This metabolic liability was prevented by replacing the piperazine ring with piperidine, an approach that contributed to the discovery of ceritinib, an ALK inhibitor approved by the FDA to treat small cell lung cancer. The discovery of the potent TSSK2 inhibitor 18 prompted the synthesis of compound 19 with the single point substitution of nitrogen at position Y by methine. structure will facilitate structure-based discovery of Rabbit Polyclonal to Cyclin D2 selective TSSK inhibitors from these pyrrolopyrimidine and pyrimidine scaffolds. reported that TSSK2 is usually localized to the equatorial segment of human sperm, whereas Shang found that TSSK2, TSSK1, and their common endogenous substrate, TSKS, accumulate in a ring-shaped structure around the base of the flagellum and in a cytoplasmic satellite derived from the chromatoid body.[3d] In contrast, Li reported that TSSK2 is usually expressed mainly in the sperm head in the postacrosomal regions and the acrosome tip, regions involved in sperm-oocyte fusion, where Izumo migrates following the acrosome reaction. TSSK2 is usually apparently expressed throughout the sperm depending on the stage of spermiogenesis, as it has been localized to the centrioles of post-meiotic spermatids, the tail and acrosomal regions of mouse epididymal sperm, and the midpiece of human spermatozoa. TSSK2 phosphorylates itself as well as TSKS and SPAG16L. TSSK2 co-immunoprecipitates with TSKS in human sperm[3a] and SPAG16L in mouse testis extracts. Human TSSK2 phosphorylates the N-terminal region of TSKS and displays robust autophosphorylation.[3c] Targeted deletion of TSSK1 and TSSK2 resulted in chimeric mice which fail to form elongated spermatids, possess apoptotic spermatocytes and spermatids, and accumulate numerous round cells in the epididymal lumen, which are likely immature spermatocytes.[3a] TSSK4 knockout mice have reduced fertility, whereas deletion of TSSK6 and the double TSSK1/TSSK2 knockout[3a] resulted in sterility suggesting that these members of the TSSK family Pantoprazole (Protonix) can be targeted with selective kinase inhibitors for male contraception. A triallelic polymorphism of the gene is usually associated with spermatogenic impairment in humans and two single-nucleotide polymorphisms of the TSSK2 gene are associated with spermatogenesis impairment and may be associated with male idiopathic infertility in humans.[3b] We recently expressed soluble, full-length human TSSK2 in baculovirus and purified the enzyme by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography.[3c] A mobility shift assay developed using purified, full-length human TSSK2 was used to screen focused compound libraries. Two series of potent inhibitors were recognized and their structure-activity associations are explained herein. Results and Conversation High-Throughput Screening A previously explained mobility shift assay detecting full-length, human TSSK2 phosphorylation of a synthetic substrate[3c] was used to screen compound libraries for inhibitors. The broad-spectrum kinase inhibitor staurosporine, previously shown to be a potent TSSK2 inhibitor (IC50 20 nM),[3c] was used Pantoprazole (Protonix) as a positive control on every screening plate. Vertical validation experiments using alternating columns of full reaction, no enzyme, and an IC50 concentration of staurosporine as positive control yielded Z values 0.85, substantially higher than the generally accepted minimum value of 0.5 for high-throughput screening (HTS). A pilot screen of the Library of Pharmacologically Active Compounds (LOPAC) produced a similar pattern of apparent inhibitors on a repeat test, indicating that the assay was reproducible. About 17,000 compounds were screened, including drug and pharmacologically active selections (Johns Hopkins FDA collection, NIH Clinical Selections 1 and 2, Prestwick Chemical Pantoprazole (Protonix) Library, Tocris Screening Collection, and Microsource Spectrum) and kinase inhibitor libraries (SelleckChem kinase inhibitor library, GSK PKIS 1 and 2, Tocris kinase inhibitor toolbox, and a ChemDiv kinase scaffold collection). Of 48 compounds that produced 20% inhibition (0.3% hit rate), 14 were confirmed as inhibitors in concentration-response experiments conducted with cherry-picked compounds from DMSO stocks. Confirmed hits included several pyrrolopyrimidines from your GSK PKIS and SelleckChem selections and the pyrimidine NVP-TAE684 (hereafter referred to as TAE684) from SelleckChem. Other active compounds included CHIR-124, PF-03814735, and hesperidin (SelleckChem), staurosporine (SelleckChem, Tocris), and the oxadiazol-imidazopyridines SK1392956A and GSK1007102B (GSK PKIS). The commercially available confirmed hits were re-purchased and the GSK PKIS confirmed hits were resupplied by GSK (solid sample) and the University or college of North Pantoprazole (Protonix) Carolina (DMSO stock). Five of these confirmed, resupplied hits inhibited TSSK2 with IC50 values 100 nM: staurosporine, TAE684, and three pyrrolopyrimidines. SAR and kinase selectivity of pyrrolopyrimidine TSSK2 inhibitors.