ABSTRACT
Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Biomarkers , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Repressor Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacokinetics , Antineoplastic Agents, Immunological/pharmacology , Arginine/metabolism , Cells, Cultured , Chromatography, Liquid , Drug Monitoring , Enzyme Activation , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Gene Expression Regulation, Neoplastic/drug effects , Heterogeneous Nuclear Ribonucleoprotein A1/blood , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Mass Spectrometry , Methylation , Mice , Molecular Targeted Therapy , Neoplasms/blood , Neoplasms/drug therapy , Neoplasms/metabolism , Protein-Arginine N-Methyltransferases/genetics , Repressor Proteins/genetics , Substrate SpecificityABSTRACT
A new DNA aptamer and antibody pair was incorporated into surface plasmon resonance (SPR) sensing platform to detect heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in plasma at clinically relevant native concentrations for the diagnosis of colorectal cancer (CRC). SPR detection of hnRNP A1 was realized via formation of the surface sandwich complex of aptamer/hnRNP A1/anti-hnRNP A; the specific adsorption of hnRNP A1 onto a gold chip surface modified with a DNA aptamer followed by the adsorption of anti-hnRNP A1. Changes in the refractive unit (RU) with respect to the hnRNP A1 concentration in buffer solutions were monitored at a fixed anti-hnRNP A1 concentration of 90 nM, resulting in a dynamic range of 0.1-10 nM of hnRNP A1. The surface sandwich SPR biosensor was further applied to the direct analysis of undiluted human normal and pooled CRC patient plasma solutions. Our plasma analysis results were compared to those obtained with a commercial enzyme-linked immunosorbent assay kit.
