| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
MLKL-IN-1 (Cpd 5): Human MLKL N-terminal executioner domain (residues 2−154), dissociation constant (\(K_{D}=50 ± 4 μM\)); the enantiomer of Cpd 5 has a \(K_{D}=132 ± 7 μM\); no significant change in binding affinity (\(K_{D}=73 ± 12 μM\)) when MLKL is covalently modified by BI-8925 at Cys86 [1]
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|---|---|
| ln Vitro |
1. MLKL-IN-1 (compound 5, Cpd 5) is a reversible noncovalent binder of the human MLKL N-terminal executioner domain, with a binding affinity (\(K_{D}\)) of 50 ± 4 μM, showing a 60-fold increase compared to the initial fragment hit Cpd 1 (\(K_{D}=933 ± 433 μM\)) [1]
2. MLKL-IN-1 (compound 5, Cpd 5) competes with the detergent nonyl-maltoside (NM) for binding to the MLKL executioner domain: at NM concentrations below the critical micelle concentration (cmc), preincubation with 1 mM Cpd 5 completely inhibits the binding of monomeric NM to MLKL, as evidenced by the absence of chemical shift perturbations (CSPs) in 2D \(^{1} H,^{15} N\) correlation spectra; however, it cannot block the transient interactions between MLKL and NM micelles at NM concentrations above cmc (12 mM) [1] 3. MLKL-IN-1 (compound 5, Cpd 5) does not affect the overall fold and backbone dynamics of the MLKL executioner domain: the root-mean-square deviation (rmsd) between the MLKL/Cpd 5 complex and the unliganded MLKL is 2.3 Å, and the molecular correlation time of the complex (16 ns) is virtually identical to that of the unliganded protein [1] 4. MLKL-IN-1 (compound 5, Cpd 5) shows no binding activity to the mouse MLKL executioner domain, as no CSPs are observed in 2D \(^{1} H,^{15} N\) correlation spectra in the presence of 500 μM Cpd 5 [1] 5. MLKL-IN-1 (compound 5, Cpd 5) has poor membrane permeability with a PAMPA permeability value of 3.1 × 10⁻⁸ cm/s and does not exhibit activity in the liposome leakage assay [1] 6. The binding affinity of MLKL-IN-1 (compound 5, Cpd 5) to the MLKL executioner domain decreases in the presence of 12 mM NM, with a \(K_{D}=215 ± 21 μM\) [1] |
| Enzyme Assay |
1. NMR-based binding assay for MLKL-IN-1 (compound 5, Cpd 5): The assay uses ¹³C/¹⁵N isotope-labeled human MLKL N-terminal executioner domain (residues 2−154) at a concentration of 40 μM in 20 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl and 1 mM TCEP. The compound is titrated at concentrations of 25, 50, 125, 250, 1000, and 2500 μM. TROSY-HSQC experiments are performed with 24 complex points in the indirect dimension and 64 scans, with an interscan delay of 1.2 s. Chemical shift perturbation (CSP) is calculated using the formula \(δ = \sqrt{(δ_H - δ_{Href})^2 + (δ_N - δ_{Nref})^2} × 100\), and nonlinear curve fitting of the titration curve is performed to determine the dissociation constant (\(K_{D}\)) [1]
2. NMR costructure determination assay for MLKL-IN-1 (compound 5, Cpd 5): Triple resonance NMR experiments (HNCACB, HNccH TOCSY, HNCC TOCSY, HCCh TOCSY, HcCH TOCSY) are used to transfer backbone and side chain assignments from the MLKL/Cpd 3 complex to the MLKL/Cpd 5 complex. Distance restraints are derived from ¹⁵N-edited NOESY (H₂O buffer) and two separate ¹³C-edited NOESY spectra (D₂O buffer) for aliphatic and aromatic carbon-bound protons (mixing time 120 ms). A filtered-¹³C-edited NOESY spectrum (mixing time 200 ms) is recorded to identify intermolecular NOEs. Torsion angle restraints are calculated from chemical shifts using TALOS software, and structure calculation is performed with CYANA software. 100 structures are calculated, and the 20 structures with the best target function are selected for the NMR structure bundle [1] 3. NM competition binding assay for MLKL-IN-1 (compound 5, Cpd 5): NMR measurements are performed with the MLKL executioner domain in the absence and presence of Cpd 5 (1 mM) and NM at concentrations of 4 mM (below cmc) and 12 mM (above cmc). 2D \(^{1} H,^{15} N\) correlation spectra are recorded to compare CSPs, and filtered/edited 3D NOESY spectra are used to analyze intermolecular interactions between MLKL, NM, and Cpd 5 [1] |
| References | |
| Additional Infomation |
1. MLKL-IN-1 (compound 5, Cpd 5) is a benzimidazole-class compound with a molecular formula of \(C_{19}H_{21}N_{2}O_{3}\) and a molecular weight of 325.15 (m/z [M+H]⁺); its enantiopure form is obtained via chiral supercritical fluid chromatography (SFC), with a specific rotation \([α]_{D}^{20}+33.5^{\circ}\) (c 1.0, MeOH) [1]
2. The chemical structure of MLKL-IN-1 (compound 5, Cpd 5) contains iso-propyl, methoxy, and carboxy substituents; the carboxy group points toward the solvent and contributes to binding affinity through interactions with water molecules at the protein surface, showing a synergistic effect with other substituents [1] 3. MLKL-IN-1 (compound 5, Cpd 5) binds to the N-terminal pole of the MLKL executioner domain, a pocket opposite to the Cys86 modification site of covalent inhibitors (NSA, xanthine class); the binding pocket is mainly composed of hydrophobic residues (Leu4, Ile7, Leu42, Leu45, Leu58, Met62, Leu116) and is partially formed upon compound binding [1] 4. The binding of MLKL-IN-1 (compound 5, Cpd 5) to MLKL is independent of the modification state of Cys86, as its \(K_{D}\) does not change significantly when MLKL is covalently saturated with the xanthine-class inhibitor BI-8925 [1] 5. MLKL-IN-1 (compound 5, Cpd 5) is optimized through NMR-guided structure-based design: starting from the indole core-containing Cpd 1, the core is swapped to benzimidazole (Cpd 2) for stability, followed by introduction of iso-propyl (Cpd 3), methoxy (Cpd 4), and carboxy (Cpd 5) groups to improve binding affinity [1] |
| Molecular Formula |
C19H20N2O3
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|---|---|
| Molecular Weight |
324.4
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| CAS # |
2837019-30-2
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| PubChem CID |
156599552
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| Appearance |
Typically exists as solid at room temperature
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
24
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| Complexity |
434
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC(C)C1=CC=C(C=C1)[C@@H](C2=NC3=C(N2)C=C(C=C3)C(=O)O)OC
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| Synonyms |
MLKL-IN-1; MLKL-IN1; MLKL-IN 1; CHEMBL5086911; 2-[(~{S})-methoxy-(4-propan-2-ylphenyl)methyl]-3~{H}-benzimidazole-5-carboxylic acid; BDBM50581530; ...; 2837019-30-2;
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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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0826 mL | 15.4131 mL | 30.8261 mL | |
| 5 mM | 0.6165 mL | 3.0826 mL | 6.1652 mL | |
| 10 mM | 0.3083 mL | 1.5413 mL | 3.0826 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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