No abstract

Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.

No abstract

Focal adhesion kinase (FAK) is ak ey mediator of tumour progression and metastasis.T od ate,c linical trials of FAKi nhibitors have reported disappointing efficacy for oncology indications.W er eport the design and characterisation of GSK215, ap otent, selective,F AK-degrading Proteolysis Targeting Chimera (PROTAC)b ased on ab inder for the VHL E3 ligase and the knownF AK inhibitor VS-4718. Xray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice,asingle dose of GSK215 induced rapid and prolonged FAKd egradation, giving al ong-lasting effect on FAKl evels ( % 96 h) and am arked PK/PD disconnect. This tool PROTACmolecule is expected to be useful for the study of FAK-degradation biology in vivo,a nd our results indicate that FAKd egradation may be ad ifferentiated clinical strategy versus FAKi nhibition for the treatment of cancer.

Sphingosine kinase 1 (SK1) plays an important role in estrogen-dependent breast tumorigenesis, but its regulation is poorly understood. A subset of microRNAs (miRNA, miR) is regulated by estrogen and contributes to cellular proliferation and cancer progression. Here, we describe that miR-515-5p is transcriptionally repressed by estrogen receptor a (ERa) and functions as a tumor suppressor in breast cancer. Its downregulation enhances cell proliferation and estrogen-dependent SK1 activity, mediated by a reduction of miR-515-5p posttranscriptional repression. Enforced expression of miR-515-5p in breast cancer cells causes a reduction in SK1 activity, reduced cell proliferation, and the induction of caspase-dependent apoptosis. Conversely, opposing effects occur with miR-515-5p inhibition and by SK1 silencing. Notably, we show that estradiol (E2) treatment downregulates miR-515-5p levels, whereas the antiestrogen tamoxifen causes a decrease in SK1, which is rescued by silencing miR-515-5p. Analysis of chromatin immunoprecipitation sequencing (ChIP-Seq) data reveals that miR-515-5p suppression is mediated by a direct interaction of ERa within its promoter. RNA-sequencing (RNA-Seq) analysis of breast cancer cells after overexpressing miR-515-5p indicates that it partly modulates cell proliferation by regulating the Wnt pathway. The clinical implications of this novel regulatory system are shown as miR-515-5p is significantly downregulated in ER-positive (n ¼ 146) compared with ER-negative (n ¼ 98) breast cancers. Overall, we identify a new link between ERa, miR-515-5p, proliferation, and apoptosis in breast cancer tumorigenesis.