of the CYP2D44 structure was minimized using the CHARM22 force field.
Results and discussion Analysis of the genomic DNA samples from 78 cynomolgus macaques and 40 rhesus macaques identified a total of 68 variants, including 64 non-synonymous variants, 1 nonsense mutation, 2 frameshift mutations, and 1 gene conversion. Of the 64 non-synonymous variants, 11 were located in substrate recognition sites (SRS) potentially important for protein function, including Y107N, G113R, R115H, Y124C, and G125R in SRS1, A209P, Q210E, and E211D in SRS2, R242G and R245 K in SRS3, and M297I in SRS4 (Table 1). All amino-acid variants were biallelic. Among the 68 variants identified, 35 (52 %) were unique to cynomolgus macaques; 14 (22 %) and 19 (27 %) were found only in Indochinese and Indonesian cynomolgus macaques, respectively, while 15 (22 %) were unique to rhesus macaques (Table 1). Seventeen variants were shared by cynomolgus and rhesus macaques. Similarly, uneven distribution of P450 gene alleles has been described in Indochinese and Indonesian cynomolgus macaques, and rhesus macaques [5, 6, 8, 9]. We note that some of those variants showed the significant deviation from the expectation under Hardy–Weinberg equilibrium. In all cases, the deviations were due to the excess of homozygous minor alleles (data not shown), which some degree of inbreeding possibly accounted for, considering that the animals analyzed were not wild-caught individuals but were purpose bred for research use. Notably, the variant c.128T[del was found predominantly in Indonesian cynomolgus macaques (Table 1).
123
Mol Biol Rep Table 1 CYP2D44 variants identified in cynomolgus and rhesus macaques Variant
c.69G[A
Exon
Amino acid change
Allele frequency
Site
Cynomolgus
Rhesus
Indochina
Indonesia
CTGAT(G[A)CACCG
M23I
0/74
0/76
1/70
c.79C[G
GGCGC(C[G)AACGC
Q27E
0/74
0/76
3/70
c.101C[T c.128T[del
CTACC(C[T)GCCAG GGGGC(T[del)GGGC
P34L L43fs91X
0/74 0/74
1/76 52/76
0/70 2/70
c.164C[T
CACAC(C[T)ATACT
P55L
1/74
0/76
2/70
c.175G[C
GCTTC(G[C)ACCAG
D59H
0/74
2/76
1/70
CTGGA(C[T)GCCGG
T76M
0/74
1/74
0/74
c.254C[A
GCTGG(C[A)GGCCG
A85E
0/74
4/76
0/74
c.269C[T
CGAGG(C[T)GCTGG
A90V
0/74
0/76
1/74
c.293C[T
GGACA(C[T)TGCCG
T98I
0/74
0/76
2/74
c.298G[A
CTGCC(G[A)ACCGC
D100N
1/74
0/76
0/74
c.227C[T
1
Nucleotide changea
2
c.319T[A
CCATC(T[A)ACCAG
Y107N
0/74
2/76
0/74
SRS1
c.337G[A
GCATC(G[A)GGCCG
G113R
0/74
0/76
1/74
SRS1
c.344G[A
GCCGC(G[A)CTCCC
R115H
0/74
1/76
0/74
SRS1
c.371A[G
GCGCT(A[G)TGGCC
Y124C
0/72
5/80
0/78
SRS1
c.373G[C
GCTAT(G[C)GCCCC
G125R
1/72
0/80
0/78
SRS1
c.395G[A
GCAGA(G[A)GCGCT
R132K
5/72
0/80
0/78
c.413C[A c.469G[A
GTCTA(C[A)CTTGC AGGAG(G[A)CCGCC
T138N A157T
6/72 2/72
0/80 0/80
0/78 0/78
c.496G[A
TCGCC(G[A)ACCAA
D166N
2/70
2/78
0/78
c.502G[A
ACCAA(G[A)CCGGA
A168T
2/70
0/78
0/78
c.505G[A
AAGCC(G[A)GATGC
G169R
1/70
0/78
0/78
CCCCA(G[A)TGGCC
S175N
13/72
28/78
79/80
c.529C[T
GTGGC(C[T)TCCTG
L177F
1/72
0/78
0/80
c.553G[T
GCAAC(G[T)TAATT
V185L
1/72
9/78
1/80
c.587A[T
CTTTG(A[T)GTACG
E196V
1/72
0/78
0/80
c.595G[C
ACGAC(G[C)ACCCT
D199H
1/72
0/78
1/78
c.601C[G
ACCCT(C[G)GCTTT
R201G
0/72
0/78
1/78
c.612G[T
CTCAG(G[T)CTGCT
R204S
0/72
1/78
0/78
c.625G[C
ACCTA(G[C)CACAG
A209P
0/72
2/78
4/78
c.628C[G
TAGCA(C[G)AGGAG
Q210E
0/72
3/78
1/78
SRS2
c.633G[C
CAGGA(G[C)GGATT
E211D
0/72
1/78
1/78
SRS2
TGCGC(G[T)AGGTG
E222X
0/72
5/78
0/78
ATGCC(G[A)TCCCC TCCTG(C[T)GCATC
V227I R232C
5/76 1/76
0/78 0/78
8/80 0/80
c.704C[G
CCCAG(C[G)GCTGG
A235G
14/76
14/78
63/80
c.710C[T
GCTGG(C[T)TGGCA
A237V
0/76
0/78
1/80
c.724C[G
TCCTA(C[G)GTTCC
R242G
0/76
8/78
0/80
SRS3
c.734G[A
CCAAA(G[A)GGCTT
R245K
0/76
8/78
67/80
SRS3
c.524G[A
3
4
c.664G[T c.679G[A c.694C[T
5
c.769G[A
TGACC(G[A)AGCAC
E257K
0/76
1/76
0/80
c.806G[C
ACCCC(G[C)AGACC
R269P
0/74
0/76
1/80
c.819C[G
ACTGA(C[G)GCCTT
D273E
0/74
0/76
1/80
GAAGG(C[A)CAAGG
A282D
2/76
0/80
0/80
