Novel Cellularly Active Inhibitor Regresses DDAH1 Induced Prostate Tumor Growth by Restraining Tumor Angiogenesis through Targeting DDAH1/ADMA/NOS Pathway

Dimethylarginine dimethylaminohydrolasel (DDAH1) inhibitors are important therapeutics by virtue of their ability to control nitric oxide (NO) production by elevating asymmetric dimethylarginine (ADMA) levels. In a screening campaign, we identified that DD1E5 (3-amino-6-tert-butyl-N-(1,3-thiazol-2-yl)-4-(trifluoromethyl)thieno[2,3-b]pyridine-2-carboxamide) inhibits the DDAH1 activity both in vitro and in cultured cells. Mechanistic studies found that DD1E5 is a competitive inhibitor (dissociation constant (K-i) of 2.05 +/- 0.15 mu M). Enzyme kinetic assays showed time and concentration dependent inhibition of DDAH1 with DD1E5, which shows tight binding with an inactivation rate constant of 0.2756 +/- 0.015 M-1 S-1. Treatment of cancer cells with DDAH1 inhibitors shows inhibition of cell proliferation and a subsequent decrease in NO production with ADMA accumulation. DD1E5 reversed the elevated VEGF, c-Myc, HIF-1 alpha, and iNOS levels induced by exogenous DDAH1 overexpression in PCa cells. Moreover, DD1E5 significantly increased intracellular levels of ADMA and reduced NO production, suggesting its therapeutic potential for cancers in which DDAH1 is upregulated. In in vitro assays, DD1E5 abrogated the secretion of angiogenic factors (bFGF and IL-8) into conditional media, indicating its antiangiogenic potential. DD1E5 inhibited in vivo growth of xenograft tumors derived from PCa cells with DDAH1 overexpression, by reducing tumor endothelial content represented with low CD31 expression. VEGF, HIF-1 alpha, and iNOS expression were reversed in DD1E5 treated tumors compared to respective control tumors. In this work, integrating multiple approaches shows DD1E5 is a promising tool for the study of methylarginine-mediated NO control and a potential therapeutic lead compound against pathological conditions with elevated NO production such as cancers and other diseases.