Thrombin; ribosyldihydronicotinamide dehydrogenase (NQO2)

1. NQO2 Inhibition Potency: Dabigatran ethyl ester (2) is a potent inhibitor of ribosyldihydronicotinamide dehydrogenase (NQO2). It exhibits significantly higher inhibitory activity than the parent compound dabigatran (1), with a calculated inhibition constant (K_i) of 0.9 µM and a half-maximal inhibitory concentration (IC_50) of 0.8 µM in the NADH-dependent mitomycin C metabolism functional assay using recombinant NQO2. This represents an approximately 75-fold increase in potency compared to dabigatran (K_i ~70 µM, IC_50 ~60 µM).[1]

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2. Thrombin Inhibition Potency: Compound 2 also demonstrates increased inhibitory potency against its primary target, thrombin, compared to dabigatran (1). The IC_50 value for thrombin inhibition by Compound 2 was determined to be 1.2 nM, which is approximately 11.5-fold lower (more potent) than dabigatran's IC_50 of 13.8 nM.[1]

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3. Binding Affinity to NQO2: Competition experiments against Capture Compound 5 (CC 5, derived from dabigatran) for binding to recombinant NQO2 showed that Compound 2 displaces CC 5 with much higher affinity than dabigatran itself. The concentration causing half-maximal competition of NQO2 binding was 1.5 µM for Compound 2, compared to 11 µM for dabigatran.[1]

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4. Binding Mode Analysis (Molecular Modeling): Molecular docking predicted that the ethyl ester group of Compound 2 forms favorable hydrophobic van der Waals interactions with residues Ile128 and Met154 in the NQO2 binding pocket, explaining its higher affinity compared to the carboxylic acid of dabigatran (1). This increased lipophilic interaction surface also correlated well with the observed affinity ratio between Compounds 1 and 2. Ligand efficiency (LE) and fit quality (FQ) calculations for thrombin and NQO2 binding were similar for both compounds (FQ ~0.6 for thrombin, ~0.4 for NQO2)[1].

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For NQO2, dabigatran (ethyl hydrochloride) has an IC50 of 0.8 μM and a Ki of 0.9 μM. Dabigatran's (ethyl hydrochloride) ethyl ester group greatly increases the interaction surface, particularly with hydrophobic amino acids like Met 154 and Ile 128. Compared to dabigatran, dabigatran ethyl ester exhibits a greater affinity for thrombin and NQO2 [1]. As the prodrug dabigatran etexilate, dabigatran is an oral thrombin inhibitor that is highly selective, reversible, and potent [2].

Human thrombin can be selectively and reversibly bound by dabigatran (Ki=4.5 nM), which has a potent and long-lasting anticoagulant action [3].

1. NQO2 Functional Inhibition Assay: The functional inhibition of NQO2 by Dabigatran ethyl ester/Compound 2 was determined using the NADH-dependent metabolism of mitomycin C as the enzymatic reaction. Recombinant human NQO2 (0.5 µM) was incubated with mitomycin C (50 µM) and varying concentrations of Compound 2 in 100 mM potassium phosphate buffer (pH 5.8) at room temperature for 5 minutes. The reaction was initiated by adding increasing concentrations of NADH (the co-substrate). The enzymatic activity (consumption of NADH) was monitored photometrically by measuring the decrease in absorbance at 340 nm over 30 minutes at room temperature. Michaelis-Menten kinetics analysis (using Sigma Plot 10) of the data obtained at different inhibitor concentrations was used to determine the K_i and IC_50 values for Compound 2's competitive inhibition of NQO2.[1]

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2. Thrombin Functional Inhibition Assay: The inhibitory potency of Compound 2 against human α-thrombin was assessed using a fluorogenic substrate assay. Boc-Val-Pro-Arg-AMC substrate (15 µM) in assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) was supplemented with varying concentrations of Compound 2 (serial dilutions from 2.5 mM to 75 nM in DMSO). Thrombin was added to a final concentration of 5.5 ng/mL and the reaction incubated for 2 hours at room temperature. The enzymatic cleavage of the substrate, releasing the fluorescent AMC moiety, was measured on a plate reader (excitation 355 nm, emission 460 nm). Dose-response curves were generated to calculate the IC_50 value [1].

