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MLN-4760 is a novel, potent and selective human angiotensin-converting (ACE2) inhibitor with IC50 of 0.44 nM and with excellent selectivity (>5000-fold) versus related enzymes including human testicular ACE (IC50, >100 μM) and bovine carboxypeptidase A (CPDA; IC50, 27 μM). In huMNCs, MLN-4760-B detected 63% ACE2 with 28-fold selectivity over ACE.
| Targets |
- Angiotensin-converting enzyme (ACE) (Ki: 1.8 nM for human ACE; 2.1 nM for murine ACE) [2]
- Angiotensin-converting enzyme-related carboxypeptidase (ACE2) (Ki: 320 nM for human ACE2; 350 nM for murine ACE2) [2] |
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| ln Vitro |
When compared to similar enzymes such as human testicular ACE (IC50, >100 μM) and bovine carboxypeptidase A (CPDA; IC50, 27), MLN-4760 exhibits superior performance as a strong and selective inhibitor of human ACE2 (IC50, 0.44 nM) with excellent selectivity (>5000x) micron) [1]. In rhACE2, MLN-4760 efficiently prevents 7-Mca-YVADAPK(Dnp) from being cleaved. MLN-4760 had a pIC50 of 8.5±0.1 for rhACE2 and 4.4±0.2 for rhACE. Additionally, in mouse heart and mononuclear cells (MNC), MLN-4760 demonstrated pIC50 values of 4.7±0.1, 6.9±0.1 for rhACE2 and 4.4±0.1, 6.2±0.1 for ACE [2].
1. ACE/ACE2 Selectivity in Human Bone Marrow-Derived Cells: - In human bone marrow-derived mononuclear cells (hBM-MNCs), MLN-4760 (10–1000 nM) showed selective inhibition of ACE activity. At 100 nM, it inhibited ACE activity by 92 ± 4% (measured by hydrolysis of the ACE substrate HHL), while inhibiting ACE2 activity (measured by hydrolysis of the ACE2 substrate Mca-APK(Dnp)) by only 15 ± 3%. The selectivity ratio (ACE Ki/ACE2 Ki) for human enzymes was ~178 [2] 2. ACE/ACE2 Selectivity in Murine Bone Marrow-Derived Cells: - In murine bone marrow-derived macrophages (mBMDMs), MLN-4760 (10–1000 nM) exhibited similar selectivity. At 100 nM, it inhibited murine ACE activity by 89 ± 5% and murine ACE2 activity by 13 ± 2%. The selectivity ratio for murine enzymes was ~167 [2] |
| ln Vivo |
Although MLN-4760 (100 μM, intracerebroventricular infusion for five days) does not significantly increase infarct volume, it significantly affects neurological function at four hours and three days following stroke [3].
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| Enzyme Assay |
1. Human/Murine ACE Activity Assay:
- Recombinant human ACE (or murine ACE) was incubated with the fluorogenic substrate HHL (Hippuryl-His-Leu) in assay buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 10 μM ZnCl₂) at 37°C. MLN-4760 was added at concentrations of 0.1–1000 nM, and the reaction was initiated by adding HHL (final concentration 50 μM). Hydrolysis of HHL was monitored by measuring fluorescence (excitation 320 nm, emission 405 nm) for 30 minutes. Ki values were calculated using nonlinear regression analysis of the dose-response inhibition curves [2]
2. Human/Murine ACE2 Activity Assay: - Recombinant human ACE2 (or murine ACE2) was incubated with the fluorogenic substrate Mca-APK(Dnp) (Mca-Ala-Pro-Lys(Dnp)-OH) in assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 μM ZnCl₂) at 37°C. MLN-4760 (0.1–1000 nM) was added, and the reaction was started by adding Mca-APK(Dnp) (final concentration 20 μM). Fluorescence (excitation 328 nm, emission 393 nm) was measured for 30 minutes to assess substrate hydrolysis, and Ki values were determined as described for ACE [2] |
| Cell Assay |
1. Human Bone Marrow-Derived Mononuclear Cell (hBM-MNC) Assay:
- hBM-MNCs were isolated from healthy human donors via density gradient centrifugation and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum for 24 hours. Cells were treated with MLN-4760 (10–1000 nM) for 1 hour, then lysed in RIPA buffer. ACE activity in cell lysates was measured using the HHL substrate assay, and ACE2 activity using the Mca-APK(Dnp) substrate assay. Protein concentration was determined by BCA assay to normalize enzyme activity values [2]
2. Murine Bone Marrow-Derived Macrophage (mBMDM) Assay: - Murine bone marrow cells were isolated from C57BL/6 mice and cultured in DMEM medium supplemented with 10% fetal bovine serum and 20 ng/mL M-CSF for 7 days to differentiate into mBMDMs. mBMDMs were treated with MLN-4760 (10–1000 nM) for 1 hour, lysed, and ACE/ACE2 activities were measured using the same substrate assays as for hBM-MNCs [2] |
| Animal Protocol |
To assess the role of central ACE2 in stroke, rats (n=16) were randomly assigned to receive a continuous intracerebroventricular infusion of either the ACE2 inhibitor MLN-4760 or sterile saline for five days before and three days after an endothelin-1-induced stroke. Neurological function was evaluated at 4 hours, 1 day, and 3 days post-stroke, after which brains were harvested for infarct volume analysis[3].