ACCCC(G[A)AGAGC
E287K
0/76
3/80
0/80
c.845C[A c.859G[A
123
6
SRS2
Mol Biol Rep Table 1 continued Variant
Exon
Nucleotide changea
Amino acid change
Allele frequency
Site
Cynomolgus
Rhesus
Indochina
Indonesia
c.891G[A
CGCAT(G[A)GTGGT
M297I
8/76
19/80
0/80
c.964G[A
TCATG(G[A)TCCTG
M322I
0/74
59/80
2/80
c.971A[G c.974C[T
CCTGC(A[G)CCCGG GCACC(C[T)GGATG
H324R P325L
1/74 0/74
0/80 2/80
24/80 0/80 8/76
AGCGC(C[T)GTGTC
R330C
1/76
0/80
c.989G[C
c.988C[T
7
GCGCC(G[C)TGTCC
R330P
0/76
0/80
1/76
c.1009A[G
TCGAC(A[G)ACGTG
N337D
0/76
0/80
27/76
c.1042C[G
AGATG(C[G)GTGAC
R348G
5/76
0/80
0/76
c.1063T[A
TGCCC(T[A)ACACC
Y355N
0/76
2/80
0/76
c.1147G[A
ACATC(G[A)AAGTG
E383K
0/76
1/80
0/76
c.1153C[A
AAGTG(C[A)AGGGC
Q385K
1/76
0/80
0/76
c.1178C[T
8
c.1222G[A
GGGGA(C[T)GACGC
T393M
3/64
0/64
4/62
AGGCC(G[A)TCTGG
V408I
0/64
0/64
3/62
c.1234T[C
AGAAG(T[C)CCTTC
S412P
63/64
64/64
62/62
c.1240C[T
CCTTC(C[T)GCTTC
R414C
0/64
1/64
0/62
c.1267G[A
TGGAT(G[A)CTCAG
A423T
0/64
1/64
0/62
c.1276C[T
AGGGC(C[T)GCTTT
R426C
0/64
0/64
5/62
c.1277G[A c.1280T[G
GGGCC(G[A)CTTTG CCGCT(T[G)TGTGA
R426H F427C
4/64 0/64
40/64 0/64
18/62 1/62 0/42
c.1388G[A
GCAGC(G[A)CTTCA
R463H
0/64
4/54
c.1403T[C
CTCGG(T[C)GCCCG
V468A
0/64
33/54
0/42
c.1415G[A
CGGAC(G[A)GCCCC
R472Q
0/64
37/54
4/42
c.1489C[del
TGCCC(C[del)GCTAG
R497Ab
0/64
0/54
5/42
a
9
SRS4
Nucleotide changes were detected by comparing with CYP2D44 cDNA sequence (GenBank accession no. DQ297684) as a reference sequence
b
c.1489C[del is the frameshift mutation resulting in the longer coding region because termination codon would be shifted to the 30 untranslated region. Due to the unknown 30 untranslated region sequence of CYP2D44, amino acid change at codon 497 is shown
In the animals carrying this allele, CYP2D44 protein, even if translated, would be truncated due to frameshift (in exon 1) and premature termination codon. This null allele was highly prevalent in Indonesian cynomolgus macaques: allele frequency was 0.55, and 16 and 10 of the 32 animals analyzed were the homozygotes and heterozygotes of the deletion, respectively. Only 6 of the 38 animals genotyped for this allele did not possess c.128T[del. Similarly, c.664G[T (E222X) is expected to be a null allele due to nonsense mutation in the coding region (Table 1). This allele was found only in Indonesian cynomolgus macaques (allele frequency 0.06), two homozygotes and one heterozygote. Therefore, CYP2D44 might not contribute substantially to a CYP2D-dependent drug metabolism in Indonesian cynomolgus macaques. In exon 9, the frameshift mutation c.1489C[del was found only in rhesus macaques (Table 1). This mutation would account for the longer coding region because the termination codon is expected to shift to the 30 untranslated
region, the sequence of which has not been reported. In the same exon 9, the substitution of 18 bases (TCTCATGTC GTTGGCTTT) with those (CATGGTGTCTTTGCTTTC) of CYP2D6 (formerly known as cynomolgus CYP2D17 and rhesus CYP2D42) was found only in eight Indonesian cynomolgus macaques as the heterozygotes. This substitution is most likely due to gene conversion, which has been found in other P450 genes such as human CYP2A6 and CYP2D6 [13]. This mutation (p.478-482SHVVG[HGVFA) does not accompany a frameshift or a shift in codon usage, and thus the mutant protein is expected to be functionally intact. Because of the relatively large amino acid changes, these two mutations in exon 9 might influence CYP2D44 enzyme activity in rhesus macaques or Indonesian cynomolgus macaques. To assess functional importance, the non-synonymous variants with minor allele frequency [10 %, including S175N, V185L, A235G, R242G, R245K, M322I, and N337D, were analyzed for bufuralol 10 -hydroxylation using
123
Mol Biol Rep
123
100 µM
2
N337D
N.D.
M322I
R245K
R242G
V185L
S175N
WT
N.D. N.D. N.D. N.D.