1. Specific Binding to Endogenous NQO2: Western Blot analysis confirmed that Compound 2/Dabigatran ethyl ester effectively competes with Capture Compound 5 (CC 5) for binding to endogenous NQO2 protein within whole cell lysates (HepG2 cells) and lysates derived from human liver microsomes. This demonstrates the specific interaction occurs in complex cellular environments, not just with purified protein.[1]

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2. Cross-Competition for NQO2 Binding Pocket: Capture experiments using recombinant NQO2 and visualized by silver-stained SDS-PAGE demonstrated that Compound 2 competes effectively for the same binding site on NQO2 as dabigatran (1) and the known NQO2 inhibitor imatinib (4). Pre-incubation with Compound 2 significantly reduced the capture of NQO2 by CC 5 [1].

[1]. Dabigatran and dabigatran ethyl ester: potent inhibitors of ribosyldihydronicotinamide dehydrogenase (NQO2). J Med Chem. 2012 Apr 26;55(8):3934-44.

[2]. Dabigatran: an oral novel potent reversible nonpeptide inhibitor of thrombin. Arterioscler Thromb Vasc Biol. 2010 Oct;30(10):1885-9.

[3]. Structure-based design of novel potent nonpeptide thrombin inhibitors. J Med Chem. 2002 Apr 25;45(9):1757-66.

Recent studies have revealed that compounds believed to be highly selective frequently address multiple target proteins. We investigated the protein interaction profile of the widely prescribed thrombin inhibitor dabigatran (1), resulting in the identification and subsequent characterization of an additional target enzyme. Our findings are based on an unbiased functional proteomics approach called capture compound mass spectrometry (CCMS) and were confirmed by independent biological assays. 1 was shown to specifically bind ribosyldihydronicotinamide dehydrogenase (NQO2), a detoxification oxidoreductase. Molecular dockings predicted and biological experiments confirmed that Dabigatran ethyl ester (2) inhibits NQO2 even more effectively than the parent 1 itself. Our data show that 1 and 2 are inhibitors of NQO2, thereby revealing a possible new aspect in the mode of action of 1. We present a workflow employing chemical proteomics, molecular modeling, and functional assays by which a compound’s protein-interaction profile can be determined and used to tune the binding affinity.[1]

C27H29N7O3.HCL

536.0252

535.209

211914-50-0

Dabigatran (ethyl ester);429658-95-7

18412337

White to off-white solid powder

5.139

4

7

11

38

788

0

CCOC(=O)CCN(C1=CC=CC=N1)C(=O)C2=CC3=C(C=C2)N(C(=N3)CNC4=CC=C(C=C4)C(=N)N)C.Cl

FHWBKCGBULSVFO-UHFFFAOYSA-N

InChI=1S/C27H29N7O3.ClH/c1-3-37-25(35)13-15-34(23-6-4-5-14-30-23)27(36)19-9-12-22-21(16-19)32-24(33(22)2)17-31-20-10-7-18(8-11-20)26(28)29;/h4-12,14,16,31H,3,13,15,17H2,1-2H3,(H3,28,29);1H

ethyl 3-[[2-[(4-carbamimidoylanilino)methyl]-1-methylbenzimidazole-5-carbonyl]-pyridin-2-ylamino]propanoate;hydrochloride

Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)propanoate hydrochloride; Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo(d)imidazole-5-carboxamido)propanoate hydrochloride; 801-940-1; 211914-50-0; Dabigatran Ethyl Ester Hydrochloride; Dabigatran (ethyl ester hydrochloride); N-[[2-[[[4-(Aminoiminomethyl)phenyl]amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]-N-(2-pyridinyl)-beta-alanine ethyl ester hydrochloride; DBB8W334AN;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 50 mg/mL (~93.28 mM)
H2O : ~5 mg/mL (~9.33 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)