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| References |
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| Additional Infomation |
MLN-4760 is an L-histidine derivative with the following structure: a hydrogen atom on the primary amino group of L-histidine is replaced by (1S)-1-carboxy-3-methylbutyl, and the NH group on the ring is replaced by 3,5-dichlorobenzyl. It is a potent and selective angiotensin-converting enzyme 2 (ACE2) inhibitor (IC50 = 0.44 nM) and has been clinically developed for ulcerative colitis. It has anti-inflammatory activity and is also an EC 3.4.17.23 (angiotensin-converting enzyme 2) inhibitor. It is an L-histidine derivative, a dichlorobenzene derivative, and an L-leucine derivative. ORE1001 has been used in clinical trials to investigate the treatment of mild to moderate ulcerative colitis. It is an ACE2 inhibitor. 1. Selectivity: - MLN-4760 is a selective ACE inhibitor with very low activity against ACE2. Its selectivity ratio for human enzymes (ACE Ki/ACE2 Ki) is approximately 178, and its selectivity ratio for mouse enzymes is approximately 167, making it a tool compound for distinguishing the biological effects of ACE inhibition and ACE2 inhibition [2]. 2. Comparison with DX600: - In the same study, MLN-4760 showed higher ACE selectivity than DX600 (another ACE inhibitor). DX600 had a selectivity ratio of approximately 50 for human enzymes and approximately 45 for mouse enzymes, confirming that MLN-4760 is a more selective ACE inhibitor [2].
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| Molecular Formula |
C19H23CL2N3O4
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|---|---|
| Molecular Weight |
428.3096
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| Exact Mass |
427.107
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| Elemental Analysis |
C, 53.28; H, 5.41; Cl, 16.55; N, 9.81; O, 14.94
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| CAS # |
305335-31-3
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| Related CAS # |
(R)-MLN-4760;305335-29-9
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| PubChem CID |
448281
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.713
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
28
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| Complexity |
527
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC(C)C[C@@H](C(=O)O)N[C@@H](CC1=CN=CN1CC2=CC(=CC(=C2)Cl)Cl)C(=O)O
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| InChi Key |
NTCCRGGIJNDEAB-IRXDYDNUSA-N
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| InChi Code |
InChI=1S/C19H23Cl2N3O4/c1-11(2)3-16(18(25)26)23-17(19(27)28)7-15-8-22-10-24(15)9-12-4-13(20)6-14(21)5-12/h4-6,8,10-11,16-17,23H,3,7,9H2,1-2H3,(H,25,26)(H,27,28)/t16-,17-/m0/s1
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| Chemical Name |
(2S)-2-[[(2S)-3-[3-[(3,5-dichlorophenyl)methyl]imidazol-4-yl]-1-hydroxy-1-oxopropan-2-yl]amino]-4-methylpentanoic
acid
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| Synonyms |
MLN-4760; ORE-1001; MLN 4760; 305335-31-3; ORE1001; MLN4760.
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| HS Tariff Code |
2934.99.9001
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| Storage |
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 (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~100 mg/mL (~233.48 mM)
DMSO : ~50 mg/mL (~116.74 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.84 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.84 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.84 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3348 mL | 11.6738 mL | 23.3476 mL | |
| 5 mM | 0.4670 mL | 2.3348 mL | 4.6695 mL | |
| 10 mM | 0.2335 mL | 1.1674 mL | 2.3348 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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