Discovery of Genetic Variants in CYP1D1: Implication for Functional Integrity of CYP1D1 in Cynomolgus Macaques and Rhesus Macaques

doi:10.2133/dmpk.DMPK-11-NT-026

Drug Metabolism and Pharmacokinetics

Volume 26, Issue 6, 2011, Pages 627-631

https://doi.org/10.2133/dmpk.DMPK-11-NT-026 Get rights and content

Summary:

The cytochrome P450 (CYP) 1 family consists of the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. In humans, CYP1A1, CYP1A2, and CYP1B1 are expressed and encode functional enzymes, whereas CYP1D1P (formerly known as CYP1A8P) is present as a pseudogene as a result of five nonsense mutations in exon 2 and exon 7 of the putative coding region. We previously identified CYP1D1 in macaques and found that it was expressed and functional in liver. Moreover, the nonsense mutations in exon 2 and exon 7 were not found in the 20 cynomolgus macaques and 10 rhesus macaques analyzed in that previous study. These results raised the possibility that CYP1D1 is a functional gene in macaques; however, the possibility that nonsense mutations are present in other exons cannot be excluded. In this study, we sought to identify genetic variants of CYP1D1 in 63 cynomolgus macaques and 30 rhesus macaques; we did not find nonsense mutations in any coding exon of the animals analyzed. Moreover, 15 of the 63 cynomolgus macaques were analyzed by quantitative polymerase chain reaction, confirming hepatic expression of CYP1D1 in all 15 animals. These results suggest that CYP1D1 is most likely functional in cynomolgus macaques and rhesus macaques.

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Cited by (14)

