A Novel Compound Targeting Protease Receptor 1 Activators for the Treatment of Glioblastoma

Abstract

Data from human biopsies, in-vitro and in-vivo models, strongly supports the role of thrombin, and its protease-activated receptor (PAR1) in the pathology and progression of glioblastoma (GBM), a high-grade glial tumor. Activation of PAR1 by thrombin stimulates vasogenic edema, tumor adhesion and tumor growth. We here present a novel six amino acid chloromethyl-ketone compound (SIXAC) which specifically inhibits PAR1 proteolytic activation and counteracts the over-activation of PAR1 by tumor generated thrombin. SIXAC effects were demonstrated in-vitro utilizing 3 cell-lines, including the highly malignant CNS-1 cell-line which was also used as a model for GBM in-vivo. The in-vitro effects of SIXAC on proliferation rate, invasion and thrombin activity were measured by XTT, wound healing, colony formation and fluorescent assays, respectively. The effect of SIXAC on GBM in-vivo was assessed by measuring tumor and edema size as quantified by MRI imaging, by survival follow-up and brain histopathology. SIXAC was found in-vitro to inhibit thrombin-activity generated by CNS-1 cells (IC50 = 5 × 10−11M) and significantly decrease proliferation rate (p < 0.03) invasion (p = 0.02) and colony formation (p = 0.03) of these cells. In the CNS-1 GBM rat animal model SIXAC was found to reduce edema volume ratio (8.8 ± 1.9 vs. 4.9 ± 1, p < 0.04) and increase median survival (16 vs. 18.5 days, p < 0.02 by Log rank Mental-Cox test). These results strengthen the important role of thrombin/PAR1 pathway in glioblastoma progression and suggest SIXAC as a novel therapeutic tool for this fatal disease.

Higher expression of PAR-1 may be associated with severity of Glioblastomas

“…The involvement of PAR-1 was further con rmed by use of a speci c inhibitor SCH-79797. It was observed that use of speci c inhibitor SCH-79797 signi cantly reduced the TFLLR induced increase in the migration and invasion of D-54 MG. Our results are in line with many previous studies both in rodents and human tissues that have reported active involvement of PAR-1 receptors in the progression of Glioblastoma multiforme [44]. These results along with ours suggest an association between PAR-1 expression and glioma migration.…”

Treatment of Diabetic Neuropathy with A Novel PAR1-Targeting Molecule

“…We chose to modulate this pathway using the specific N-terminal PAR1 sequence recognized by thrombin as a template for the unique PARIN5 molecule, aiming to specifically inhibit PAR1 activation by thrombin. The PARIN5 molecule was previously designed as part of a set of molecules which differ in their backbone length, based on the thrombin recognition site sequence of PAR1, and found to be potent PAR1 inhibitors [ 21 ]. Based on the specificity of the protease-binding site on PAR1 in comparison to PAR3 and PAR4, it is reasonable to attribute the specificity of PARIN5 to PAR1.…”

Extracellular proteolysis in glioblastoma progression and therapeutics

“…This interaction induces resistance to apoptosis and to cytotoxic agents via increased level of Bcl and inhibition of Bim [95]. Recently a specific inhibitor, which prevents activation of PAR1 at thrombin (and thrombin-like protease) cleavage site, resulted in decreased proliferation and improved survival in vivo in glioblastoma [96]. This inhibitor was not designed to block KLK6-mediated activation of PAR1; however, this study highly supports that the KLK6/PAR1 pathway could be a potential target in glioma.…”