Discovery of BDK inhibitors with stabilizing or destabilizing pharmacology (Allan Reyes) AlphaLISA SureFire technology for the elucidation of cellular signaling cascades and mechanism of action in drug discovery programs (Antony Sheehan)

Recent studies have shown that elevated branched chain amino acid (BCAA) levels are strongly associated with metabolic disorders, such as obesity, metabolic syndrome, and type 2 diabetes mellitus. Emerging data also suggests that this is likely due to a catabolic defect and promoting BCAA catabolism could improve cardiometabolic disease. Branched chain ketoacid (BCKA) dehydrogenase kinase (BDK) is a mitochondrial protein kinase that regulates the activity of branched-chain α-ketoacid dehydrogenase complex (BCKD) through phosphorylation and inactivation. Inactivation of the BCKD complex leads to decreased flux of BCAA through the complex and increased circulating BCAA levels. Inhibition of BDK using a tool small molecule lowers glucose and insulin levels, ameliorates hepatic steatosis in a rat model and improves heart function in a mouse heart failure model. Here we share our efforts to understand the divergent pharmacology observed in our lead chemical matter and the design and implementation of an AlphaLISA Surefire based robust and predictive in-vitro screening cascade for the identification of efficacious small molecule BDK inhibitors.

Protein phosphorylation and total target characterization within cellular pathways are commonly analyzed for measurement of drug effectiveness and mechanism of action studies. The AlphaLISA SureFire Ultra panel of protein phosphorylation kits provide such rapid, homogeneous (no-wash), and highly sensitive assays allowing signaling pathways and drug effectiveness to be analysed from concept generation through high throughput screening campaigns. Utilization of these kits has become widely adopted across various therapeutic areas and modalities, from small molecule screens to biologics, for projects ranging from neuroinflammation, oncology, immunology, PROTACs for targeted protein degradation, and metabolic diseases.

To maximize data generation per sample, we have also recently expanded the SureFire Ultra assay portfolio to include the analysis of two targets per sample in a fully mix-and-read multiplexing format, termed Alpha SureFire Ultra Multiplex. This technology can measure either two different phosphoproteins, the phospho and total levels of the same protein or two different total proteins. We are continually expanding the development of new phospho/total assay kits to allow a greater selection of pathways for analysis, where normalization of the phospho signal with the total protein is achieved.

Data will be presented on numerous recently developed assays and therapeutic area applications, highlighting the utility of Alpha SureFire technology as a platform of choice for both high and low throughput screening projects in drug discovery.