Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia telangiectasia and rad3 related protein (ATR).

The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer cell progression. Herein we describe the discovery of 19, a pyrazolopyrimidine-containing inhibitor of ATR via a strategic repurposing of compounds targeting PI3K.

Targeting prostate cancer with compounds possessing dual activity as androgen receptor antagonists and HDAC6 inhibitors.

While enzalutamide and abiraterone are approved for treatment of metastatic castration-resistant prostate cancer (mCRPC), approximately 20-40% of patients have no response to these agents. It has been stipulated that the lack of response and the development of secondary resistance to these drugs may be due to the presence of AR splice variants. HDAC6 has a role in regulating the androgen receptor (AR) by modulating heat shock protein 90 (Hsp90) acetylation, which controls the nuclear localization and activation of the AR in androgen-dependent and independent scenarios. With dual-acting AR-HDAC6 inhibitors it should be possible to target patients who don't respond to enzalutamide. Herein, we describe the design, synthesis and biological evaluation of dual-acting compounds which target AR and are also specific towards HDAC6. Our efforts led to compound 10 which was found to have potent dual activity (HDAC6 IC50=0.0356µM and AR binding IC50=<0.03µM). Compound 10 was further evaluated for antagonist and other cell-based activities, in vitro stability and pharmacokinetics.

hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature.

To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis.

Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti-miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti-miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery of anti-miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti-miR-132 inhibits neovascularization by maintaining vessels in the resting state.

Derivation of vasculature from embryonic stem cells.

The formation of the multicellular vascular system is critical to the growth, development, and viability of an organism, and many embryonic lethal mouse knockouts are due to vascular defects. Unfortunately, the complex nature, and many cell types involved in vasculogenesis and angiogenesis has stymied in vitro models of vascular formation. This unit describes a system that allows human embryonic stem cells to differentiate and spontaneously form vascular networks via both vasculogenesis and angiogenesis in the context of the three germ layers.

Cooperation between VEGF and beta3 integrin during cardiac vascular development.

In the developing myocardium, vascular endothelial growth factor (VEGF)-dependent neovascularization occurs by division of existing vessels, a process that persists for several weeks following birth. During this remodeling phase, mRNA expression of beta3 integrin in the heart decreases significantly as vessel maturation progresses. However, in male mice lacking beta3, coronary capillaries fail to mature and continue to exhibit irregular endothelial thickness, endothelial protrusions into the lumen, and expanded cytoplasmic vacuoles. Surprisingly, this phenotype was not seen in female beta3-null mice. Enhanced VEGF signaling contributes to the beta3-null phenotype, because these vessels can be normalized by inhibitors of VEGF or Flk-1. Moreover, intravenous injection of VEGF induces a similar angiogenic phenotype in hearts of adult wild-type mice. These findings show a clear vascular phenotype in the hearts of mice lacking beta3 and suggest this integrin plays a critical role in coronary vascular development and the vascular response to VEGF.

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

Regulation of alpha1(I) collagen messenger RNA decay by interactions with alphaCP at the 3'-untranslated region.

Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs). Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein alphaCP binds to the alpha1(I) collagen 3'-untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of alphaCP in the decay of transcripts containing the collagen 3'-UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, alphaCP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of alphaCP binding activity increased the rate of decay of wild type transcripts containing the alphaCP 3'-UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no alphaCP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for alphaCP. The addition of recombinant alphaCP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain alphaCP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting alphaCP showed a reduction of the binding activity of alphaCP and a concomitant reduction in intracellular levels of alpha1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of alphaCP in the stabilization of alpha1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs.

Hepatitis C virus core and nonstructural proteins induce fibrogenic effects in hepatic stellate cells.

BACKGROUND & AIMS: The mechanisms by which hepatitis C virus (HCV) induces liver fibrosis are unknown. Hepatocytes secrete HCV proteins, which may interact with hepatic stellate cells (HSCs). Our aims were to investigate whether HCV proteins induce fibrogenic effects on HSCs. METHODS & RESULTS: Human-activated HSCs expressed messenger RNA (mRNA) for the putative HCV receptors CD81, LDL receptor, and C1q receptor as assessed by RT-PCR. Incubation of activated but not quiescent human HSCs with recombinant core and NS3 protein increased intracellular calcium concentration and reactive oxygen species production, as well as stimulated intracellular signaling pathways. Adenoviruses encoding core and nonstructural proteins (NS3-NS5) were used to express HCV proteins in HSCs. Expression of core protein increased cell proliferation in a Ras/ERK and PI3K/AKT dependent manner. In contrast, NS3-NS5 protein expression preferentially induced proinflammatory actions, such as increased chemokine secretion and expression of intercellular cell adhesion molecule type 1 (ICAM-1) through the NF-kappa B and c-Jun N-terminal kinase pathways. These effects were attenuated by antioxidants. Infection of freshly isolated rat HSCs with adenovirus-encoding core protein resulted in accelerated cell activation, as assessed by alpha-smooth muscle actin expression. Moreover, adenovirus-encoding core and NS3-NS5 proteins increased the secretion of bioactive TGF beta 1 and the expression of procollagen alpha1(I) in early cultured rat HSCs, as assessed by ELISA and RNase protection assay, respectively. CONCLUSIONS: HCV core and nonstructural proteins regulate distinct biologic functions in HSCs. A direct interaction between HCV proteins and HSCs may contribute to HCV-induced liver fibrosis.