| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
| Targets |
Ac-KQKLR-AMC TFA targets the active site of Cathepsin S, a cysteine protease belonging to the papain family. Cathepsin S is primarily located in lysosomes and is highly expressed in antigen-presenting cells (dendritic cells, macrophages, B cells). The peptide sequence KQKLR (Lys-Gln-Lys-Leu-Arg) is specifically recognized and cleaved by Cathepsin S between the leucine and arginine residues (Leu-Arg bond). The acetyl group at the N-terminus enhances stability against exopeptidases. Cathepsin S has optimal activity at acidic pH (5.0-6.0), matching the lysosomal environment. The substrate can also be used to measure activity of other cysteine proteases (e.g., Cathepsin L, Cathepsin K) but with lower efficiency; it is considered a selective Cathepsin S substrate.
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| ln Vitro |
In vitro, Ac-KQKLR-AMC TFA is used to measure Cathepsin S activity in enzyme kinetic and inhibitor screening assays. Recombinant human Cathepsin S (activated with DTT) cleaves the substrate with a Km of 5-20 microM and kcat of 0.5-2 s-¹. The increase in fluorescence (Ex/Em 354/442 nm) is linear with time and enzyme concentration. The substrate is stable in acidic buffers and does not auto-hydrolyze. In the presence of potent Cathepsin S inhibitors (IC50 values as low as sub-nanomolar), the fluorescence signal is reduced in a concentration-dependent manner. The substrate is also used to measure Cathepsin S activity in cell lysates or tissue homogenates. It has been used in studies of inflammatory diseases, cancer invasion, and pain, where Cathepsin S activity is upregulated. No direct cytotoxicity of the substrate itself has been observed.
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| ln Vivo |
In vivo, Ac-KQKLR-AMC TFA is not typically administered in animals as a drug; it is a laboratory reagent for ex vivo activity assays. However, Cathepsin S activity measured using this substrate has been correlated with disease progression in mouse models. In a mouse model of chronic pain (spinal nerve ligation), Cathepsin S activity in spinal cord extracts measured with Ac-KQKLR-AMC TFA is elevated, and treatment with a Cathepsin S inhibitor reduces pain behavior. In a mouse model of atherosclerosis (ApoE-/- mice), Cathepsin S activity in aortic tissue is increased, correlating with plaque instability. The substrate can also be used in in vivo imaging after modification (not with AMC, which is not suitable for deep tissue imaging). For ex vivo use, after animal sacrifice, tissues are homogenized and assayed with the substrate. Thus, the compound serves as a tool to evaluate target engagement of Cathepsin S inhibitors in vivo.
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| Enzyme Assay |
The cell-free enzyme activity assay for Ac-KQKLR-AMC TFA is performed in 96-well or 384-well black plates. Recombinant human Cathepsin S (active site titrated) is activated in activation buffer: 50 mM sodium acetate (pH 5.5), 150 mM NaCl, 2.5 mM DTT, 2 mM EDTA. Pre-incubate the enzyme in activation buffer for 15-30 minutes at room temperature. The substrate Ac-KQKLR-AMC TFA is dissolved in DMSO (10 mM stock) and diluted in assay buffer (100 mM MES, pH 5.5, 2 mM DTT, 0.01% Brij-35) to working concentrations (0.5-200 microM). In a 96-well plate, mix 50 microL of activated Cathepsin S (final concentration 0.1-10 nM) and 40 microL assay buffer. Add 10 microL of substrate (final concentration 10-20 microM). For inhibition assays, add 5 microL of inhibitor (serial dilutions) and pre-incubate for 10-15 minutes before adding substrate. Final assay volume is 100 microL. Incubate at 37degC for 30-60 minutes. Fluorescence is measured at Ex/Em 354/442 nm using a microplate reader. The increase in relative fluorescence units (RFU) per minute is proportional to enzyme activity. The Km is determined by fitting the initial rate vs substrate concentration to the Michaelis-Menten equation. For inhibitor IC50 determination, the percent inhibition is calculated and fitted to a four-parameter logistic curve. Control wells: no enzyme (background) and no inhibitor (full activity). Z' factors >0.7 indicate assay suitability for HTS. The assay is robust and can be miniaturized to 384-well format.
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| Cell Assay |
For cellular Cathepsin S activity assays, cells are seeded in 6-well or 96-well plates. Human cell lines (e.g., THP-1 macrophages, A549, HeLa) or primary cells (human dendritic cells, murine macrophages) are used. For macrophages, THP-1 cells are differentiated with PMA (100 ng/mL for 48 hours). For Cathepsin S upregulation, cells can be stimulated with IFN-gamma (50 ng/mL) or LPS (100 ng/mL) for 24-48 hours. After treatment with inhibitors (e.g., Cathepsin S inhibitors, 0.1-1000 nM, 2-6 hours), cells are washed with cold PBS and lysed in extraction buffer (0.1 M sodium acetate pH 5.5, 150 mM NaCl, 0.1% Triton X-100, 2.5 mM DTT, protease inhibitors). Lysates are centrifuged (10,000g, 10 min, 4degC) to remove debris. Protein concentration is determined by BCA assay. In a 96-well black plate, add 50 microg (20-50 microL) of cell lysate, add assay buffer to 90 microL, then add 10 microL of Ac-KQKLR-AMC TFA substrate (final concentration 10-20 microM). Incubate at 37degC for 1-4 hours. Fluorescence is measured at Ex/Em 354/442 nm. Activity is normalized to protein concentration. To confirm specificity, parallel wells are pre-incubated with selective Cathepsin S inhibitor (e.g., 10 microM LHVS) for 10 minutes before adding substrate; residual activity is non-specific. Cathepsin S activity in lysates from diseased tissues (e.g., atherosclerotic aortas, arthritic joints) is similarly measured. This cellular/tissue assay is widely used for pharmacodynamic (PD) studies to assess target engagement of Cathepsin S inhibitors in preclinical models.
