PT  - JOURNAL ARTICLE
AU  - Elisabeth M. Rothweiler
AU  - Jakub Stefaniak
AU  - Jennifer A. Ward
AU  - Catherine Rogers
AU  - Esra Balikci
AU  - Kilian V. M. Huber
TI  - A chemical biology toolbox to investigate in-cell target engagement and specificity of PRMT5-inhibitors
AID  - 10.1101/2022.01.20.477145
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.20.477145
4099  - http://biorxiv.org/content/early/2022/01/27/2022.01.20.477145.short
4100  - http://biorxiv.org/content/early/2022/01/27/2022.01.20.477145.full
AB  - Increasing evidence suggests the protein arginine methyltransferase PRMT5 as a contributor to tumorigenesis in various cancer types and several inhibitors have entered clinical trials. Robust assays to determine cellular target engagement and selectivity are an important asset for the optimisation of inhibitors and the design of relevant in vivo studies. Here we report a suite of chemical biology assays enabling quantitative assessment of PRMT5 inhibitor in-cell target engagement and global selectivity profiling using a representative set of inhibitors. With the help of a bespoke cellular probe, we assess inhibitor target occupancy in cells in relation to biochemical and functional cellular assays. Investigating the influence of SAM, the natural cofactor of PRMT5, our results support the hypothesis that SAM positively contributes to the engagement of substrate-competitive inhibitors via a PRMT5:SAM:inhibitor ternary complex. Extensive proteomic profiling studies by drug affinity chromatography and thermal profiling further indicate high specificity of the clinical PRMT5 inhibitor GSK3326595 (pemrametostat).Competing Interest StatementThe authors have declared no competing interest.