0
N337D
N337D
M322I
R245K
R242G
A235G
0
V185L
0
S175N
1.5
WT
1.7
4
100 µM
3.0
M322I
10 µM
3.4
Dextromethorphan O-demethylation (nnol/min/nmol CYP2D44)
Fig. 2 Catalytic activities of CYP2D44 non-synonymous variants. Bufuralol 10 hydroxylation and dextromethorphan Odemethylation were measured using 10- or 100-lM substrate with heterologously expressed proteins (wild type, S175N, V185L, A235G, R242G, R245K, M322I, or N337D) as described in Materials and Methods. Values are average of duplicate determinations
Bufuralol 1'-hydroxylation (nnol/min/nmol CYP2D44)
the variant proteins heterologously expressed in E. coli. Other non-synonymous variants (M297I, R330C, R426H, V468A, and R472Q) failed to be expressed in E. coli and thus were excluded from the analysis. It should be noted that CYP2D44 protein model based on a crystal structure of reported human CYP2D6 showed the typical P450 conformation (Fig. 1). The analysis showed that all these variants except for M322I showed substantially reduced activity, 6.1to 24-fold and 3.3- to 24-fold less than wild type at 10 and 100 lM of substrate, respectively (Fig. 2). Interestingly,
R245K
Fig. 1 CYP2D44 protein structure. The structure was established based on reported human CYP2D6 structure as described in Materials and Methods. The non-synonymous variants analyzed for their drugmetabolizing activities are indicated in the figure
R242G
N-terminus
A235G
A235
V185L
R242
A235G
R245
N337 M322
S175N
V185 S175
these variants were more prevalent in rhesus macaques than in cynomolgus macaques, including A235G, R245K, and N337D, of which N337D was found only in rhesus macaques (Table 1). The results raised the possibility that CYP2D44 might be a defective enzyme in rhesus macaques. In cynomolgus macaques, S175N, V185L, R242G, and R245K were predominantly found in Indochinese cynomolgus macaques (Table 1), indicating the potential differences of CYP2D44-dependent drug metabolism between Indochinese and Indonesian cynomolgus macaques. A high prevalence of null or defective alleles in Indonesian cynomolgus macaques and rhesus macaques might imply pseudogenization of CYP2D44 in these populations or lineages. However, a final conclusion must be drawn from further investigation using a large number of animals from various populations, because only limited numbers of animals were analyzed in the present study. We previously revealed that CYP2D44 identified in Indochinese cynomolgus macaques was a functional enzyme [10], raising the possibility that CYP2D44 is functional in Indochinese cynomolgus macaques, but is under pseudogenization in Indonesian cynomolgus macaques and rhesus macaques. P450 genes relevant to detoxification of xenobiotics are characterized by frequent gene duplications and losses even between closely related species [14]. Indeed, we previously found that CYP2C93, encoding the drug-metabolizing enzyme, is functional in rhesus macaques, but appears to be pseudogenized in cynomolgus macaques [15].
WT
C-terminus
Mol Biol Rep
In conclusion, the screening of 78 cynomolgus and 40 rhesus macaque genomes successfully identified 64 nonsynonymous variants, 1 nonsense mutation, 2 frameshift mutations, and 1 gene conversion in CYP2D44. Importantly, two null alleles (c.128T[del and c.664G[T) were largely found in Indonesian cynomolgus macaques, but absent or scarce in Indochinese cynomolgus macaques or rhesus macaques. Seven CYP2D44 variants, including S175N, V185L, A235G, R242G, R245K, and N337D, showed reduced rates of bufuralol 10 -hydroxylation. Some of these variants were more prevalent in Indonesian cynomolgus macaques and/or rhesus macaques than in Indochinese cynomolgus macaques. CYP2D44 appears to be under pseudogenization in Indonesian cynomolgus macaques, and possibly rhesus macaques. Therefore, genetic polymorphisms might account for the variability of CYP2D44-dependent metabolism in macaques. The data presented in the present study provides useful information when one conducts drug metabolism studies using cynomolgus or rhesus macaques. Acknowledgments We greatly thank Mr. Masahiro Utoh for his support of this work, and Mr. Lance Bell for his advice on English writing.
References 1. Baillie TA, Rettie AE (2011) Role of biotransformation in druginduced toxicity: influence of intra- and inter-species differences in drug metabolism. Drug Metab Pharmacokinet 26(1):15–29 2. Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, Ball SE (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32(11):1201–1208 3. Ingelman-Sundberg M (2005) Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity. Pharmacogenomics J 5(1):6–13 4. Jacqz E, Billante C, Moysan F, Mathieu H (1988) The non-human primate: a possible model for human genetically determined polymorphisms in oxidative drug metabolism. Mol Pharmacol 34(2):215–217
5. Uehara S, Murayama N, Yamazaki H, Uno Y (2012) CYP2C76 non-synonymous variants in cynomolgus and rhesus macaques. Drug Metab Pharmacokinet 27(3):344–348 6. Uno Y, Matsushita A, Osada N, Uehara S, Kohara S, Nagata R, Fukuzaki K, Utoh M, Murayama N, Yamazaki H (2010) Genetic variants of CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques. Drug Metab Dispos 38(2):209–214 7. Uno Y, Osada N (2011) CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2. BMC Evol Biol 11:283 8. Uno Y, Sakuraba H, Uehara S, Kumano T, Matsuno K, Nakamura C, Kito G, Kamataki T, Nagata R (2009) A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism. Drug Metab Dispos 37(1):14–17 9. Uno Y, Uehara S, Yamazaki H (2011) A discovery of genetic variants in CYP1D1: an implication for functional integrity of CYP1D1 in cynomolgus macaques and rhesus macaques. Drug Metab Pharmacokinet 26(6):627–631 10. Uno Y, Uehara S, Kohara S, Murayama N, Yamazaki H (2010) Cynomolgus monkey CYP2D44 newly identified in liver, metabolizes bufuralol and dextromethorphan. Drug Metab Dispos 38(9):1486–1492 11. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378 12. Yamazaki H, Nakamura M, Komatsu T, Ohyama K, Hatanaka N, Asahi S, Shimada N, Guengerich FP, Shimada T, Nakajima M, Yokoi T (2002) Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein Expr Purif 24(3):329–337 13. Zhou SF, Liu JP, Chowbay B (2009) Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab Rev 41(2):89–295 14. Thomas JH (2007) Rapid birth–death evolution specific to xenobiotic cytochrome P450 genes in vertebrates. PLoS Genet 3(5):e67 15. Uno Y, Uehara S, Kohara S, Iwasaki K, Nagata R, Fukuzaki K, Utoh M, Murayama N, Yamazaki H (2011) Newly identified CYP2C93 is a functional enzyme in rhesus monkey, but not in cynomolgus monkey. PLoS One 6(2):e16923
