Stefaniak, J. | Search bioRxiv

bioRxiv2022

A chemical biology toolbox to investigate in-cell target engagement and specificity of PRMT5-inhibitors

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). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/477145v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@11e448org.highwire.dtl.DTLVardef@a27194org.highwire.dtl.DTLVardef@cec270org.highwire.dtl.DTLVardef@3f76aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Comparing short and long batteries to assess deficits and their neural bases in stroke aphasia

Multiple language assessments are necessary for diagnosing, characterising and quantifying the multifaceted deficits observed in many patients post-stroke. Current language batteries, however, tend to be an imperfect trade-off between time and sensitivity of assessment. There have hitherto been two main types of battery. Extensive batteries provide thorough information but are impractically long for application in clinical settings or large-scale research studies. Clinically-targeted batteries tend to provide superficial information about a large number of language skills in a relatively short period of time by reducing the depth of each test but, consequently, can struggle to identify mild deficits, qualify the level of each impairment or reveal the underlying component structure. In the current study, we compared these batteries across a large group of individuals with chronic stroke aphasia to determine their utility. In addition, we developed a data-driven reduced version of an extensive battery that maintained sensitivity to mild impairment, ability to grade deficits and the component structure. The underlying structure of these three language batteries (extensive, shallow and data-reduced) was analysed using cross-validation analysis and principal component analysis. This revealed a four-factor solution for the extensive and data-reduced batteries, identifying phonology, semantic skills, fluency and executive function in contrast to a two-factor solution using the shallow battery (phonological/language severity and cognitive severity). Lesion symptom mapping using participants factor scores identified convergent neural structures based on existing language models for phonology (superior temporal gyrus), semantics (inferior temporal gyrus), speech fluency (precentral gyrus) and executive function (lateral occipitotemporal cortex) based on the extensive and data-reduced batteries. The two components in the shallow battery converged with the phonology and executive function clusters. In addition, we show that multivariate prediction models could be utilised to predict the component scores using neural data, however not for every component score within every test battery. Overall, the data-reduced battery appears to be an effective way to save assessment time yet retain the underlying structure of language and cognitive deficits observed in post stroke aphasia.

neuroscience↗