A Comprehensive Functional Assessment of CES1 Nonsynonymous Variants

Carboxylesterase 1 (CES1), encoded by CES1 gene, is the primary hepatic hydrolase responsible for metabolizing (activating or deactivating) many clinically important medications. Significant interindividual variability in therapeutic outcomes of drugs metabolized by CES1 has been consistently documented. Genetic variants of CES1 are increasingly recognized as a major factor affecting the responses to CES1 substrate medications. CES1 gene is highly polymorphic with over 2000 single nucleotide polymorphisms (SNPs). However, the functions of most of the SNPs remain undetermined. In the current study, we examined 17 CES1 nonsynonymous SNPs for their effects on CES1 activity and expression using several complementary in vitro methods. The selection of the 17 candidate SNPs was based on the criteria that the SNP has a minor allele frequency (MAF) greater than 0.5% in a population or is located within 5Å distance from the active site of CES1. The mutant CES1 plasmids were generated by site‐directed mutagenesis and transfected to human embryonic kidney cells (Flp‐InTM‐293). The CES1 mRNA and protein expression levels in the transfected cells were measured by real‐time PCR and LC‐MS/MS based targeted quantitative proteomics, respectively. The catalytic activity of these SNPs was determined using four CES1 substrates, i.e. enalapril, ramipril, trandolapril, and clopidogrel. Among the 17 candidate SNPs, a total of six variants (L40Ter, G142E, G147C, Y170D, R171C and E220G) were found to be loss‐of‐function variants of the CES1 gene. The L40Ter failed to produce a functional CES1 protein due to a premature stop codon in the exon 2 of the mutant gene. Except for the L40Ter, all the remaining loss‐of‐function variants reside near the CES1 active site. The G147C displayed dramatic decrease in CES1 mRNA and protein expressions, indicating that this mutation could result in instable mRNA products and subsequently decrease the protein expression. Similar to the previously reported loss‐of‐function CES1 variant G143E, the G142E and E220G variants both led to null CES1 activity without altering mRNA and protein expressions. Interestingly, CES1 protein expressions of the Y170D and R171C variants were substentially lower than that of wild type CES1 gene whereas mRNA levels were comparable between the variants and the wild type gene. We suspect that the Y170D and R171C proteins are more susceptible to protein degradation. In addition to the loss‐of‐function variants, nine SNPs, including D203E, R186P, T167S, T290M, A158V, R199H, N340K, Q169P and G173D, showed significantly impaired CES1 activities in vitro, ranging from 18% to 65% of the wild type enzyme, although their mRNA and protein expressions were comparable to the wild type CES1. In conclusion, this study represents the first comprehenisve functional assessment of CES1 nonsynonymous SNPs. With further clinical validation studies, the SNPs with deficient CES1 function can be used as genetic biomarkers to individulize pharmacotherapy for patients treated with CES1 substrate drugs. Support or Funding Information the National Heart, Lung, and Blood Institute [R01HL126969] CES1 mRNA and protein expression levels in seventeen CES1 nonsynonymous variantsThe null activity of G147C was in accordance with its dramatically decreased CES1 mRNA and protein expression levels. CES1 SNPs L40Ter, Y170D and R171C led to significant decrease in protein expression without changing mRNA expression levels.Activities of seventeen CES1 nonsynonymous variants on hydrolyzing four CES1 substratesi.e. enalapril, ramipril, trandolapril, and clopidogrel. Six variants (L40Ter, G142E, G147C, Y170D, R171C and E220G) were found to be loss‐of‐function variants. In addition, nine SNPs, including D203E, R186P, T167S, T290M, A158V, R199H, N340K, Q169P and G173D, showed significantly impaired CES1 activities in vitro.