Genome-Wide Association Study for the Genetic Determinants of Thiopurine Methyltransferase Protein Expression in Human Livers and Racial Differences.

INTRODUCTION: Polymorphisms in the Thiopurine S-Methyltransferase (TPMT) gene are associated with decreased TPMT activity, but little is known about their impact on TPMT protein expression in the liver. This project is to conduct a genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with altered TPMT protein expression in human livers and to determine if demographics affect hepatic TPMT protein expression. METHODS: Human liver samples (n = 287) were genotyped using a whole genome genotyping panel and quantified for TPMT protein expression using a Data-Independent Acquisition proteomics approach. RESULTS AND DISCUSSION: Thirty-one SNPs were found to be associated with differential expression of TPMT protein in the human livers. Subsequent analysis, conditioning on rs1142345, a SNP associated with the TPMT*3A and TPMT*3C alleles, showed no additional independent signals. Mean TPMT expression is significantly higher in wildtype donors compared to those carrying the known TPMT alleles, including TPMT*3A, TPMT*3C, and TPMT*24 (0.107 ± 0.028 vs. 0.052 ± 0.014 pmol/mg total protein, P = 2.2 × 10-16). After removing samples carrying the known TPMT variants, European ancestry donors exhibited significantly higher expression than African ancestry donors (0.109 ± 0.026 vs. 0.090 ± 0.041 pmol/mg total protein, P = 0.020). CONCLUSION: The GWAS identified 31 SNPs associated with TPMT protein expression in human livers. Hepatic TPMT protein expression was significantly lower in subjects carrying the TPMT*3A, TPMT*3C, and TPMT*24 alleles compared to non-carriers. European ancestry was associated with significantly higher hepatic TPMT protein expression than African ancestry, independent of known TPMT variants.

Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms.

It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants.

Tissue- and cell-expression of druggable host proteins provide insights into repurposing drugs for COVID-19.

Several human host proteins play important roles in the lifecycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many drugs targeting these host proteins have been investigated as potential therapeutics for coronavirus disease 2019 (COVID-19). The tissue-specific expressions of selected host proteins were summarized using proteomics data retrieved from the Human Protein Atlas, ProteomicsDB, Human Proteome Map databases, and a clinical COVID-19 study. Protein expression features in different cell lines were summarized based on recent proteomics studies. The half-maximal effective concentration or half-maximal inhibitory concentration values were collected from in vitro studies. The pharmacokinetic data were mainly from studies in healthy subjects or non-COVID-19 patients. Considerable tissue-specific expression patterns were observed for several host proteins. ACE2 expression in the lungs was significantly lower than in many other tissues (e.g., the kidneys and intestines); TMPRSS2 expression in the lungs was significantly lower than in other tissues (e.g., the prostate and intestines). The expression levels of endocytosis-associated proteins CTSL, CLTC, NPC1, and PIKfyve in the lungs were comparable to or higher than most other tissues. TMPRSS2 expression was markedly different between cell lines, which could be associated with the cell-dependent antiviral activities of several drugs. Drug delivery receptor ICAM1 and CTSB were expressed at a higher level in the lungs than in other tissues. In conclusion, the cell- and tissue-specific proteomics data could help interpret the in vitro antiviral activities of host-directed drugs in various cells and aid the transition of the in vitro findings to clinical research to develop safe and effective therapeutics for COVID-19.

Data-independent acquisition (DIA): An emerging proteomics technology for analysis of drug-metabolizing enzymes and transporters.

Data-independent acquisition (DIA) proteomics is a recently-developed global mass spectrometry (MS)-based proteomics strategy. In a DIA method, precursor ions are isolated into pre-defined isolation windows and fragmented; all fragmented ions in each window are then analyzed by a high-resolution mass spectrometer. DIA proteomics analysis is characterized by a broad protein coverage, high reproducibility, and accuracy, and its combination with advances in other techniques such as sample preparation and computational data analysis could lead to further improvements in assay performances. DIA technology has been increasingly utilized in various proteomics studies, including quantifying drug-metabolizing enzymes and transporters. Quantitative proteomics study of drug-metabolizing enzymes and transporters could lead to a better understanding of pharmacokinetics and pharmacodynamics and facilitate drug development. This review summarizes the application of DIA technology in proteomic analysis of drug-metabolizing enzymes and transporters.

Effect of CES1 genetic variation on enalapril steady-state pharmacokinetics and pharmacodynamics in healthy subjects.

AIMS: Enalapril is a prodrug and needs to be activated by carboxylesterase 1 (CES1). A previous in vitro study demonstrated the CES1 genetic variant, G143E (rs71647871), significantly impaired enalapril activation. Two previous clinical studies examined the impact of G143E on single-dose enalapril PK (10 mg); however, the results were inconclusive. A prospective, multi-dose, pharmacokinetics and pharmacodynamics (PK/PD) study was conducted to determine the impact of the CES1 G143E variant on enalapril steady-state PK and PD in healthy volunteers. METHODS: Study participants were stratified to G143E non-carriers (n = 15) and G143E carriers (n = 6). All the carriers were G143E heterozygotes. Study subjects received enalapril 10 mg daily for seven consecutive days prior to a 72 hour PK/PD study. Plasma concentrations of enalapril and its active metabolite enalaprilat were quantified by an established liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: The CES1 G143E carriers had 30.9% lower enalaprilat Cmax (P = 0.03) compared to the non-carriers (38.01 vs. 55.01 ng/mL). The carrier group had 27.5% lower AUC0-∞ (P = 0.02) of plasma enalaprilat compared to the non-carriers (374.29 vs. 515.91 ng*h/mL). The carriers also had a 32.3% lower enalaprilat-to-enalapril AUC0-∞ ratio (P = 0.003) relative to the non-carriers. The average maximum reduction of systolic blood pressure in the non-carrier group was approximately 12.4% at the end of the study compared to the baseline (P = 0.001). No statistically significant blood pressure reduction was observed in the G143E carriers. CONCLUSIONS: The CES1 loss-of-function G143E variant significantly impaired enalapril activation and its systolic blood pressure-lowering effect in healthy volunteers.