Abstract 4750: Development of potent NADPH oxidase inhibitors with significant activity against colon cancer

Abstract

Abstract The NADPH oxidase (NOX) gene family (NOX1-5 and Duox1/2) plays an important role in host defense and the development of inflammation-induced carcinogenesis. We recently found that two iodonium-class NOX inhibitors, diphenyleneiodonium (DPI) and di-2-thienyliodonium (DTI), block NOX-dependent reactive oxygen species (ROS) formation, NOX1 mRNA expression, and growth of human colon cancer cells in vitro and in vivo (Free Rad. Biol. Med. 57: 162, 2013). To improve the efficacy and bioavailability of DPI and DTI, we synthesized 48 new analogs of these compounds (21 DPI analogs, 12 DTI analogs, and 15 DPI and/or DTI hybrids; structures to be presented at the meeting) and prioritized the agents for further study based on inhibition of: HT-29 growth (MTT and clonogenic assay), ROS generation (by chemiluminescence and flow cytometry), and NOX1 mRNA expression (by qRT-PCR). The compounds were also characterized for lack of altered mitochondrial ROS production, and inhibitory effects on ROS generated by cell-based assays specific for NOX1, NOX2, NOX4, NOX5, and Duox2. DPI and five DPI analogs, NSCs 740104, 734428, 742837, 751140, 737392, strongly inhibited HT-29 cell growth in a dose dependant manner (IC50's after 48 hr drug exposure of 300, 50, 74, 87, 148, and 202 nM, respectively). Treatment for 24 hr with 10 nM DPI, 740104, 734428, or 751140, or with 100 nM 737392 significantly decreased ROS production in HT-29 cells. In addition, 60% or greater inhibition of NOX1 mRNA expression was observed following treatment with 250 nM 740104 and 734428 for 4 hr; 751140 and DPI for 24 hr; and 737392 for 48 hr. Analog 742837 demonstrated no significant inhibition of either ROS production or NOX1 expression in HT-29 cells; it was not analyzed further. Growth inhibitory properties of 734958, a DTI analog, were similar to the parent molecule. To evaluate compound specificity, a luminescence assay for O2•¯ was used with: HEK293 cells expressing all components of the NOX1 complex; fully differentiated HL-60 cells expressing active NOX2; and UACC257 melanoma cells that exclusively express active NOX5. H2O2 was measured by Amplex Red assay to determine NOX4 or Duox2 activity in fully reconstituted HEK293 cells. DPI, 734428, 740104, 737392, or 751140 exposure for 30 min inhibited NOX1, 2, 4, and 5 with low nM IC50's; analogs 740104, 734428, and 751140 were more potent inhibitors of NOX1, 2, and 4, respectively, than DPI; each was broadly active against these NOX isoforms. The DTI analog 734958 was more potent than its parent molecule. NSC737392 and 734428 were >10-fold more active than the other compounds examined against Duox2 (200-400 nM IC50's). In summary, we developed novel iodonium analogs with anticancer activity that possess enhanced potency against human NOX species, including the first agents with specificity against Duox2. These tool compounds will be evaluated in vivo for their therapeutic and anti-inflammatory activities in NOX-containing tumor xenograft models. Citation Format: Jiamo Lu, Prabhakar Risbood, Charles T. Kane, Md Tafazzal Hossain, Yongzhong Wu, Smitha Antony, Jennifer L. Meitzler, Agnes Juhasz, Han Liu, Guojian Jiang, Krishnendu Roy, James H. Doroshow. Development of potent NADPH oxidase inhibitors with significant activity against colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2014-4750