High-throughput kinetic system for small-molecule drug discovery - Abstract Evidence is accumulating for higher efficacy of small molecule drugs that have off-rates slower than the pharmacokinetic clearance rate. Despite the importance of kinetics, the standard approach is low throughput Biacore testing in de novo drug discovery/screening. But we offer a new way that outruns commercial plate readers. Capienda Biotech is developing the first instrument offering rapid kinetics for competitive inhibitors at high throughput for drug discovery and optimization. Advancing upon our NSF SBIR Phase I success in building a prototype instrument for highly parallel assays and sensitive detection for binding kinase targets (128 tests simultaneously), we propose to upgrade the instrument (up to 384 tests simultaneously) with hardware upgrade for higher-throughput kinetics. The innovative instrument design and assay chemistry enables us to characterize compounds that target human Cyclin-Dependent Kinases for oncology. The approach is expected to apply broadly across a growing portfolio of >500 kinase targets. Kinase pathways have complex roles in cells. Therapeutic kinase inhibitors are approved to treat cancer or to modulate the immune system. As of November 2023, the FDA has approved 80 kinase inhibitors: 64 (80%) for cancer [31]. Twenty cyclin-dependent kinases comprise an important family of targets for oncology, and CDK subfamilies are committed to either progression through the cell cycle or transcriptional regulation [100]. Unfortunately, despite their potential as oncology targets, CDKs are under-exploited. Only four CDK-specific drugs have gained FDA approval (abemaciclib, palbociclib, ribociclib, trilaciclib) and all against the same target subfamily: CDK4/6 [31]. This proposal will expand the members of CDK subfamilies that can be targeted using high throughput kinetics. The approach will lead Medicinal Chemists to find drugs with greater efficacy and fewer side-effects due to poor target selectivity. Selectivity of kinase inhibitors is a major issue in addressing the kinase class of targets. Drugs for auto- immune and anti-inflammatory indications have been approved by the FDA. Sixteen small molecule inhibitors target kinases regulating the immune system. Ruxolitinib, baricitinib, momelotinib, tofacitinib and fostamatinib specifically bind the intended kinases in immune signaling (JAKs or SYK) and have little unintended binding to typical kinases targeted by oncology drugs [42], all without hematologic abnormalities (neutropenia, thrombocytopenia or anemia) [51-53,55-56]. By contrast, cancer drugs ponatinib, dasatinib and danusertib bind their intended targets (BRC-ABL and aurora kinases) and also multiple off-target kinases that regulate the immune system including BTK, LYN, LCK, FYN and MAPK p38α [42], but in the clinic patients experience adverse events often including neutropenia, thrombocytopenia, anemia and hypertension [44,46,48]. In the longer run, kinome-wide examination of compounds early on and throughout the drug discovery process may minimize polypharmacology and the unintended adverse effects in the clinic. These issues would be ameliorated using a solution for high-throughput selectivity including kinetic characterization. We will measure several key kinetic parameters: kon, koff, drug residence time, dissociation half-life and determine Kd. The information we gain will distinguish between promising vs. unpromising compounds. Measurements like these are needed to find drugs like Gleevec (imatinib), which bind kinase followed by conformational change of the kinase to lock the inhibitor into a stable inhibitor-kinase complex for long duration of action and improved pharmacokinetics. Overall, this project addresses an unmet analytical need in drug discovery, which will open a bottleneck in the path to new, highly specific medicines. The project will enable kinetic understanding across a large number of compounds that has been difficult or impractical to attain. We will establis
