IL-4-STAT6 axis amplifies histamine-induced vascular endothelial dysfunction and hypovolemic shock.

BACKGROUND: Mast cell-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We previously revealed a synergistic interaction between the cytokine IL-4 and the mast cell-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis is unknown. OBJECTIVE: We sought to identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions. METHODS: RNA sequencing, Western blot, Ca2+ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock. RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4-stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia. CONCLUSIONS: These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.

Discovery of SMD-3040 as a Potent and Selective SMARCA2 PROTAC Degrader with Strong in vivo Antitumor Activity.

SMARCA2 is an attractive synthetic lethality target for human cancers with SMARCA4 deficiency. Herein, we report the design, synthesis, and biological evaluation of selective SMARCA2 protein degraders developed using the proteolysis targeting chimera (PROTAC) technology. Our efforts have led to the discovery of a series of potent and selective SMARCA2 degraders, exemplified by SMD-3040. SMD-3040 degrades SMARCA2 protein with a low nanomolar DC50 and Dmax > 90% and demonstrates an excellent degradation selectivity for SMARCA2 protein over SMARCA4 protein. It displays potent cell growth inhibitory activity in a panel of SMARCA4-deficient cancer cell lines and has much weaker activity in SMARCA4 wild-type cancer cell lines. SMD-3040 achieves strong tumor growth inhibition in two SMARCA4-deficient xenograft models at well-tolerated dose schedules. Further optimization of SMD-3040 may lead to the discovery of new therapies for the treatment of human cancers with SMARCA4 deficiency.

A selective small-molecule STAT5 PROTAC degrader capable of achieving tumor regression in vivo.

Signal transducer and activator of transcription 5 (STAT5) is an attractive therapeutic target, but successful targeting of STAT5 has proved to be difficult. Here we report the development of AK-2292 as a first, potent and selective small-molecule degrader of both STAT5A and STAT5B isoforms. AK-2292 induces degradation of STAT5A/B proteins with an outstanding selectivity over all other STAT proteins and more than 6,000 non-STAT proteins, leading to selective inhibition of STAT5 activity in cells. AK-2292 effectively induces STAT5 depletion in normal mouse tissues and human chronic myeloid leukemia (CML) xenograft tissues and achieves tumor regression in two CML xenograft mouse models at well-tolerated dose schedules. AK-2292 is not only a powerful research tool with which to investigate the biology of STAT5 and the therapeutic potential of selective STAT5 protein depletion and inhibition but also a promising lead compound toward ultimate development of a STAT5-targeted therapy.

Discovery of a Potent and Selective STAT5 PROTAC Degrader with Strong Antitumor Activity In Vivo in Acute Myeloid Leukemia.

STAT5 is an attractive therapeutic target for human cancers. We report herein the discovery of a potent and selective STAT5 degrader with strong antitumor activity in vivo. We first obtained small-molecule ligands with sub-micromolar to low micromolar binding affinities to STAT5 and STAT6 SH2 domains and determined co-crystal structures of three such ligands in complex with STAT5A. We successfully transformed these ligands into potent and selective STAT5 degraders using the PROTAC technology with AK-2292 as the best compound. AK-2292 effectively induces degradation of STAT5A, STAT5B, and phosphorylated STAT5 proteins in a concentration- and time-dependent manner in acute myeloid leukemia (AML) cell lines and demonstrates excellent degradation selectivity for STAT5 over all other STAT members. It exerts potent and specific cell growth inhibitory activity in AML cell lines with high levels of phosphorylated STAT5. AK-2292 effectively reduces STAT5 protein in vivo and achieves strong antitumor activity in mice at well-tolerated dose schedules.

Consecutive cyclization of allylaminoalkene by intramolecular aminolithiation-carbolithiation.

Consecutive cyclization of allylaminoalkenes by tandem aminolithiation-carbolithiation proceeded smoothly by using a lithium amide as a lithiating agent as well as protonating agent to give bicyclic amines, octahydroindolizine and hexahydro-1 H-pyrrolizine, in reasonably high yield and diastereoselectivity.