(M1330-01-04) High-Throughput and Selective LC-MS Quantitation of SMARCA2/4 for SMARCA2 Targeted Protein Degrader Cross-Species Pharmacodynamics Analysis

Purpose: Targeted protein degrader (TPD) is revolutionizing the way to tackle undruggable targets by degrading target proteins. Quantifying target protein is critical to understand the pharmacodynamics of TPD compounds. Traditional assays such as western blot and ELISA are facing challenges in terms of throughput, cross-species capability, and specificity. The purpose of this work is to develop a targeted LC-MS assay for SMARCA2/4 quantitation to support cross-species pharmacodynamics analysis of a SMARCA2 TPD compound.

Methods: Tissues were lysed in IP lysis buffer with protease inhibitor and nuclease. Lysates were aliquoted into 96-well plate and anti-SMARCA2/4 antibody-conjugated beads were added and vortexed overnight at 4 °C. Beads were washed using the KingFisher Flex Processor with IP, TBST and HBSN buffer. Captured proteins were denatured in 8 M urea with TCEP followed by alkylation with iodoacetamide. Samples were then 6-fold diluted and digested with trypsin. Digestion was quenched and samples were pre-fractionated at high pH condition using Oasis 96-well µElution plate. Desired fractions were collected, dried and resuspended. An orbitrap Fusion Lumos mass spectrometer coupled with an Ultimate 3000 nano LC was used for PRM acquisition and the data analysis was performed with Skyline.

Results: SMARCA2 and SMARCA4 are interchangeable core subunits of SWI/SNF chromatin remodeling complex which plays important roles in controlling gene expression. SMARCA4 mutant cancer cells are highly dependent on its paralog SMARCA2 for survival and selectively degrading SMARCA2 can therefore block tumor growth while sparing normal tissues. Targeted protein degraders (TPD) can target disease-causing proteins for destruction. They function by recruiting an E3 ligase to the proximity of target proteins, leading to their subsequent ubiquitination and proteasomal degradation. A TPD compound was designed to target SMARCA2 and quantifying SMARCA2/4 for pharmacodynamic assessment is therefore a critical component in the compound development process. However, traditional assays suffer from challenges including low throughput, lack of cross-species capability, and poor specificity partly due to the high sequence homology between SMARCA2/4. Benefiting from targeted LC-MS analysis of unique signature peptides, our assay enables highly specific detection of SMARCA2/4 simultaneously across multiple species (mouse, rat, monkey, dog, human) and various tissue matrices (liver, lung, spleen, kidney, stomach, PBMCs). A combined use of several capture antibodies and offline high pH fractionation greatly improves the detection sensitivity. The entire workflow is further optimized towards a high-throughput manner, for instance, by taking advantage of KingFisher Flex 96 Deep-Well Magnetic Particle Processor and Oasis 96-well µElution plate. The assay is employed for SMARCA2/4 quantitation after administration of the TPD compound in a few PK/PD and Tox studies. Tissue-dependent degradation profiles are observed with great selectivity at lower doses and increased SMARCA4 degradation at higher doses. The results provide reference and guidance for compound optimization and pre-clinical animal species selection.

Conclusion: We develop a multiplexed LC-MS assay by coupling immunocapture, offline high pH fractionation and targeted acquisition for a SMARCA2 TPD compound cross-species pharmacodynamics analysis. This assay features boosted sensitivity, 100% specificity towards SMARCA2/4, cross-species capability and high-throughput in a 96-well plate format. The utility of the assay was demonstrated by supporting several PK/PD and tox studies in different species and tissue matrices. This assay can serve as a generic and powerful tool for TPD compound pharmacodynamics analysis.