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

Abstract

Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme involved in the metabolism of asymmetric dimethylarginine (ADMA) and N-monomethyl arginine (NMMA), which are endogenous inhibitors of the nitric oxide synthase (NOS) family of enzymes. Two isoforms of DDAH have been identified in humans, DDAH-1 and DDAH-2. DDAH-1 inhibition represents a promising strategy to limit the overproduction of NO in pathological states without affecting the homeostatic role of this important messenger molecule. Here we describe the design and synthesis of 12 novel DDAH-1 inhibitors and report their derived kinetic parameters, IC50 and Ki. Arginine analogue 10a, characterized by an acylsulfonamide isosteric replacement of the carboxylate, showed a 13-fold greater inhibitory potential relative to the known DDAH-1 inhibitor, L-257. Compound 10a was utilized to study the putative binding interactions of human DDAH-1 inhibition using molecular dynamics simulations. The latter suggests that several stabilizing interactions occur in the DDAH-1 active-site, providing structural insights for the enhanced inhibitory potential demonstrated by in vitro inhibition studies.

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

“…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.…”

“…More recently, the potential therapeutic benefit of inhibiting DDAH1 was demonstrated for breast cancer (55). DDAH1 activity was inhibited in triple negative breast cancer cell lines by the potent DDAH1 inhibitors, arginine analogs ZST316 and ZST152 (244,249), as identified by increased intracellular ADMA concentrations and decreased intracellular L-citrulline concentrations (55). In an in vitro Matrigel tube formation model of VM, both ZST316 and ZST152 significantly inhibited the number of vessel-like networks formed at concentrations above 1 µM (55).…”

Human dimethylarginine dimethylaminohydrolase 1 inhibition by proton pump inhibitors and the cardiovascular risk marker asymmetric dimethylarginine: in vitro and in vivo significance

“…2). Inhibition with the known DDAH1 inhibitor ZST316 was 72% 18 .

Figure 2Concentration dependent inhibition of DDAH1 by ZST316 (positive control), esomeprazole (EPZ), lansoprazole (LPZ), omeprazole (OPZ), pantoprazole (PPZ) and rabeprazole (RPZ). Residual DDAH1 activity is reported as percentage of control activity.

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“…DDAH1 over-expression was performed as reported by Tommasi et al . 18 A single batch of lysate prepared from DDAH1-expressing HEK293Tcells was used for all in vitro experiments.…”

“…Incubation mixtures contained DDAH1-expressing HEK293T cell lysate (0.4 mg/mL total protein), phosphate buffer (0.1 mol/L, pH 7.4), PPI (0–100 µmol/L) and ADMA (0 to 500 µmol/L). The DDAH1 inhibitor ZST316 (compound 10a) 18 was used as a positive control for DDAH1 inhibition at a concentration of 1 µmol/L. Following pre-incubation (0 to 240 min), reactions were initiated by the addition of substrate (ADMA).…”