Indolyl‐quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation‐mediated control of tumor vasculature

Abstract

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.

Redox modulation of Ecto-NOX1 in human platelets

“…In some experiments, NADH oxidation was monitored after heating to 50 C for 10 min and removing heat-denatured proteins by centrifugation at 6000 rpm for 5 min (Yantiri and Morré 2001). NAD(P)H oxidase activity was also measured by monitoring the reduction of WST-1, a cellimpermeable artificial electron acceptor (Geng et al 2009). Briefly, intact platelets (1 Â 10 8 ) were incubated with 0.5 mM WST-1 and 0.2 mM NADH or 0.2 mM NADPH.…”

“…Platelets also showed an electron donor preference, since the rate of NADHdependent WST-1 reduction was significantly higher than that of NADPH-dependent WST1 reduction (Figure 2A). It has been reported that the Ecto-NOX proteins may be responsible for cell surface reduction of WST-1 (Geng et al 2009); none the less, this tetrazolium salt has also been used for detection of superoxide or superoxide-generating enzymes (Oritani et al 2004, Tan andBerridge 2010). In order to circumvent the misunderstanding that could come from the use of WST-1, we also monitored the disappearance of NAD(P)H at 340 nm.…”

Fine mapping of whole RB1 gene deletions in retinoblastoma patients confirms PCDH8 as a candidate gene for psychomotor delay

“…Inhibition of ENOX1 has been reported to decrease angiogenesis in tumour growth. 17 ENOX1 was found with a high but not exclusive expression level in fetal brain (see http://biogps.org), but no other data are available in the literature to incriminate this gene in the context of psychomotor delay. Comparative analysis of deleted intervals in delayed and non-delayed patients identified PCDH8 as a candidate gene for psychomotor delay (Figure 1).…”

Perspectives on the History and Evolution of Tumor Models

“…In combination with other results, however, endothelial cell assays can be a source of lead compounds. For instance, Sekhar et al combined observations from a chemistry-driven drug discovery screen for inhibitors of endothelial cell tubule formation with biochemical pathway screening and shRNA suppression to identify compounds to pursue as drug leads, and also validate ENOX1 as a target for enhancing radiation response of tumors [49]. Other endothelial cell strategies have looked to capitalize on differences between tumor and normal endothelial cells.…”