123
CYP2D44 polymorphisms in cynomolgus and rhesus macaques Yasuhiro Uno • Shotaro Uehara • Sakae Kohara • Naoki Osada • Norie Murayama • Hiroshi Yamazaki
Received: 3 July 2014 / Accepted: 10 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Macaques, including cynomolgus and rhesus macaques, are important animal species used in drug metabolism studies. CYP2D44 is expressed in cynomolgus macaque liver and encodes a functional drug metabolizing enzyme, metabolizing typical human CYP2D substrates such as bufuralol and dextromethorphan. CYP2D44 is highly homologous to human CYP2D6 that is known to be polymorphic with a large inter-individual variation in metabolic activities, however, genetic polymorphisms have not been investigated in macaque CYP2D44. In the present study, screening of 78 cynomolgus and 40 rhesus macaques found a total of 67 variants, including 64 non-synonymous variants, 1 nonsense mutation, and 2 frameshift mutations, and 1 gene conversion, of which 14, 19, and 15 variants were unique to Indochinese cynomolgus macaques, Indonesian cynomolgus macaques, and Chinese rhesus macaques, respectively. Eleven of the 64 non-synonymous variants were located in substrate recognition sites, the regions important for protein function. By site-directed mutagenesis and
Y. Uno (&) S. Uehara S. Kohara Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minami Akasaka, Kainan, Wakayama 642-0017, Japan e-mail: [email protected] S. Uehara N. Murayama H. Yamazaki Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo, Japan N. Osada Department of Population Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan N. Osada Department of Genetics, Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan
metabolic assays, S175N, V185L, A235G, R242G, R245K, and N337D showed substantially decreased activity in bufuralol 10 -hydroxylation as compared with wild-type proteins. Moreover, two null alleles (c.128T[del and c.664G[T) were found in Indonesian cynomolgus macaques, but not in Indochinese cynomolgus macaques or Chinese rhesus macaques. These results suggest that genetic polymorphisms might account for the variability of CYP2D44-dependent metabolism in macaques. Keywords Cytochrome P450 Genetic polymorphism Metabolic activity Monkey
Introduction Animal studies are conducted in the preclinical drug development to predict metabolism of the newly developed drugs in humans; however, intra- and inter-species differences are occasionally seen in the studies, making it difficult to extrapolate the animal data to humans [1]. Such differences are partly accounted for by cytochrome P450 (P450), a family of important drug-metabolizing enzymes, which is involved in metabolism of a large number of drugs [2]. In the preclinical studies, rodent and non-rodent species are used, and the non-rodent species frequently used are dogs and monkeys. Therefore, it is important to understand the intra- and inter-species differences in a P450mediated drug metabolism in monkeys. CYP2D6, one of P450s, is involved in the metabolism of approximately 20–25 % of the prescribed drugs, including bufuralol and dextromethorphan [3]. CYP2D6 is highly polymorphic with a number of alleles (see http://www. cypalleles.ki.se). A large inter-individual variation that has been noted for CYP2D6-dependent metabolic activities is
123
Mol Biol Rep
partly explained by the genetic polymorphisms at CYP2D6 locus [3]. For example, CYP2D6*4 and CYP2D6*10 result in a defective or unstable enzyme, and the entire gene is deleted in some human genomes (CYP2D6*5). Moreover, these alleles are distributed differently in populations; CYP2D6*4 and CYP2D6*10 are mainly found in Caucasians and Asians, respectively, making these alleles important when prescribing drugs at hospital or in clinical studies during drug development. Cynomolgus macaques (Macaca fascicularis), and also rhesus macaques (Macaca mulatta), are frequently used in drug metabolism studies due to their evolutionary closeness to humans. In vivo analysis using CYP2D probe drug (dextromethorphan) revealed inter-animal variability in pharmacokinetics [4], which could be accounted for by genetic polymorphisms. In cynomolgus macaques, a number of genetic variants have been identified, including P450 genes [5–9]. However, investigation on involvement of genetic polymorphisms in the inter-animal variability of CYP2D-dependent drug metabolism has been hampered by the lack of information on the CYP2D genetic polymorphisms in this species. In cynomolgus and rhesus macaques, two CYP2D genes, CYP2D17 and CYP2D44, are expressed predominantly in liver and encode functional drug-metabolizing enzymes, metabolizing human CYP2D6 substrates, bufuralol and dextromethorphan [10]. In the present study, a screening was conducted using genome samples from 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia) and 40 Chinese rhesus macaques to identify genetic variants in CYP2D44. Distribution of the variants found was analyzed in Indochinese and Indonesian cynomolgus macaques, and Chinese rhesus macaques. For some of the variants, protein was expressed in Escherichia coli, with which metabolic assays were performed using CYP2D substrate (bufuralol).
Materials and methods Preparation of genomic DNA Whole blood samples were collected from 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia, 4–5 years of age, weighing 3–5 kg). Blood samples were also obtained from 40 rhesus macaques (from China, 7 years of age, weighing 3–5 kg). From these samples, genomic DNA was extracted using the Puregene DNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd.
123
PCR amplification, sequencing, and sequence analysis To identify genetic variants, genomic DNA samples of 78 cynomolgus macaques and 40 rhesus macaques were subjected to direct sequencing of exons 1–7. PCR was carried out in a 20-lL reaction containing 1 ng of genomic DNA, 5 pmol of forward and reverse primers, and 1 unit of LATaq DNA polymerase (Takara, Ohtsu, Japan) for exons 1–3, AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) for exons 4–7, and Ex-Taq DNA polymerase (Takara, Ohtsu, Japan) for exons 8–9. The thermal cycler conditions included an initial denaturation at 95 °C for 2 min, and 35 cycles of 20 s at 95 °C, 30 s at 62 °C, and 2 min at 72 °C, followed by a final extension step of 10 min at 72 °C. For exons 4–7, an initial denaturation was conducted at 95 °C for 10 min. The PCR primers were mmCYP2Dv (5flk1a) 50 -GCAGCCCCTCAG TCAGCTA-30 and mfCYP2Dv (3int1c) 50 -CTTTGGAAA ATCTCATCTTTCATGC-30 for exons 1–2, mfCYP2Dv (5int2x1) 50 -GGGACAGACACCTCGTTTCC-30 and mfCYP 2Dv (3int4a) 50 -GGGTCTCCATCTCTCCTGTG-30 for exons 3–4, mfCYP2Dv (5int4a) 50 -GCATAGAAGGGTTTGGGA AA-30 and mfCYP2Dv (3int5a) 50 -AGAGGGTACTTGGG GACACA-30 for exon 5, mfCYP2Dv (5int5a) 50 -GTGAG GACCAAGGGTGGTG-30 and mfCYP2Dv (3int6a) 50 TCAGGGGACCAGCCCTGACCCTCTCCT-30 for exon 6, mfCYP2Dv (5int6a) 50 -CAGCACAGGCTTGACCAGTCT -30 and mfCYP2Dv (3int7a) 50 -CACACAGCTGGACTCTG TCAA-30 for exon 7, and mmCYP2D44 (5int7a) 50 -CCA GTCTAGTAGGGAAGACAGATCA-30 and mmCYP2D44 (3flk1a) 50 -GCCACTAGATTCATTTTCTGTACAAC-30 for exons 8 and 9. Sequencing was performed using an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequencing primers were mmCYP2Dv (5flk1b) 50 -CGCTATGCTCCCAGTACACA-30 or mfCYP 2Dv (3int1d) 50 -GGAAGTCCCTCAAATCTGCTTC-30 for exon 1, mfCYP2Dv (5ex1b) 50 -GGACTTCAAGAACA CACCATACTG-30 or mf2Dv (3int2xx2) 50 -TGTGGTTTTC CTGGTCTTCC-30 for exon 2, mf2Dv (3int3xx1) 50 -CCCC ACCTTCCCAGTTCC-30 for exon 3, mfCYP2Dv (5int3a) 50 AAGGCGGGAACTGGGAAG-30 for exon 4, mfCYP2Dv (5int4a) or mfCYP2Dv (3int5a) for exon 5, mfCYP2Dv (5int5a) for exon 6, mfCYP2Dv (5int6a) for exon 7, and mmCYP2D44 (5int7b) 50 -CTGTGTGCCAGGCA GTGT-30 and/or mmCYP2D44 (3flk1a) for exons 8 and 9. Sequence analysis was conducted using Sequencher (Gene Codes, Ann Arbor, MI, USA) and DNASIS Pro (Hitachi Software, Tokyo, Japan). To determine genetic variants, the sequence was compared to cynomolgus CYP2D44 cDNA sequence (GenBank accession DQ297684).
Mol Biol Rep
Preparation of CYP2D44 proteins CYP2D44 protein variants were prepared as previously described [10], including the construction of pCW expression plasmids, protein expression, and membrane preparation, except that the pCW vector did not contain the reductase cDNA. The content of CYP2D44 protein variants in the membrane preparation was determined by Fe2?CO versus Fe2? difference spectra according to the method previously described [11]. QuikChange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used to introduce each mutation into the expression plasmid of cynomolgus CYP2D44 according to the manufacturer’s instructions. The primers used were: mfCYP2DvSNP_c128del (5qc1) 50 -CTGCCACTGC CGGGGCGGGCAACCTGCTGC-30 and mfCYP2DvSNP_ c128del (3qc1) 50 -GCAGCAGGTTGCCCGCCCCGGCAG TGGCAG-30 for c.128T[del, mfCYP2DvSNP_c524A[G (5qc1) 50 -CCCCTTTCGCCCCAaTGGCCTCCTGGACAA30 and mfCYP2DvSNP_c524A[G (3qc1) 50 -TTGTCCAGGAGGCCAtTGGGGCGAAAGGGG-30 for c.524G[A, mf CYP2DvSNP_c553G[T (5qc1) 50 -AGCGGCGAGCAACtTAATTGCCTCCCTCAC-30 mfCYP2DvSNP_c553G[T 0 (3qc1) 5 -GTGAGGGAGGCAATTAtGTTGCTCGCCGC T-30 for c.553G[T, mfCYP2DvSNP_c704G[C (5qc1) 50 CTGCGCATCCCAGgGCTGGCTGGCAAGGTC-30 mfCYP 2DvSNP_c704G[C (3qc1) 50 -GACCTTGCCAGCCAGCcC TGGGATGCGCAG-30 for c.704G[C, mfCYP2DvSNP_ c724C[G (5qc1) 50 -CTGGCAAGGTCCTAgGTTCCCAAA GGGCTT-30 mfCYP2DvSNP_c724C[G (3qc1) 50 -AAGCCC TTTGGGAACcTAGGACCTTGCCAG-30 for c.724C[G, mfCYP2DvSNP_c734A[G (5qc1) 50 -CTACGTTCCCAAA aGGCTTTCTTGACCCAG-30 mfCYP2DvSNP_c734A[G (3qc1) 50 -CTGGGTCAAGAAAGCCtTTTGGGAACGTAG30 for c.734A[G, mfCYP2DvSNP_c964G[A (5qc1) 50 GCCTCCTGCTCATGaTCCTGCACCCGGATG-30 mfCYP 2DvSNP_c964G[A (3qc1) 50 -CATCCGGGTGCAGGAtCA TGAGCAGGAGGC-30 for c.964G[A, and mfCYP2DvSNP_ c1009G[A (5qc1) 50 -CAACAGGAGATCGACgACGT GATAGGGCAG-30 mfCYP2DvSNP_c1009G[A (3qc1) 50 CTGCCCTATCACGTcGTCGATCTCCTGTTG-30 for c.1009A[G. Small letters indicate the nucleotide to be changed. The sequence of the insert was verified by sequencing. Drug-metabolizing assays Drug-metabolizing capabilities of the CYP2D44 protein variants heterologously expressed in E. coli were evaluated using a CYP2D substrate (bufuralol) as described previously [10, 12]. In brief, a typical mixture (0.25 mL) contained recombinant CYP2D44 protein (5 pmol), an NADPH-generating system (0.25 mM NADP?, 2.5 mM
glucose 6-phosphate, and 0.25 unit/mL glucose-6-phosphate dehydrogenase), and bufuralol (10 or 100 lM) in 50 mM potassium phosphate buffer (pH 7.4). The membrane preparation was fortified with NADPH-P450 reductase (0.10 nM). Reactions were incubated at 37 °C for 10 min, and subsequently terminated by adding 0.025 mL of 17 % perchloric acid. The supernatant obtained by centrifugation (900 g, 5 min) was analyzed by high-performance liquid chromatography with a fluorescence detector. CYP2D44 3D structure To establish a model of a three-dimensional structure, cynomolgus CYP2D44 amino acid sequence (wild type) was aligned with a crystal structure of human CYP2D6 (Protein Data Bank code 3TDA) using the MOE software (version 2013.10; Chemical Computing Group, Montreal, Canada) according to the manufacturer’s instructions. The energy of the CYP2D44 structure was minimized using the CHARM22 force field.
