Project summary/abstract. The emerging field of allosteric inhibition of protein kinases has the potential to provide highly targeted therapeutics for diverse diseases without undesirable side effects. Our long-term goal is the discovery and development of highly selective allosteric inhibitors for side effect-free cancer chemotherapy and for reversible non-hormonal contraception. The overall objective of this application is to identify novel allosteric inhibitors for four kinases: WEE1-like protein kinase 2 (WEE2), testis-specific serine/threonine kinase 1 (TSSK1), cyclin-depended kinase 11 (CDK11), and cyclin dependent kinase 2 (CDK2) by screening DNA encoded libraries (DEL) consisting of 2B compounds. The four kinases are validated drug targets: WEE2 for female non-hormonal contraception, TSSK1 for male non-hormonal contraception, CDK11 for cancer chemotherapy and CDK2 for cancer and male and female non-hormonal contraception. WEE2, TSSK1 and CDK11 are on the NIH list of underexplored targets. CDK2 inhibitors have been studied for cancer application but limited studies have been carried out for male contraception and none for female contraception. Hit compounds from the DEL screen will be resynthesized by conventional synthesis and then confirmed by using functional biochemical assays, orthogonal confirmatory assays, and biochemical and biophysical assays to determine allosteric binding. Validated compounds will be tested in a 24 kinome screen for kinase selectivity and in a limited set of in vitro ADMET assays to assess their drug-like properties. CDK11 and CDK2 inhibitors will be tested in cancer cell lines that express their respective targets. TSSK1 inhibitors will be tested using a computer-assisted sperm analyzer (CASA) for sperm motility measurements. Selective allosteric inhibitors identified in this project are expected to be useful probes to investigate the biology of the four kinases and to serve as early leads for drug discovery.