A Comprehensive Functional Assessment of Carboxylesterase 1 Nonsynonymous Polymorphisms

Abstract

Carboxylesterase 1 (CES1) is the predominant human hepatic hydrolase responsible for the metabolism of many clinically important medications. CES1 expression and activity vary markedly among individuals; and genetic variation is a major contributing factor to CES1 interindividual variability. In this study, we comprehensively examined the functions of nonsynonymous single nucleotide polymorphisms (nsSNPs) and haplotypes using transfected cell lines and individual human liver tissues. The 20 candidate variants include nsSNPs with a minor allele frequency >0.5% in a given population or located in close proximity to the CES1 active site. Five nsSNPs, including L40Ter (rs151291296), G142E (rs121912777), G147C (rs146456965), Y170D (rs148947808), and R171C (rs201065375), were loss-of-function variants for metabolizing the CES1 substrates clopidogrel, enalapril, and sacubitril. In addition, A158V (rs202121317), R199H (rs2307243), E220G (rs200707504), and T290M (rs202001817) decreased CES1 activity to a lesser extent in a substrate-dependent manner. Several nsSNPs, includingL40Ter (rs151291296), G147C (rs146456965), Y170D (rs148947808), and R171C (rs201065375), significantly reduced CES1 protein and/or mRNA expression levels in the transfected cells. Functions of the common nonsynonymous haplotypes D203E-A269S and S75N-D203E-A269S were evaluated using cells stably expressing the haplotypes and a large set of the human liver. Neither CES1 expression nor activity was affected by the two haplotypes. In summary, this study revealed several functional nsSNPs with impaired activity on the metabolism of CES1 substrate drugs. Clinical investigations are warranted to determine whether these nsSNPs can serve as biomarkers for the prediction of therapeutic outcomes of drugs metabolized by CES1.

Functional Study of Carboxylesterase 1 Protein Isoforms

“…The deleterious effect of the G143E variant on CES1 activity has been consistently demonstrated by several in vitro and clinical investigations for other CES1 substrate drugs, such as oseltamivir, trandolapril, clopidogrel, ACE inhibitors, dabigatran and sacubitril . Additionally, we recently conducted a comprehensive functional study of CES1 nonsynonymous variants and revealed a few CES1 SNPs which impaired CES1 activity in the hydrolysis of enalapril, clopidogrel, and sacubitril; these include L40Ter (rs151291296), A158V (rs202121317), R199H (rs2307243), E220G (rs200707504), and T290M (rs202001817) . Aside from nonsynonymous variants, two SNPs ‐816A>C (rs3785161) and ‐75T>G (rs3815583), located in the promoter regions of CES1P1 and CES1 , respectively, have been reported to be associated with responses to CES1 substrates, such as imidapril, clopidogrel, and methylphenidate, although not all studies have supported this association .…”

“…The desired sequences of the CES1 plasmids were confirmed by DNA sequencing analysis. The validated CES1 isoform plasmids were then transfected into Flp‐In‐293 cells following previously published protocol . Briefly, CES1 plasmids were co‐transfected with pOG44 plasmid at a ratio of 1:10 into Flp‐In‐293 cells with the Lipofectamine 2000 reagent.…”

“…The validated CES1 isoform plasmids were then transfected into Flp-In-293 cells following previously published protocol. [16] Briefly, CES1 plasmids were co-transfected with pOG44 plasmid at a ratio of 1:10 into Flp-In-293 cells with the Lipofectamine 2000 reagent. Six hours after transfection, cells were gently rinsed to remove the transfection reagents and cultured in complete medium (DMEM containing 10% FBS).…”

“…DOI: 10.1002/pmic.201800288 CES1 expression and activity are affected by both non-genetic (e.g., age and gender) [14] and genetic factors. [7,9,10,15,16] CES1 is encoded by the CES1 gene located on chromosome 16. In addition, the non-functional pseudogene CES1P1 coincides with CES1 in the reverse orientation and has a functional variant CES1P1VAR that can produce functional CES1 proteins.…”

“…Isoform 2 (NM 001025194.1) is considered the reference mRNA, and its protein product has been widely used as the wild-type CES1 in most CES1 pharmacogenetic studies. [4][5][6][7]9,10,16,17,24] Isoforms 1, 3, and 4 are generated from the AS events +GCA X2, -CAG X10, and both +GCA X2 and −CAG X10, respectively (Figure 1). It is plausible that these differences in splicing may contribute to the variability in CES1 expression and/or activity observed among individuals and consequently affect responses to CES1 substrate medications.…”