Results and discussion Analysis of the genomic DNA samples from 78 cynomolgus macaques and 40 rhesus macaques identified a total of 68 variants, including 64 non-synonymous variants, 1 nonsense mutation, 2 frameshift mutations, and 1 gene conversion. Of the 64 non-synonymous variants, 11 were located in substrate recognition sites (SRS) potentially important for protein function, including Y107N, G113R, R115H, Y124C, and G125R in SRS1, A209P, Q210E, and E211D in SRS2, R242G and R245 K in SRS3, and M297I in SRS4 (Table 1). All amino-acid variants were biallelic. Among the 68 variants identified, 35 (52 %) were unique to cynomolgus macaques; 14 (22 %) and 19 (27 %) were found only in Indochinese and Indonesian cynomolgus macaques, respectively, while 15 (22 %) were unique to rhesus macaques (Table 1). Seventeen variants were shared by cynomolgus and rhesus macaques. Similarly, uneven distribution of P450 gene alleles has been described in Indochinese and Indonesian cynomolgus macaques, and rhesus macaques [5, 6, 8, 9]. We note that some of those variants showed the significant deviation from the expectation under Hardy–Weinberg equilibrium. In all cases, the deviations were due to the excess of homozygous minor alleles (data not shown), which some degree of inbreeding possibly accounted for, considering that the animals analyzed were not wild-caught individuals but were purpose bred for research use. Notably, the variant c.128T[del was found predominantly in Indonesian cynomolgus macaques (Table 1).
123
Mol Biol Rep Table 1 CYP2D44 variants identified in cynomolgus and rhesus macaques Variant
c.69G[A
Exon
Amino acid change
Allele frequency
Site
Cynomolgus
Rhesus
Indochina
Indonesia
CTGAT(G[A)CACCG
M23I
0/74
0/76
1/70
c.79C[G
GGCGC(C[G)AACGC
Q27E
0/74
0/76
3/70
c.101C[T c.128T[del
CTACC(C[T)GCCAG GGGGC(T[del)GGGC
P34L L43fs91X
0/74 0/74
1/76 52/76
0/70 2/70
c.164C[T
CACAC(C[T)ATACT
P55L
1/74
0/76
2/70
c.175G[C
GCTTC(G[C)ACCAG
D59H
0/74
2/76
1/70
CTGGA(C[T)GCCGG
T76M
0/74
1/74
0/74
c.254C[A
GCTGG(C[A)GGCCG
A85E
0/74
4/76
0/74
c.269C[T
CGAGG(C[T)GCTGG
A90V
0/74
0/76
1/74
c.293C[T
GGACA(C[T)TGCCG
T98I
0/74
0/76
2/74
c.298G[A
CTGCC(G[A)ACCGC
D100N
1/74
0/76
0/74
c.227C[T
1
Nucleotide changea
2
c.319T[A
CCATC(T[A)ACCAG
Y107N
0/74
2/76
0/74
SRS1
c.337G[A
GCATC(G[A)GGCCG
G113R
0/74
0/76
1/74
SRS1
c.344G[A
GCCGC(G[A)CTCCC
R115H
0/74
1/76
0/74
SRS1
c.371A[G
GCGCT(A[G)TGGCC
Y124C
0/72
5/80
0/78
SRS1
c.373G[C
GCTAT(G[C)GCCCC
G125R
1/72
0/80
0/78
SRS1
c.395G[A
GCAGA(G[A)GCGCT
R132K
5/72
0/80
0/78
c.413C[A c.469G[A
GTCTA(C[A)CTTGC AGGAG(G[A)CCGCC
T138N A157T
6/72 2/72
0/80 0/80
0/78 0/78
c.496G[A
TCGCC(G[A)ACCAA
D166N
2/70
2/78
0/78
c.502G[A
ACCAA(G[A)CCGGA
A168T
2/70
0/78
0/78
c.505G[A
AAGCC(G[A)GATGC
G169R
1/70
0/78
0/78
CCCCA(G[A)TGGCC
S175N
13/72
28/78
79/80
c.529C[T
GTGGC(C[T)TCCTG
L177F
1/72
0/78
0/80
c.553G[T
GCAAC(G[T)TAATT
V185L
1/72
9/78
1/80
c.587A[T
CTTTG(A[T)GTACG
E196V
1/72
0/78
0/80
c.595G[C
ACGAC(G[C)ACCCT
D199H
1/72
0/78
1/78
c.601C[G
ACCCT(C[G)GCTTT
R201G
0/72
0/78
1/78
c.612G[T
CTCAG(G[T)CTGCT
R204S
0/72
1/78
0/78
c.625G[C
ACCTA(G[C)CACAG
A209P
0/72
2/78
4/78
c.628C[G
TAGCA(C[G)AGGAG
Q210E
0/72
3/78
1/78
SRS2
c.633G[C
CAGGA(G[C)GGATT
E211D
0/72
1/78
1/78
SRS2
TGCGC(G[T)AGGTG
E222X
0/72
5/78
0/78
ATGCC(G[A)TCCCC TCCTG(C[T)GCATC
V227I R232C
5/76 1/76
0/78 0/78
8/80 0/80
c.704C[G
CCCAG(C[G)GCTGG
A235G
14/76
14/78
63/80
c.710C[T
GCTGG(C[T)TGGCA
A237V
0/76
0/78
1/80
c.724C[G
TCCTA(C[G)GTTCC
R242G
0/76
8/78
0/80
SRS3
c.734G[A
CCAAA(G[A)GGCTT
R245K
0/76
8/78
67/80
SRS3
c.524G[A
3
4
c.664G[T c.679G[A c.694C[T
5
c.769G[A
TGACC(G[A)AGCAC
E257K
0/76
1/76
0/80
c.806G[C
ACCCC(G[C)AGACC
R269P
0/74
0/76
1/80
c.819C[G
ACTGA(C[G)GCCTT
D273E
0/74
0/76
1/80
GAAGG(C[A)CAAGG
A282D
2/76
0/80
0/80
ACCCC(G[A)AGAGC
E287K
0/76
3/80
