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Prout-Holm, Riley Austin

University of British Columbia

Target engagement assays detect and quantify the direct physical interaction of a protein of interest and its ligand, typically done by measuring subtle changes in a target protein’s stability induced upon ligand binding. To perturb protein stability, an external stressor is typically applied. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Current techniques often use slow-growing and expensive cell lines, exogenous visualizing agents, or exceptionally pure protein samples. Conventional target engagement techniques are useful but expensive and have low throughput. Here we describe three variations of target engagement that utilize robust prokaryotic expression systems, internal fluorescent probes, and alternative methods to measure target stability all with minimal sample manipulation. Prokaryotic Lysate–Thermal Shift Assay (ProLys-TSA) detects the melting behavior of target proteins using an internal fluorescent reporter across a temperature range in E. coli lysates. GFP-PRMT1 constructs treated with SAH cofactor and inhibitor EPZ020411 were used to validate ProLys-TSA. Isothermal Ligand-induced Resolubilization Assay (ILIRA) utilizes lyotropic solubility stress to measure target engagement through changes in target protein solubility. ILIRA was validated using 4 target proteins and inhibitor pairings: PRMT1 with SAH cofactor and MS023, DHFR with pyrimethamine, NUDT5 with TH5427, and PARP1 with olaparib. Ligand Inclusion Body Rescue-Target Engagement Assay (LIBR-TEA) utilizes the polar aprotic solvent DMSO to cause protein solubility stress in whole-cell E. coli samples. Fluorescent measurements of recombinant GFP-PRMT1 expressed in E. coli cells treated with MS023 were the basis of measurement. Stress methods used in the described assays can be classified as: lyotropic stress, solvent stress and thermal stress. Internal GFP reporters provide a direct quantification of soluble state protein. Preliminary results suggest that all three stressors chosen were able to destabilize target proteins, decreasing soluble protein levels regardless of measurement method. After addition of target-specific cofactor or small molecule inhibitor, statistically significant increases in soluble protein were observed in all three assay types. ILIRA represents our most developed assay concept. Initial data obtained suggest all three methods have potential to be further developed into robust target engagement assays, representing novel contributions to the field.

Text

2024-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83830

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/