Background: Whether ligand-induced clusters of DR5 have a specific structural organization is unknown. Results: Ligand binding results in the formation of death receptor dimers that exist within high molecular weight networks. Conclusion: Ligand-induced DR5 clusters are highly organized networks formed through dimerization of receptor trimers. Significance: The biophysical character of DR5 networks may have implications for future rational design of DR5-targeted therapeutics.

Testis-specific serine/threonine kinase 2 (TSSK2) is an important target for reversible male contraception. A high-throughput screen of ~17,000 compounds using a mobility shift assay identified two potent series of inhibitors having a pyrrolopyrimidine or pyrimidine core. The pyrrolopyrimidine 10 (IC50 22 nM; GSK2163632A) and the pyrimidine 17 (IC50 31 nM; ALK inhibitor 1) are the most potent TSSK2 inhibitors in these series, which contain the first sub-100 nanomolar inhibitors of any TSSK isoform reported, except for the broad kinase inhibitor staurosporine. The novel, potent pyrimidine TSSK2 inhibitor compound 19 (IC50 66 nM; 2-[[5-chloro-2-[2-methoxy-4-(1-methylpiperidin-4-yl)anilino]pyrimidin-4-yl]amino]-N-methylbenzenesulfonamide) lacks the potential for metabolic activation. Compound 19 had a rank order potency TSSK1 > TSSK2 > TSSK3 > TSSK6, indicating that potent dual inhibitors of TSSK1/2 can be identified, which may be required for a complete contraceptive effect. The future availability of a TSSK2 crystal structure will facilitate structure-based discovery of selective TSSK inhibitors from these pyrrolopyrimidine and pyrimidine scaffolds.

WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells.

volume (8M urea minus 0M urea) as a function of voltage, we observe an exponential decay as the electric field effects override the chemical effects until finally the presence of urea does not affect the translocation properties (Protein: PDZ2 Mutants). Nanopores are a unique and powerful tool since they can accurately produce transient denaturing conditions (using electric fields) while measuring the protein's response.