Effects of purine-scaffold inhibitors on HUVECs: Involvement of the purinergic pathway and interference with ATP. Implications for preventing the adverse effects of extracellular Grp94

BACKGROUND: Extracellular Glucose-regulated protein94 (Grp94) is linked to pathological conditions disrupting the obligatory intracellular location of this Heat Shock Protein (HSP). In plasma, Grp94 is linked to IgG in complexes that drive adverse effects on vascular cells and are biomarker of gastro-intestinal cancer. By blocking ATP site in different HSPs, purine-scaffold inhibitors are used as promising anti-cancer compounds, but their effects on vasculature are not known. METHODS: We tested the capacity of two purine-scaffold inhibitors, PU-H71 and PU-WS13, to prevent the binding of Grp94 to IgG and to antagonize the effects of Grp94 and native Grp94-IgG complexes on HUVECs in different experimental conditions. RESULTS: PU-H71 and PU-WS13 blocked Grp94 and the formation of Grp94-IgG complexes in absence of cells. Instead, in presence of HUVECs rather than Grp94 PU-inhibitors targeted cells causing stimulation of Akt and VEGF pathways and displaying angiogenic-like effects similar to, although less intense than that provoked by Grp94 and Grp94-IgG complexes. Unlike Grp94 and Grp94-IgG complexes, PU-inhibitors also activated the purinergic pathway and increased the expression of the ATP receptor P2X7. Effects of PU-inhibitors on HUVECs were reversed by ATP and in presence of ATP PU-inhibitors were again able to block Grp94. CONCLUSIONS: PU-inhibitors can display direct effects on endothelial cells by targeting the ATP receptor P2X7. In absence of ATP, PU-inhibitors preferentially bind to cells rather than Grp94. ATP antagonizes the PU-inhibitor binding to cells thus restoring the capacity to block Grp94 and Grp94-IgG complex formation. Results have implications for enhancing the therapeutic efficacy of PU-inhibitors against circulating pathogenic Grp94.