Dengue virus or NS1 protein induces trans-endothelial cell permeability associated with VE-Cadherin and RhoA phosphorylation in HMEC-1 cells preventable by Angiopoietin-1.

A transient increase in trans-endothelial cell permeability in dengue patients leads to vascular leakage and shock syndrome. Here, we analysed the molecular mechanisms that cause permeability changes in human dermal microvascular endothelial cells (HMEC-1) using a direct dengue virus (DENV) infection model or treatment with NS1, a secreted DENV non-structural protein. In HMEC-1 cells, both treatments increase permeability with a concordant increase in the secretion of angiopoietin-2 (Ang-2). There is phosphorylation and loss of the junction protein VE-Cadherin from the inter-endothelial cell junctions and phosphorylation of RhoA. Direct virus infection results in activation of Src by phosphorylation, whereas NS1 treatment alone does not lead to Src activation. Furthermore, treatment with recombinant Ang-1, a physiological antagonist of Ang-2, prevents Ang-2 release, VE-Cadherin phosphorylation and internalization, and phosphorylation of RhoA and Src, resulting in restoration of barrier function. The permeability increase could also be prevented by blocking the Ang1/2 signalling receptor, Tie-2, or using a Rho/ROCK-specific inhibitor. Dasatinib, a Src-family kinase (SFK) inhibitor that inhibits Src phosphorylation, prevents enhanced permeability induced by direct DENV infection whereas in NS1 protein-treated cells its effect is less significant. The results provide important insights on the mechanisms of increased trans-endothelial permeability in DENV infection, and suggest the therapeutic potential of using recombinant Ang-1 or targeting these key molecules to prevent vascular leakage in dengue.

Optimization of Aminoimidazole Derivatives as Src Family Kinase Inhibitors.

Protein kinases have emerged as crucial targets for cancer therapy over the last decades. Since 2001, 40 and 39 kinase inhibitors have been approved by FDA and EMA, respectively, and the majority are antineoplastic drugs. Morevoer, many candidates are currently in clinical trials. We previously reported a small library of 4-aminoimidazole and 2-aminothiazole derivatives active as Src family kinase (SFK) inhibitors. Starting from these results, we decided to perform an optimization study applying a mix and match strategy to identify a more potent generation of 4-aminoimidazoles. Firstly, a computational study has been performed, then compounds showing the best predicted docking scores were synthesized and screened in a cell-free assay for their SFK inhibitory activity. All the new chemical entities showed IC50s in the nanomolar range, with 2⁻130 fold increased activities compared to the previously reported inhibitors. Finally, the most active compounds have been tested on three cancer cell lines characterized by Src hyperactivation. Compounds 4k and 4l showed an interesting antiproliferative activity on SH-SY5Y neuroblastoma (NB) cell line. In this assay, the compounds resulted more potent than dasatinib, a tyrosine kinase inhibitor approved for the treatment of leukemias and in clinical trials for NB.

Src family kinase inhibitor bosutinib enhances retinoic acid-induced differentiation of HL-60 leukemia cells.

The acute promyelocytic leukemia (APL) has been treated with all-trans retinoic acid (RA) for decades. While RA has largely been ineffective in non-APL AML subtypes, co-treatments combining RA and other agents are currently in clinical trials. Using the RA-responsive non-APL AML cell line HL-60, we tested the efficacy of the Src family kinase (SFK) inhibitor bosutinib on RA-induced differentiation. HL-60 has been recently shown to bear fidelity to a subtype of AML that respond to RA. We found that co-treatment with RA and bosutinib enhanced differentiation evidenced by increased CD11b expression, G1/G0 cell cycle arrest, and respiratory burst. Expression of the SFK members Fgr and Lyn was enhanced, while SFK activation was inhibited. Phosphorylation of several sites of c-Raf was increased and expression of AhR and p85 PI3K was enhanced. Expression of c-Cbl and mTOR was decreased. Our study suggests that SFK inhibition enhances RA-induced differentiation and may have therapeutic value in non-APL AML.

Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells.

The hematopoietic cell kinase (HCK) is a member of the SRC family of cytoplasmic tyrosine kinases (SFKs), and is expressed in cells of the myeloid and B-lymphocyte cell lineages. Excessive HCK activation is associated with several types of leukemia and enhances cell proliferation and survival by physical association with oncogenic fusion proteins, and with functional interactions with receptor tyrosine kinases. Elevated HCK activity is also observed in many solid malignancies, including breast and colon cancer, and correlates with decreased patient survival rates. HCK enhances the secretion of growth factors and pro-inflammatory cytokines from myeloid cells, and promotes macrophage polarization towards a wound healing and tumor-promoting alternatively activated phenotype. Within tumor associated macrophages, HCK stimulates the formation of podosomes that facilitate extracellular matrix degradation, which enhance immune and epithelial cell invasion. By virtue of functional cooperation between HCK and bona fide oncogenic tyrosine kinases, excessive HCK activation can also reduce drug efficacy and contribute to chemo-resistance, while genetic ablation of HCK results in minimal physiological consequences in healthy mice. Given its known crystal structure, HCK therefore provides an attractive therapeutic target to both, directly inhibit the growth of cancer cells, and indirectly curb the source of tumor-promoting changes in the tumor microenvironment.