Genetic variants of N-acetyltransferases 1 and 2 (NAT1 and NAT2) in cynomolgus and rhesus macaques
2020, Biochemical Pharmacology
Citation Excerpt :
Only two variants were common to both cynomolgus and rhesus macaques. A similar uneven distribution of cytochrome P450 variants has been described between cynomolgus and rhesus macaques and between different groups of cynomolgus macaques [19–21]. To assess the functional significance of the identified variants, three NAT1 variant proteins (I114T, D122N, and T257I) and six NAT2 variant proteins (HIV58PLEREHEFYC, T98A, I149V, Q155E, T98A/Q155E, and K282E) were heterologously expressed in E. coli (Fig. 1).
In humans, polymorphic N-acetyltransferases NAT1 and NAT2 are important enzymes that metabolize endogenous and exogenous compounds, including drugs. These enzymes exhibit considerable inter-individual variability in humans. The cynomolgus macaque is a nonhuman primate species that is widely used in drug metabolism studies. NAT1/2 in these macaques have molecular and enzymatic similarities to their human orthologs; however, genetic polymorphisms in NAT1/2 have not been fully investigated in this species. In this study, the resequencing of NAT1 and NAT2 in 114 cynomolgus macaques and 19 rhesus macaques found 15 non-synonymous variants for NAT1 and 11 non-synonymous variants and 1 insertion/deletion variant for NAT2. Nine (60%) and five (33%) NAT1 variants and seven (67%) and three (25%) NAT2 variants were unique to cynomolgus and rhesus macaques, respectively. Functional characterization of the mutant enzymes was carried out using cynomolgus NAT1 and NAT2 proteins heterologously expressed in Escherichia coli. Compared with wild-type NAT1, the D122N NAT1 variant showed substantially lower acetylation activities toward p-aminobenzoic acid but had higher acetylation activities toward isoniazid. Moreover, liver cytosolic fractions from cynomolgus macaques homozygous for T98A NAT2 showed significantly lower acetylation activities toward isoniazid than wild-type NAT2; similar results were obtained for recombinant T98A NAT2. Interestingly, all the rhesus macaques analyzed were homozygous for T98A. These findings indicate that polymorphic NAT1/2 variants in cynomolgus and rhesus macaques, especially the T98A NAT2 variant, could account for the inter-animal and/or inter-lineage variabilities of NAT-dependent drug metabolism in macaques.
Genetic polymorphisms of drug-metabolizing cytochrome P450 enzymes in cynomolgus and rhesus monkeys and common marmosets in preclinical studies for humans
2018, Biochemical Pharmacology
Citation Excerpt :
These include 7 nonsense mutations (4 in P450 1A2, 1 in 2D8, 1 in 2G2, and 1 in 3A5), 5 frameshift mutations (1 in P450 1A2, 1 in 2C76, 2 in 2D8, and 1 in 3A4), and 1 gene conversion (P450 2D8) with the remaining as non-synonymous mutations. The frequencies of some P450 genetic variants are different between the origins of cynomolgus monkeys and/or between cynomolgus and rhesus monkeys [3–18]. Monkey P450 variants which have been studied in terms of in vitro and/or in vivo function, and were nonsense or frameshift mutations and gene conversion, have been identified and summarized in Table 2.
Cynomolgus monkeys (Macaca fascicularis, Old World Monkeys) and common marmosets (Callithrix jacchus, New World Monkeys) have been widely, and expectedly, used as non-human primate models in drug development studies. Major drug-metabolizing cytochrome P450 (P450) enzymes information is now available that supports these primate species as animal models, and it is established that multiple forms of cynomolgus monkey and common marmoset P450 enzymes have generally similar substrate recognition functionality to human P450 enzymes. This research update provides information on genetic polymorphisms of P450 enzymes in cynomolgus monkey and common marmoset like human P450 enzymes. Information on rhesus monkeys (Macaca mulatta), another macaque species used in drug metabolism studies, is also included for comparison. Among a variety of cynomolgus monkey P450 variants investigated, typical examples include individual pharmacokinetic data for efavirenz and R-warfarin associated with cynomolgus monkey P450 2C9 (formerly 2C43) and 2C19 (2C75) variants, respectively, and for R-omeprazole and S-warfarin associated with marmoset P450 2C19 variants. These findings provide a foundation for understanding the individual pharmacokinetic and toxicological results in non-human primates as preclinical models and will help to further support understanding of molecular mechanisms of human P450 function. In addition to these polymorphic P450 enzymes, effects of aging on some drug clearances mediated by cynomolgus monkey and common marmoset P450 enzymes were found in elder animals or animals pretreated with rifampicin. This review describes genetic and acquired individual differences in cynomolgus monkey and common marmoset P450 enzymes involved in drug oxidation associated with pharmacological and/or toxicological effects.
Genetic polymorphism of cynomolgus and rhesus macaque CYP2C9
2015, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
Seven variants were shared by both lineages. Similarly, uneven distribution of alleles has been described in P450 genes between cynomolgus and rhesus macaques or between Indochinese and Indonesian cynomolgus macaques [9–12]. Because four non-synonymous variants, c.317C > T (A106V), c.334A > C (I112L), c.887C > G (A296G), and c.1421A > G (N474S), were located in SRS, important for protein function, these variants are worth investigation on their influence on CYP2C9 metabolic activity.
Cynomolgus and rhesus macaques are non-human primate species widely used in drug metabolism studies. Cynomolgus CYP2C9 (formerly known as CYP2C43) is predominantly expressed in liver and encodes a drug-metabolizing enzyme that metabolizes human CYP2C substrates such as S-mephenytoin and progesterone. In addition, cynomolgus CYP2C9 also metabolizes caffeine, resulting in the formation of the metabolite that is not generated efficiently in humans. Genetic variants of human CYP2C genes account for the inter-individual variability in drug metabolism: however, CYP2C9 variants have not been found in macaques. To see if CYP2C9 is polymorphic in macaques, in this study, CYP2C9 was re-sequenced in 78 cynomolgus and 36 rhesus macaques. A total of 27 non-synonymous variants were found, among which 4 were located in substrate recognition sites, the domain important for protein function. Thirteen and seven variants were unique to cynomolgus and rhesus macaques, respectively. This study revealed the polymorphic nature of cynomolgus and rhesus CYP2C9, similar to human CYP2C genes, by identification of numerous genetic variants including non-synonymous variants.
CYP2C19 polymorphisms account for inter-individual variability of drug metabolism in cynomolgus macaques
2014, Biochemical Pharmacology
Citation Excerpt :
The previous study reported that pharmacokinetics of S-mephenytoin was highly variable among 64 cynomolgus macaques [5], and such inter-animal variations might be accounted for by genetic variants. Genetic polymorphisms have been found in macaque P450 genes [6–10]: however, the genetic variants have not been investigated in macaque CYP2C19. In the present study, the genomes of 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia) and 36 rhesus macaques were analyzed to identify CYP2C19 genetic variants.
CYP2C19 (formerly known as CYP2C75), highly homologous to human CYP2C19, has been identified in cynomolgus and rhesus macaques, non-human primate species widely used in drug metabolism studies. CYP2C19 is predominantly expressed in liver and encodes a functional drug-metabolizing enzyme. Genetic variants in human CYP2C genes account for the inter-individual variability in drug metabolism; however, genetic variants have not been investigated in macaque CYP2C19. In the present study, re-sequencing of CYP2C19 in 78 cynomolgus and 36 rhesus macaques identified 34 non-synonymous variants. Among these, 6 were located in substrate recognition sites, the domains important for protein function. Eighteen and 6 variants were unique to cynomolgus and rhesus macaques, respectively. Four variants were characterized by site-directed mutagenesis and metabolic assays, and 3 variants (p.Phe100Asn, p.Ala103Val, and p.Ile112Leu) showed substantially reduced activity as compared with wild type in flurbiprofen 4′-hydroxylation, omeprazole 5-hydroxylation, and R-/S-warfarin 7-hydroxylation. These variants, co-segregating in the animals analyzed, influenced metabolic activities because the homozygotes and/or heterozygotes showed significantly reduced catalytic activities in liver toward flurbiprofen 4′-hydroxylation and omeprazole 5-hydroxylation as compared with wild type. Kinetic analysis for R-warfarin 7-hydroxylation and docking simulation indicated that CYP2C19 Ala103Val would change the function and conformation of this enzyme. Ala103Val variation diminished homotropic cooperativity of CYP2C19 with R-warfarin yielding low metabolic capacity. These results indicated that the interindividual variability of CYP2C-dependent drug metabolism is at least partly accounted for by CYP2C19 variants in cynomolgus macaques.
Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: Implications for cytochrome P450 genes
2012, Drug Metabolism and Pharmacokinetics
Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (> 2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (> 2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (> 2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (< 1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.
Functionally relevant genetic variants of glutathione S-transferase GSTM5 in cynomolgus and rhesus macaques
2019, Xenobiotica

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NoteDiscovery of Genetic Variants in CYP1D1: Implication for Functional Integrity of CYP1D1 in Cynomolgus Macaques and Rhesus Macaques

Summary:

Lancet

Biochem. Pharmacol.

Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants

Pharmacogenetics

Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man

Xenobiotica

Comparisons of phase I and phase II in vitro hepatic enzyme activities of human, dog, rhesus monkey, and cynomolgus monkey

Drug Metab. Dispos.

Note
Discovery of Genetic Variants in CYP1D1: Implication for Functional Integrity of CYP1D1 in Cynomolgus Macaques and Rhesus Macaques