Abstract 4540: Development of a biochemical High Throughput Screening (HTS) assay for chemical inhibitors of MALT1, a target for lymphoma therapeutics.

Abstract

Abstract MALT1 plays critical role in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 acts both as a scaffold protein to recruit upstream signal transducers and as an intracellular cysteine protease with proteolytic activity that cleaves substrate proteins involved in NF-kB induction. Dysregulation of MALT1 activity occurs in lymphoma as a result of chromosomal translocations that fuse the catalytic domain of MALT1 with portions of BirC3 (c-IAP2). Previous studies have shown that MALT1’s proteolytic activity is required for its participation in NF-kB induction and for promoting cell viability of certain types of lymphomas, particularly the activated B-cell (ABC) type of diffuse large B-cell lymphoma. To lay a foundation for discovery of drug-like chemical inhibitors of MALT1, we developed a fluorescence-based, biochemical assay for measuring protease activity of MALT1 in vitro in 384 and 1,536 well formats. The assay components included recombinant purified full-length MALT1 protein, the MALT1-binding protein, Bcl-10, and the fluorigenic substrate Ac-LRSR-AMC (Ac-Leu-Arg-Ser-Arg-7-amino-4-methylcoumarin). Experiments showed that addition of Bcl-10 increases MALT1‘s protease activity up to 20-40 fold. The high throughput screening (HTS) assay was validated with excellent Z’ factors (>0.7) in both 384 and 1,536 well formats, with performance remaining robust for more than 6 hours, and thus suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with two small collections of compounds enriched in bioactive molecules (n = 1280 for LopacTM and 2000 for SpectrumTM library), yielding confirmed hit rates of 0.2 % and 0.7%, respectively. Counter-screening assays were also generated using other types of intracellular cysteine proteases, specifically Caspase-3 and Autophagin-1 (ATG4B), for eliminating non-specific inhibitors. Various supporting cell-based assays have also been generated to support hit characterization. In conclusion, we have generated a robust biochemical HTS assays for detection of chemical inhibitors of the MALT1 protease. We plan to utilize the HTS assay for screening large compound libraries as a starting point for discovery of novel therapeutics for treatment of lymphoma, autoimmunity, and allograft rejection. Citation Format: Dayong Zhai, Chih-Wen Shu, Paul Diaz, John C. Reed. Development of a biochemical High Throughput Screening (HTS) assay for chemical inhibitors of MALT1, a target for lymphoma therapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2013-4540