Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase.

Necroptosis and signaling regulated by RIP1 kinase activity is emerging as a key driver of inflammation in a variety of disease settings. A significant amount has been learned about how RIP1 regulates necrotic cell death through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1). Nec-1 has been a transformational tool for exploring the function of RIP1 kinase activity; however, its utility is somewhat limited by moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have driven an effort to identify next-generation tools to study RIP1 function, and have led to the identification of 7-Cl-O-Nec-1 (Nec-1s), which has improved pharmacokinetic properties and lacks IDO inhibitory activity. Here we describe the characterization of GSK'963, a chiral small-molecule inhibitor of RIP1 kinase that is chemically distinct from both Nec-1 and Nec-1s. GSK'963 is significantly more potent than Nec-1 in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK'963 is >10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK'962, which can be used to confirm on-target effects. The increased in vitro potency of GSK'963 also translates in vivo, where GSK'963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK'963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.

Partial agonist/antagonist properties of androstenedione and 4-androsten-3beta,17beta-diol.

Androgens play important endocrine roles in development and physiology. Here, we characterize activities of two "Andro" prohormones, androstenedione (A-dione) and 4-androsten-3beta,17beta-diol (A-diol) in MDA-MB-453 (MDA) and LNCaP cells. A-dione and A-diol, like cyproterone acetate, were partial agonists of transfected mouse mammary tumor virus (MMTV) and endogenous prostate-specific antigen (PSA) promoters. Different from bicalutamide but similar to CPA, both are inducers of LNCaP cell proliferation with only mild suppression of 5alpha-dihydrotestosterone (DHT)-enhanced cell growth. Like bicalutamide and cyproterone acetate, A-dione and A-diol significantly antagonized DHT/R1881-induced PSA expression by up to 30% in LNCaP cells. Meanwhile, in MDA cells, EC(50)s for the MMTV promoter were between 10 and 100nM. Co-factor studies showed GRIP1 as most active for endogenous androgen receptor (AR), increasing MMTV transcription by up to five-fold, without substantially altering EC(50)s of DHT, A-dione or A-diol. Consistent with their transcriptional activities, A-dione and A-diol bound full-length endogenous AR from MDA or LNCaP cells with affinities of 30-70nM, although binding to expressed ligand-binding domain (LBD) was >20-fold weaker. In contrast, DHT, R1881, and bicalutamide bound similarly to LBD or aporeceptor. Together, these data suggest that A-dione and A-diol are ligands for AR with partial agonist/antagonist activities in cell-based transcription assays. Binding affinities for both are most accurately assessed by AR aporeceptor complex. In addition to being testosterone precursors in vivo, either may impart its own transcriptional regulation of AR.

A diagnostic approach to identifying submicroscopic 7p21 deletions in Saethre-Chotzen syndrome: fluorescence in situ hybridization and dosage-sensitive Southern blot analysis.

PURPOSE: To report on the use of fluorescence in situ hybridization (FISH) and dosage-sensitive Southern blot analysis in the molecular diagnosis of patients with Saethre-Chotzen syndrome. METHODS: FISH and dosage-sensitive Southern blot analysis utilizing TWIST gene probes were performed on patients with Saethre-Chotzen syndrome but without an identifiable TWIST sequence variation. RESULTS: Four unrelated patients with a deletion of the TWIST gene were identified by Southern blot; one of them had a complex chromosomal rearrangement involving 7p21 and no apparent deletion by FISH, suggesting a smaller deletion in the region including the TWIST gene. A fifth patient had an abnormal TWIST gene fragment on Southern blot analysis that segregated with the disease in the family; FISH was normal in this patient, suggesting a partial deletion or rearrangement in or near the gene. CONCLUSION: FISH and dosage-sensitive Southern blot analysis are useful diagnostic tools in Saethre-Chotzen syndrome without TWIST sequence variation.

Molecular analysis of the von hippel-lindau disease gene.

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that predisposes the affected individual to develop characteristic tumors. These include CNS hemangioblastoma, retinal angiomas, endolymphatic sac tumors, pancreatic cysts and tumors, epididymal cystadenomas, pheochromocytomas, renal cysts, and clear-cell renal carcinoma. The VHL gene was localized to 3p25 and then isolated by Latif et al. (1). The gene contains three exons with an open reading frame of 852 nucleotides, which encode a predicted protein of 284 amino acids. The VHL protein is believed to have several functions. It is involved in transcription regulation through its inhibition of elongation by binding to the B and C subunits of elongin. Mutations of VHL allow the B and C subunits to bind with the A subunit. This complex then overcomes "pausing" of RNA polymerase during mRNA transcription (2,3). Several studies suggest that the VHL protein is also involved in regulation of hypoxia-inducible transcripts, particularly vascular endothelial growth factor (VEGF), by altering mRNA stability (4,5). Therefore, VHL gene mutations permit the overexpression of VEGF under normoxic conditions, which leads to the angiogenesis believed to be required for tumor growth. The VHL-elongin BC complex (VBC) also binds two other proteins-CUL2 and Rbx1-in a complex that has structural similarity to other E3 ubiquitin ligase complexes (6). Such complexes mediate the degradation of cell-cycle regulatory proteins.