Author: Jing Xie
Xie, Jing, 2023 Rational Identification of Novel Human Dimethylarginine Dimethylaminohydrolase-1 (hDDAH-1) Inhibitors , Flinders University, College of Medicine and Public Health
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Nitric oxide works as a versatile messenger with significant physiological, biochemical and pathological functions. However, nitric oxide concentration needs to be maintained in a tight range as any imbalance (excess or reduction) can trigger pathological states. At present, there is no drug in the market to prevent excessive production of nitric oxide, therefore, there is an increasing interest in screening and developing novel inhibitors.
L-arginine is catalysed by nitric oxide synthase to produce nitric oxide and L-citrulline. Asymmetric dimethylarginine (ADMA) works as a competitive inhibitor of nitric oxide synthase by binding to the active site. However, ADMA can be degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH-1) into L-citrulline and dimethylamine (DMA). Therefore, the inhibition of DDAH-1 will cause accumulation of ADMA and then decrease nitric oxide synthase and lower nitric oxide concentration. This shows that DDAH-1 is an indirect modulator of nitric oxide.
In the last 30 years, different groups have designed multiple DDAH-1 inhibitors. Some of them have low side effects and cytotoxicity. L-257, an arginine analogue methyl ester synthesized based on 4124W (a weak DDAH inhibitor), was proven to have high activity and non-cytotoxicity. The compound structure was visualized in silico and used as a reference for novel DDAH inhibitor design, selection and docking in different groups for the treatment of diseases characterized by excessive nitric oxide. Compounds ZST316 and ZST152 are arginine analogues which were synthesized by our group using L-257 side-chain derivatives. Based on the non-cytotoxicity and no off-target effects of L-257, both compounds showed higher inhibition than L-257. Both compounds are competitive and potent DDAH-1 inhibitors, however their inhibition remains in the micromolar range and their half-life is relatively short. Therefore, this project is focused on optimization of specific measurement for human DDAH-1 (hDDAH-1) activity, docking and selecting in silico, and screening novel chemicals with better properties in vitro to target DDAH-1.
Based on the minimum effect of experimental reagents on color development studied by Train & Bavlia (2007), specific parameters and conditions were further optimized, including the validation of intra- and inter-day variability; calibrators, protein and time linearity; lower limit of detection (LOD) and quantification (LOQ); characterization of DDAH-1-ADMA kinetics; and specificity for the matrix to analyte. According to the standards published by FDA in 2018, the %CV and accuracy should be in the range of ± 15 % and 100 ± 20% respectively. The optimized colorimetric assay presented high precision (the highest %CV was 9 %), accuracy (105 %) and robustness (similar results between different operators under same experimental conditions). LOD and LOQ were 9 μM and 27 μM respectively. Calibration curves were always linear and the matrix for citrulline was irreplaceable. The relationship between citrulline formation and enzyme concentration and incubation time showed strong linearity and Michaelis Menten regression was proved as the best model fitting DDAH-1-ADMA Kinetics. Km was 105.6 ± 7.6 μM and Vmax was 844.7 ± 19.3 pmol*mg-1*min-1 respectively.
The hDDAH-1 inhibitory potency of 5 potential inhibitors selected from the novel in silico method was reported by measuring the efficiency of ADMA-catalyzed formation of citrulline through the optimized colorimetric assay. The novel in silico screening method of potential hDDAH-1 inhibitors was based on L-257 (pose overlap and LFvscore) and ZST316 (major interact residues Asp78, Arg97 and Asp268). A total of 54 novel compounds were filtered from the database Zinc 20 and eMolecule (total of 4,950,701 compounds) but only 5 compounds were treated to hDDAH-1 overexpressed HEK293T cells to test DDAH-1 activity. We hypothesized that at least one of these compounds would exhibit inhibitory effects on hDDAH-1. According to the in vitro screening, compounds H-Arg-NH2.2HCI and H-D-Arg(NO2)-OMe HCI were shown to be weak hDDAH-1 inhibitors. These results support the efficacy of the approach used. The remaining compounds should also be explored for hDDAH-1 activity in future experiments, including further derivation of parameters Ki and IC50 for complete kinetic characterization and investigation of their in vivo effects.
Keywords: DDAH-1, hDDAH-1, ADMA, NOS, Nitric Oxide, NO
Subject: Clinical Pharmacology thesis
Thesis type: Masters
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Pramod Nair