Developing an Irreversible Inhibitor of Human DDAH‐1, an Enzyme Upregulated in Melanoma

Abstract

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. Herein, N5-(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI = 1.3 ± 0.6 μM; kinact = 0.34 ± 0.07 min−1), with > 500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the “in cell” IC50 value (6.6 ± 0.2 μM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50 / 64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 in comparison to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO represents a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production such as septic shock and idiopathic pulmonary fibrosis.

Inhibition of Dimethylarginine Dimethylaminohydrolase (DDAH) Enzymes as an Emerging Therapeutic Strategy to Target Angiogenesis and Vasculogenic Mimicry in Cancer

“…The research group demonstrated that DDAH1 is overexpressed in melanoma cell lines compared to normal human epidermal melanocytes and that cellular inhibition of DDAH1 by N 5 -(1imino-2-chloroethyl)-L-ornithine (Cl-NIO) resulted in reduced nitric oxide production in the A375 melanoma cell line. The reduction in NO synthesis was measured by quantifying 3nitrotyrosine levels and total nitrate and nitrite in the cell culture supernatant and it was independent of changes in DDAH1 or iNOS expression (183). Unfortunately, this study did not assess the effects of DDAH1 inhibition by Cl-NIO on specific tumor parameters, such as tumor cell viability and proliferation in vitro and/or in vivo growth of xenograft tumors derived from A375 cells, or assess the impact on angiogenesis.…”

“…Studies to date have demonstrated an increase in DDAH1 protein expression in human glioma, meningioma, prostate cancer, and hepatocellular carcinoma, primarily by means of large-scale proteomic analysis. An upregulation of DDAH1 protein has also been observed in cohorts of melanoma and breast cancer cell lines, relative to normal melanocyte, and mammary epithelial cells, respectively (183,187). Aside from the identification that DDAH1 expression is significantly altered in these cancers, only a handful of these studies undertook further analysis into the specific role and function of DDAH1 within each cancer context.…”

“…Over the last two decades various different classes of DDAH1 inhibitors have been synthesized; these exhibit different structures, features and mechanisms of action, and have been previously extensively reviewed (244). Whilst some of these molecules have structural similarity with the DDAH substrates (methylated arginines) (183,(245)(246)(247)(248)(249), others bear a very different chemical structure (56, [250][251][252]. A comprehensive discussion on all DDAH inhibitors synthesized to date and their impact on endothelial cells falls outside the scope of this review, however, here we summarize a small body of evidence that identifies the therapeutic potential for pharmacological inhibition of DDAH1 in cancer.…”

“…The first study to show some potential for DDAH1 inhibition by a small molecule in cancer was published by (183). The research group demonstrated that DDAH1 is overexpressed in melanoma cell lines compared to normal human epidermal melanocytes and that cellular inhibition of DDAH1 by N 5 -(1imino-2-chloroethyl)-L-ornithine (Cl-NIO) resulted in reduced nitric oxide production in the A375 melanoma cell line.…”

Arginine analogues incorporating carboxylate bioisosteric functions are micromolar inhibitors of human recombinant DDAH-1

“…As such, several classes of DDAH inhibitors have been described in the literature. 32,34,35 Conversely, modifications of the carboxylic acid functional group have not been extensively investigated. 2).…”