A Novel Assay Platform for Targeted Protein Degradation Monitoring: Case Study of Antibody Free Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) for MALT1 Target Quantification.

The mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a central regulator of inflammatory signaling downstream of immunoreceptor tyrosine-based activation motif-containing receptors and G protein-coupled receptors (GPCRs). MALT1 has been investigated as a drug target in inflammation and cancer by inhibitors and targeted degradation. Accurate quantification of MALT1 protein is a critical unmet need for drug discovery. However, traditional ligand binding assays (LBAs), such as Enzyme-Linked Immunosorbent Assay (ELISA) or Meso Scale Discovery Assay (MSD), have been limited by the lack of suitable reagents. To address this limitation, we developed LC-MS/MS targeted MALT1 degradation monitoring assays which have enabled effective monitoring of target degradation in vivo and in vitro. A customized, reagent-independent one dimensional (1D)-LC-MS/MS assay was initially used to facilitate early-phase drug discovery screening. Furthermore, the continued progress of the discovery chemistry resulted in molecules with a higher percentage of MALT1 degradation, resulting in lower remaining MALT1 concentrations within sample matrices and the need for a highly sensitive quantitation. Consequently, a 2D-LC-MS/MS assay with a novel instrument configuration was developed. This assay has proven crucial for early drug optimization and dose selection in both in vitro and in vivo studies. The examples of assay application include the measurement of the DC50 of a lead compound across multiple species, with DC50 value of 5.2 nM in monkeys, 57.0 nM in rats, and 86.9 nM in dog splenocytes. This assay was also used to help select one of the best lead compounds with degradation showing 77.9% with in vivo treatment.