A novel and potent inhibitor of dimethylarginine dimethylaminohydrolase: a modulator of cardiovascular nitric oxide

Abstract

PD 404182 [6H-6-imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine], a heterocyclic iminobenzothiazine derivative, is a member of the Library of Pharmacologically Active Compounds (LOPAC) that is reported to possess antimicrobial and anti-inflammatory properties. In this study, we used biochemical assays to screen LOPAC against human dimethylarginine dimethylaminohydrolase isoform 1 (DDAH1), an enzyme that physiologically metabolizes asymmetric dimethylarginine (ADMA), an endogenous and competitive inhibitor of nitric oxide (NO) synthase. We discovered that PD 404182 directly and dose-dependently inhibits DDAH. Moreover, PD 404182 significantly increased intracellular levels of ADMA in cultured primary human vascular endothelial cells (ECs) and reduced lipopolysaccharide-induced NO production in these cells, suggesting its therapeutic potential in septic shock-induced vascular collapse. In addition, PD 404182 abrogated the formation of tube-like structures by ECs in an in vitro angiogenesis assay, indicating its antiangiogenic potential in diseases characterized by pathologically excessive angiogenesis. Furthermore, we investigated the potential mechanism of inhibition of DDAH by this small molecule and found that PD 404182, which has striking structural similarity to ADMA, could be competed by a DDAH substrate, suggesting that it is a competitive inhibitor. Finally, our enzyme kinetics assay showed time-dependent inhibition, and our inhibitor dilution assay showed that the enzymatic activity of DDAH did not recover significantly after dilution, suggesting that PD 404182 might be a tightly bound, covalent, or an irreversible inhibitor of human DDAH1. This proposal is supported by mass spectrometry studies with PD 404182 and glutathione.

Fig. 1.

Dose-dependent inhibition of DDAH activity by a novel small molecule, PD 404182. The biochemical conversion of the artificial substrate SMTC into methanethiol in the presence of PD 404182 is shown. The mean percent inhibition at 4 hours, in reference to vehicle control, from duplicate experiments is shown. The x-axis shows final compound concentration.

Fig. 2.

Validation of DDAH inhibition by PD 404182. In vitro production of L-citrulline from the endogenous substrate ADMA in the presence of vehicle (DMSO) control, PD 404182, or L-257 is shown. Compounds were used at 20 μM final concentration each. Data are mean ± S.E.M. from duplicate experiments (*P < 0.05).

Fig. 3.

Intracellular inhibition of DDAH activity by PD 404182. Cellular ADMA concentration in vascular ECs following treatment with vehicle or the indicated small molecules at 20 μM each. Data are mean ± S.E.M. from duplicate experiments (*P < 0.05).

Fig. 4.

The effect of PD 404182 on NO production. Human vascular endothelial cells were treated with vehicle, PD 404182, or L-257 in the presence of LPS (100 ng/ml; Sigma-Aldrich) for 24 hours. Total nitrite (NOx) was measured using Griess reaction. Data are mean ± S.E.M. from duplicate experiments. *P < 0.05 compared with non-LPS control. *⁺P < 0.05 compared with vehicle + LPS.

Fig. 5.

PD 404182 attenuated endothelial tube formation. Human microvascular endothelial cells were seeded on Matrigel and treated with vehicle, PD 404182, or L-257 for 18 hours, and formation of tube-like structures was visualized microscopically. Representative images are from duplicate experiments.

Fig. 6.

Competition assay. DDAH was incubated with PD 404182 (10 μM) or vehicle for 30 minutes, and various substrate (SMTC) concentrations were added to initiate the reaction. Fluorescence intensity was measured, and DDAH activity was calculated relative to vehicle controls. Data are mean ± S.E.M. from triplicate experiments. *P < 0.05 versus 50 μM; **P < 0.05 between 100 and 1000 μM substrate concentration. The Michaelis constant (K_M) for SMTC is ~ 3 μM (Wang et al., 2009).

Fig. 7.

Inhibitor dilution assay demonstrating low recovery of DDAH activity. PD 404182 (at 90 or 900 μM; IC₅₀ is 9 μM) was preincubated with DDAH at high concentration and then serially diluted, as described above. The recovery of DDAH activity upon dilution of the inhibitor was evaluated. Data are mean ± S.E.M. from triplicate experiments. ns, not significant.

Fig. 8.

Schematic of the structural resemblance of PD 404182 with the endogenous NOS inhibitor and DDAH substrate ADMA. The chemical similarities between the two are highlighted in bold. A putative reaction of PD 404182 with the active site cysteine (Cys273) of human DDAH-1 is also proposed. The active site cysteine first forms a covalent intermediate with PD 404182. This can fragment to release thiol 192 (a species with a molecular weight of 192), leaving the cysteine cyanylated. This could then form a mixed disulfide with thiol 192 or other thiols.