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

Ling Geng^*, Girish Rachakonda^*, D. James Morré, Dorothy M. Morré^||, Peter A. Crooks^¶, Vijayakumar N. Sonar^¶,1, Joseph L. Roti Roti^#, Buck E. Rogers^#, Suellen Greco^**, Fei Ye, Kenneth J. Salleng, Soumya Sasi^*, Michael L. Freeman^*,2 and Konjeti R. Sekhar^*,2

^* Department of Radiation Oncology,

Department of Biostatistics, and

Animal Care, Vanderbilt University School of Medicine/Vanderbilt-Ingram Cancer Center, Nashville, Tennessee, USA;

Department of Medicinal Chemistry and Molecular Pharmacology and

^|| Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana, USA;

^¶ Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky, USA; and

^# Department of Radiation Oncology and

^** Division of Comparative Medicine, Washington University School of Medicine, St. Louis, Missouri, USA

² Correspondence: B902 TVC Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232. E-mail: K.R.S., raja.konjeti{at}vanderbilt.edu; M.L.F., michael.freeman{at}vanderbilt.edu

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 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 (EC₅₀ = 10 µM). 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.—Geng, L., Rachakonda, G., Morré, D. J., Morré, D. M., Crooks, P. A., Sonar, V. N., Roti Roti, J. L., Rogers, B. E., Greco, S., Ye, F., Salleng, K. J., Sasi, S., Freeman, M. L., Sekhar, K. R. Indolyl-quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation-mediated control of tumor vasculature.

Key Words: angiogenesis • (Z)-(±)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol