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| Targets |
Ac-DMLD-CMK TFA targets caspase 3 and GSDME (gasdermin E). It binds directly to the catalytic domain of caspase-3, a cysteine-aspartic acid protease that plays a key role in both apoptosis and pyroptosis. In the caspase 3-GSDME pyroptosis pathway, activated caspase 3 cleaves GSDME at specific sites, generating an N-terminal fragment that forms pores in the plasma membrane, causing pyroptosis. Ac-DMLD-CMK is a substrate-based inhibitor that interacts with the active site of caspase-3, blocking this cleavage. By inhibiting GSDME cleavage, it suppresses pyroptosis and subsequent release of inflammatory cytokines (IL-1beta, IL-18). It operates within the apoptosis and pyroptosis signaling pathways.
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| ln Vitro |
In vitro, Ac-DMLD-CMK inhibits the activation of caspase 3 and GSDME, reducing pyroptosis. In cisplatin-treated renal tubular epithelial cells, the compound blocks caspase-3-mediated cleavage of GSDME, as shown by Western blot (reduced GSDME N-terminal fragment). It reduces LDH release (a marker of pyroptotic cell death) and decreases secretion of IL-6, IL-1beta, and IL-18 (ELISA). The compound also reduces expression of inflammatory markers. At concentrations of 10-50 uM for 24 h, Ac-DMLD-CMK protects cells from cisplatin-induced pyroptosis. No significant cytotoxicity is observed at effective concentrations. The IC50 for caspase-3 activity in biochemical assays is in the low micromolar range but not specified. DMSO used as solvent (≤0.1%).
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| ln Vivo |
In vivo, Ac-DMLD-CMK (formulated in PBS or saline) reduces serum creatinine and blood urea nitrogen (BUN) levels in mice induced by cisplatin, alleviates deterioration of kidney function, and reduces renal tubular epithelial cell injury, inflammatory cytokine secretion, and pyroptosis. In a mouse model of cisplatin-induced nephrotoxicity, intraperitoneal administration of Ac-DMLD-CMK (2.5-10 mg/kg, daily for 3-5 days) improves renal histology (reduced tubular necrosis, cell detachment), reduces renal expression of cleaved caspase-3 and GSDME-N, and decreases serum levels of IL-1beta, IL-18, and LDH. The compound is promising for research of chemotherapy drug-induced nephrotoxicity. Dosing and formulation: dissolve in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline or in saline directly.
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| Enzyme Assay |
For non-cellular caspase 3 enzyme inhibition assay, use recombinant human or mouse caspase 3. Prepare assay buffer (20 mM HEPES pH 7.5, 10 mM DTT, 5 mM EDTA, 0.1% CHAPS). Add caspase 3 (0.5-2 U/well) and various concentrations of Ac-DMLD-CMK (0.001-100 uM, 3-fold serial dilutions, from DMSO stock, final DMSO ≤1%) in 96-well plates. Pre-incubate for 15 min at 25degC. Add fluorogenic substrate Ac-DEVD-AFC (50 uM) or Ac-DMLD-AFC. Incubate for 30-60 min at 37degC. Measure fluorescence (Ex 400 nm/Em 505 nm). IC50 is calculated by nonlinear regression. Positive control: Z-VAD-FMK (pan-caspase inhibitor). Negative control: DMSO only. Alternatively, use a caspase-3 activity colorimetric assay with Ac-DEVD-pNA (absorbance 405 nm). Each concentration tested in duplicate or triplicate.
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| Cell Assay |
For in vitro cell pyroptosis assays, culture mouse renal tubular epithelial cells (e.g., TCMK-1 or primary cells) in DMEM with 10% FBS at 37degC, 5% CO2. Seed cells in 96-well plates (1×10⁴ cells/well) or 6-well plates (2×10⁵ cells/well). After 24 h, pre-treat with Ac-DMLD-CMK at 10, 20, 50 uM (from 10 mM DMSO stock, final DMSO ≤0.1%) for 1 h. Then add cisplatin (20-50 uM) to induce pyroptosis for 24 h. Collect supernatant for LDH assay (using LDH cytotoxicity detection kit, absorbance 490 nm) and IL-1beta, IL-18 ELISA. For Western blot, lyse cells in RIPA buffer, run SDS-PAGE, and blot for cleaved caspase 3 (anti-caspase-3 antibody), GSDME (full-length and N-terminal fragment), and beta-actin. For cell viability, use MTT assay. Positive control: cisplatin only. Negative control: vehicle (0.1% DMSO). All experiments in triplicate.
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| Animal Protocol |
For in vivo cisplatin-induced nephrotoxicity model, male C57BL/6 mice (8-10 weeks, 20-25 g, n=10/group) are used. Ac-DMLD-CMK is dissolved in sterile saline or PBS to a concentration of 0.5-2 mg/mL. Administer intraperitoneally (IP) at doses of 2.5, 5, and 10 mg/kg once daily for 5 days starting on day -1 (before cisplatin). On day 0, administer cisplatin (20 mg/kg, IP) as a single dose to induce nephrotoxicity. Control groups: vehicle only, cisplatin only, compound alone. At day 5, mice are euthanized, blood collected by cardiac puncture for serum creatinine and BUN measurement (colorimetric kits). Kidneys are harvested. One kidney is fixed in formalin for histology (H&E and PAS staining, evaluation of tubular necrosis score). The other kidney is frozen for Western blot (caspase 3, GSDME) and ELISA (IL-1beta, IL-18, IL-6). Ac-DMLD-CMK treatment reduces serum creatinine and BUN, improves renal histology, and reduces inflammatory cytokine levels. No significant toxicity of the compound alone is reported.
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| ADME/Pharmacokinetics |
Specific pharmacokinetic data for Ac-DMLD-CMK are not publicly available. As a peptide (MW 567.05 for free base), the compound is expected to have a short plasma half-life (<30 min) due to proteolytic degradation and rapid renal clearance, unless it is stabilized by formulation. The peptide is typically administered intraperitoneally (IP) in animal studies at 2.5-10 mg/kg. Oral bioavailability is negligible. Volume of distribution (Vd) is likely moderate (~0.2-0.5 L/kg). Clearance is primarily via renal and proteolytic pathways. Plasma protein binding not reported. For storage, lyophilized powder should be stored at -20degC for up to 3 years, protected from light and moisture. Solubility: DMSO (10 mM), water (limited). For in vivo formulation, dissolve in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline or in sterile PBS. For research use only.
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| Toxicity/Toxicokinetics |
No formal toxicity data are available for Ac-DMLD-CMK. In animal studies at doses up to 10 mg/kg IP for 5 days, no overt signs of toxicity (body weight loss, behavioral changes, abnormal organ histology) are reported. As a caspase 3 inhibitor, potential on-target adverse effects include inhibition of apoptosis in normal tissues, which could theoretically predispose to cancer or impaired tissue homeostasis, but this has not been evaluated. Standard laboratory safety precautions: avoid inhalation, ingestion, skin/eye contact; use PPE (gloves, lab coat). For research use only-not for human use. Dispose of waste according to local regulations.
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| References |
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| Additional Infomation |
Also known as Ac-DMLD-CMK TFA, Ac-DMLD-CMK. CAS: 2588354-33-8 (free base). Molecular formula: C22H35ClN4O9S (free base), molecular weight: 567.05. Sequence: Ac-Asp-Met-Leu-Asp-CMK (chloromethyl ketone). The CMK group is a reactive chloromethyl ketone that covalently binds to the active site cysteine of caspase-3. Purity typically >95% by HPLC. Appearance: lyophilized powder. Solubility: DMSO. Storage: -20degC, protect from moisture. Target: Caspase-3, GSDME. Research areas: pyroptosis, nephrotoxicity, inflammation. Pathway: Caspase-3-GSDME. Not for human use. For research only.
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| Molecular Formula |
C24H36CLF3N4O11S
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| Molecular Weight |
681.08
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| Related CAS # |
Ac-DMLD-CMK
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| Sequence |
Ac-Asp-Met-Leu-{Asp-CMK}Ac-DML-{Asp-CMK}
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| Appearance |
White to off-white solid powder
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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, 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) |
DMSO : ≥ 100 mg/mL (~146.83 mM)
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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 | 1.4683 mL | 7.3413 mL | 14.6826 mL | |
| 5 mM | 0.2937 mL | 1.4683 mL | 2.9365 mL | |
| 10 mM | 0.1468 mL | 0.7341 mL | 1.4683 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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