0/80
c.845C[A c.859G[A
123
6
SRS2
Mol Biol Rep Table 1 continued Variant
Exon
Nucleotide changea
Amino acid change
Allele frequency
Site
Cynomolgus
Rhesus
Indochina
Indonesia
c.891G[A
CGCAT(G[A)GTGGT
M297I
8/76
19/80
0/80
c.964G[A
TCATG(G[A)TCCTG
M322I
0/74
59/80
2/80
c.971A[G c.974C[T
CCTGC(A[G)CCCGG GCACC(C[T)GGATG
H324R P325L
1/74 0/74
0/80 2/80
24/80 0/80 8/76
AGCGC(C[T)GTGTC
R330C
1/76
0/80
c.989G[C
c.988C[T
7
GCGCC(G[C)TGTCC
R330P
0/76
0/80
1/76
c.1009A[G
TCGAC(A[G)ACGTG
N337D
0/76
0/80
27/76
c.1042C[G
AGATG(C[G)GTGAC
R348G
5/76
0/80
0/76
c.1063T[A
TGCCC(T[A)ACACC
Y355N
0/76
2/80
0/76
c.1147G[A
ACATC(G[A)AAGTG
E383K
0/76
1/80
0/76
c.1153C[A
AAGTG(C[A)AGGGC
Q385K
1/76
0/80
0/76
c.1178C[T
8
c.1222G[A
GGGGA(C[T)GACGC
T393M
3/64
0/64
4/62
AGGCC(G[A)TCTGG
V408I
0/64
0/64
3/62
c.1234T[C
AGAAG(T[C)CCTTC
S412P
63/64
64/64
62/62
c.1240C[T
CCTTC(C[T)GCTTC
R414C
0/64
1/64
0/62
c.1267G[A
TGGAT(G[A)CTCAG
A423T
0/64
1/64
0/62
c.1276C[T
AGGGC(C[T)GCTTT
R426C
0/64
0/64
5/62
c.1277G[A c.1280T[G
GGGCC(G[A)CTTTG CCGCT(T[G)TGTGA
R426H F427C
4/64 0/64
40/64 0/64
18/62 1/62 0/42
c.1388G[A
GCAGC(G[A)CTTCA
R463H
0/64
4/54
c.1403T[C
CTCGG(T[C)GCCCG
V468A
0/64
33/54
0/42
c.1415G[A
CGGAC(G[A)GCCCC
R472Q
0/64
37/54
4/42
c.1489C[del
TGCCC(C[del)GCTAG
R497Ab
0/64
0/54
5/42
a
9
SRS4
Nucleotide changes were detected by comparing with CYP2D44 cDNA sequence (GenBank accession no. DQ297684) as a reference sequence
b
c.1489C[del is the frameshift mutation resulting in the longer coding region because termination codon would be shifted to the 30 untranslated region. Due to the unknown 30 untranslated region sequence of CYP2D44, amino acid change at codon 497 is shown
In the animals carrying this allele, CYP2D44 protein, even if translated, would be truncated due to frameshift (in exon 1) and premature termination codon. This null allele was highly prevalent in Indonesian cynomolgus macaques: allele frequency was 0.55, and 16 and 10 of the 32 animals analyzed were the homozygotes and heterozygotes of the deletion, respectively. Only 6 of the 38 animals genotyped for this allele did not possess c.128T[del. Similarly, c.664G[T (E222X) is expected to be a null allele due to nonsense mutation in the coding region (Table 1). This allele was found only in Indonesian cynomolgus macaques (allele frequency 0.06), two homozygotes and one heterozygote. Therefore, CYP2D44 might not contribute substantially to a CYP2D-dependent drug metabolism in Indonesian cynomolgus macaques. In exon 9, the frameshift mutation c.1489C[del was found only in rhesus macaques (Table 1). This mutation would account for the longer coding region because the termination codon is expected to shift to the 30 untranslated
region, the sequence of which has not been reported. In the same exon 9, the substitution of 18 bases (TCTCATGTC GTTGGCTTT) with those (CATGGTGTCTTTGCTTTC) of CYP2D6 (formerly known as cynomolgus CYP2D17 and rhesus CYP2D42) was found only in eight Indonesian cynomolgus macaques as the heterozygotes. This substitution is most likely due to gene conversion, which has been found in other P450 genes such as human CYP2A6 and CYP2D6 [13]. This mutation (p.478-482SHVVG[HGVFA) does not accompany a frameshift or a shift in codon usage, and thus the mutant protein is expected to be functionally intact. Because of the relatively large amino acid changes, these two mutations in exon 9 might influence CYP2D44 enzyme activity in rhesus macaques or Indonesian cynomolgus macaques. To assess functional importance, the non-synonymous variants with minor allele frequency [10 %, including S175N, V185L, A235G, R242G, R245K, M322I, and N337D, were analyzed for bufuralol 10 -hydroxylation using
123
Mol Biol Rep
123
100 µM
2
N337D
N.D.
M322I
R245K
R242G
V185L
S175N
WT
N.D. N.D. N.D. N.D.
