Evaluation of Small Interfering RNA Mediated Knockdown of Drug Metabolizing Enzymes as a Tool for Reaction Phenotyping
Martell, Cydney M.
MetadataShow full item record
Identification of the individual drug metabolizing enzymes responsible for the clearance of drug molecules is important in assessing the risk of drug-drug interactions. However, this is limited by paucity of tools available for reaction phenotyping less studied noncytochrome P450 enzymes. One technique that could be used for reaction phenotyping is siRNA mediated gene knockdown. This study aimed to explore the potency and specificity of siRNA knockdown of select drug metabolizing enzymes as a preliminary investigation into the use of siRNA as a tool for reaction phenotyping. Primary hepatocytes were treated with siRNA targeting five representative drug metabolizing enzymes: CYP3A4, CYP2E1, AOX1, UGT1A1, and UGT2B7 and then the potency and specificity of gene knockdown was determined with enzyme activity assays and qPCR. Targeted knockdown of all the drug metabolizing enzymes was observed by a reduction in both the targeted enzyme’s activity and gene expression. In addition to the targeted knockdown, off-target effects were observed for all siRNA treatment at the activity level and some at the mRNA level with CYP2E1 and CYP3A4 being the most susceptible to off-target effects. With the lack of specificity of siRNA mediated gene knockdown this technique appears to be a poor tool for reaction phenotyping. Overall these results suggest that more optimization, understanding, and elimination of the off-target effects would be needed for siRNA mediated gene knockdown to be a helpful technique in reaction phenotyping.