0
N337D
N337D
M322I
R245K
R242G
A235G
0
V185L
0
S175N
1.5
WT
1.7
4
100 µM
3.0
M322I
10 µM
3.4
Dextromethorphan O-demethylation (nnol/min/nmol CYP2D44)
Fig. 2 Catalytic activities of CYP2D44 non-synonymous variants. Bufuralol 10 hydroxylation and dextromethorphan Odemethylation were measured using 10- or 100-lM substrate with heterologously expressed proteins (wild type, S175N, V185L, A235G, R242G, R245K, M322I, or N337D) as described in Materials and Methods. Values are average of duplicate determinations
Bufuralol 1'-hydroxylation (nnol/min/nmol CYP2D44)
the variant proteins heterologously expressed in E. coli. Other non-synonymous variants (M297I, R330C, R426H, V468A, and R472Q) failed to be expressed in E. coli and thus were excluded from the analysis. It should be noted that CYP2D44 protein model based on a crystal structure of reported human CYP2D6 showed the typical P450 conformation (Fig. 1). The analysis showed that all these variants except for M322I showed substantially reduced activity, 6.1to 24-fold and 3.3- to 24-fold less than wild type at 10 and 100 lM of substrate, respectively (Fig. 2). Interestingly,
R245K
Fig. 1 CYP2D44 protein structure. The structure was established based on reported human CYP2D6 structure as described in Materials and Methods. The non-synonymous variants analyzed for their drugmetabolizing activities are indicated in the figure
R242G
N-terminus
A235G
A235
V185L
R242
A235G
R245
N337 M322
S175N
V185 S175
these variants were more prevalent in rhesus macaques than in cynomolgus macaques, including A235G, R245K, and N337D, of which N337D was found only in rhesus macaques (Table 1). The results raised the possibility that CYP2D44 might be a defective enzyme in rhesus macaques. In cynomolgus macaques, S175N, V185L, R242G, and R245K were predominantly found in Indochinese cynomolgus macaques (Table 1), indicating the potential differences of CYP2D44-dependent drug metabolism between Indochinese and Indonesian cynomolgus macaques. A high prevalence of null or defective alleles in Indonesian cynomolgus macaques and rhesus macaques might imply pseudogenization of CYP2D44 in these populations or lineages. However, a final conclusion must be drawn from further investigation using a large number of animals from various populations, because only limited numbers of animals were analyzed in the present study. We previously revealed that CYP2D44 identified in Indochinese cynomolgus macaques was a functional enzyme [10], raising the possibility that CYP2D44 is functional in Indochinese cynomolgus macaques, but is under pseudogenization in Indonesian cynomolgus macaques and rhesus macaques. P450 genes relevant to detoxification of xenobiotics are characterized by frequent gene duplications and losses even between closely related species [14]. Indeed, we previously found that CYP2C93, encoding the drug-metabolizing enzyme, is functional in rhesus macaques, but appears to be pseudogenized in cynomolgus macaques [15].
WT
C-terminus
Mol Biol Rep
In conclusion, the screening of 78 cynomolgus and 40 rhesus macaque genomes successfully identified 64 nonsynonymous variants, 1 nonsense mutation, 2 frameshift mutations, and 1 gene conversion in CYP2D44. Importantly, two null alleles (c.128T[del and c.664G[T) were largely found in Indonesian cynomolgus macaques, but absent or scarce in Indochinese cynomolgus macaques or rhesus macaques. Seven CYP2D44 variants, including S175N, V185L, A235G, R242G, R245K, and N337D, showed reduced rates of bufuralol 10 -hydroxylation. Some of these variants were more prevalent in Indonesian cynomolgus macaques and/or rhesus macaques than in Indochinese cynomolgus macaques. CYP2D44 appears to be under pseudogenization in Indonesian cynomolgus macaques, and possibly rhesus macaques. Therefore, genetic polymorphisms might account for the variability of CYP2D44-dependent metabolism in macaques. The data presented in the present study provides useful information when one conducts drug metabolism studies using cynomolgus or rhesus macaques. Acknowledgments We greatly thank Mr. Masahiro Utoh for his support of this work, and Mr. Lance Bell for his advice on English writing.
References 1. Baillie TA, Rettie AE (2011) Role of biotransformation in druginduced toxicity: influence of intra- and inter-species differences in drug metabolism. Drug Metab Pharmacokinet 26(1):15–29 2. Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, Ball SE (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32(11):1201–1208 3. Ingelman-Sundberg M (2005) Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity. Pharmacogenomics J 5(1):6–13 4. Jacqz E, Billante C, Moysan F, Mathieu H (1988) The non-human primate: a possible model for human genetically determined polymorphisms in oxidative drug metabolism. Mol Pharmacol 34(2):215–217
5. Uehara S, Murayama N, Yamazaki H, Uno Y (2012) CYP2C76 non-synonymous variants in cynomolgus and rhesus macaques. Drug Metab Pharmacokinet 27(3):344–348 6. Uno Y, Matsushita A, Osada N, Uehara S, Kohara S, Nagata R, Fukuzaki K, Utoh M, Murayama N, Yamazaki H (2010) Genetic variants of CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques. Drug Metab Dispos 38(2):209–214 7. Uno Y, Osada N (2011) CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2. BMC Evol Biol 11:283 8. Uno Y, Sakuraba H, Uehara S, Kumano T, Matsuno K, Nakamura C, Kito G, Kamataki T, Nagata R (2009) A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism. Drug Metab Dispos 37(1):14–17 9. Uno Y, Uehara S, Yamazaki H (2011) A discovery of genetic variants in CYP1D1: an implication for functional integrity of CYP1D1 in cynomolgus macaques and rhesus macaques. Drug Metab Pharmacokinet 26(6):627–631 10. Uno Y, Uehara S, Kohara S, Murayama N, Yamazaki H (2010) Cynomolgus monkey CYP2D44 newly identified in liver, metabolizes bufuralol and dextromethorphan. Drug Metab Dispos 38(9):1486–1492 11. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378 12. Yamazaki H, Nakamura M, Komatsu T, Ohyama K, Hatanaka N, Asahi S, Shimada N, Guengerich FP, Shimada T, Nakajima M, Yokoi T (2002) Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein Expr Purif 24(3):329–337 13. Zhou SF, Liu JP, Chowbay B (2009) Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab Rev 41(2):89–295 14. Thomas JH (2007) Rapid birth–death evolution specific to xenobiotic cytochrome P450 genes in vertebrates. PLoS Genet 3(5):e67 15. Uno Y, Uehara S, Kohara S, Iwasaki K, Nagata R, Fukuzaki K, Utoh M, Murayama N, Yamazaki H (2011) Newly identified CYP2C93 is a functional enzyme in rhesus monkey, but not in cynomolgus monkey. PLoS One 6(2):e16923
123