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| Animal Protocol |
For ex vivo tissue activity measurement from animal models, mice are treated with Cathepsin S inhibitors or vehicle (e.g., oral gavage, 1-30 mg/kg, daily for 7 days). Two hours after the last dose, mice are euthanized. Tissues of interest (e.g., spleen, spinal cord, aorta, skin) are harvested, snap-frozen in liquid nitrogen, and stored at -80degC. For homogenization, tissues are weighed and homogenized in ice-cold lysis buffer (0.1 M sodium acetate pH 5.5, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, 5 mM DTT, protease inhibitor cocktail) using a bead mill or dounce homogenizer. Homogenates are centrifuged at 10,000g for 10 minutes at 4degC. Supernatant protein concentration is determined. Then, 50-100 microg of protein lysate is incubated with Ac-KQKLR-AMC TFA substrate (10-20 microM) in a 96-well plate at 37degC for 2-4 hours. Fluorescence is read. Cathepsin S activity in treated groups is compared to vehicle group, and the percent inhibition of enzyme activity in ex vivo tissues is calculated. This ex vivo protocol is crucial for assessing whether an inhibitor achieves sufficient tissue exposure and target engagement.
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| ADME/Pharmacokinetics |
Ac-KQKLR-AMC TFA has MW 931.05 (for the peptide TFA salt, exact MW may vary). The peptide is highly soluble in DMSO (10-20 mM) and in water (1-2 mg/mL). The TFA salt stabilizes the peptide and improves water solubility. Storage: lyophilized powder at -20degC, desiccated, protected from light. Reconstituted solutions in DMSO (10 mM) are stable for 3-6 months at -80degC; avoid repeated freeze-thaw cycles. The AMC fluorophore is stable in the dark; solutions should be stored in amber vials or foil-wrapped. The excitation maximum is 354 nm (UV range); emission 442 nm (blue). The product is for research use only, not for clinical diagnostic purposes. The substrate is stable in acidic buffers (pH 4.0-6.0) but may hydrolyze at neutral or basic pH (>7.5) due to AMC instability. Always include control wells without enzyme to subtract background fluorescence. The Km for Cathepsin S is 5-20 microM; use 10-20 microM for routine assays. For kinetic experiments, titrate substrate concentration (0.5-200 microM).
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| Toxicity/Toxicokinetics |
Ac-KQKLR-AMC TFA has CAS number 2237216-84-9. The peptide is Acetyl-Lysine-Glutamine-Lysine-Leucine-Arginine-AMC. The molecular formula is C42H64N12O11 (free peptide) plus TFA and H2O. The TFA salt form is often used commercially. The compound is also referred to as Ac-KQKLR-AMC, Cathepsin S substrate (fluorogenic), and Cathepsin S Substrate, fluorogenic. It is a sensitive and selective substrate for Cathepsin S. Research applications: screening of Cathepsin S inhibitors for treatment of autoimmune diseases (rheumatoid arthritis, psoriasis, multiple sclerosis), pain, cancer metastasis, and atherosclerosis. The substrate can be used for continuous monitoring of enzyme activity (endpoint or kinetic readouts). It is not cell-permeable; for live-cell assays, cell-permeable AMC derivatives (e.g., cell-permeable fluorogenic substrates) are required. However, the substrate can be used in cell lysates as described. Purity is typically >95% by HPLC. The compound should be handled with standard lab safety; no unusual toxicity reported.
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| References |
| Molecular Formula |
C47H69F9N12O15
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| Molecular Weight |
1213.11
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| Exact Mass |
1212.486
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| CAS # |
2237216-84-9
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| Related CAS # |
Ac-KQKLR-AMC; 1135686-31-5
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| PubChem CID |
178201541
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| Sequence |
Ac-Lys-Gln-Lys-Leu-Arg-{AMC}Ac-KQKLR-{AMC}
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
14
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| Rotatable Bond Count |
28
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| Heavy Atom Count |
83
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| Complexity |
1690
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CC1=CC(=O)OC2=C1C=CC(=C2)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)NC(=O)C.C(=O)(C(F)(F)F)O.C(=O)(C(F)(F)F)O.C(=O)(C(F)(F)F)O
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| InChi Key |
GPFYZPWDELTNTI-DHBALVKUSA-N
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| InChi Code |
InChI=1S/C41H66N12O9.3C2HF3O2/c1-23(2)20-32(40(61)51-30(12-9-19-47-41(45)46)36(57)49-26-13-14-27-24(3)21-35(56)62-33(27)22-26)53-38(59)29(11-6-8-18-43)50-39(60)31(15-16-34(44)55)52-37(58)28(48-25(4)54)10-5-7-17-42;3*3-2(4,5)1(6)7/h13-14,21-23,28-32H,5-12,15-20,42-43H2,1-4H3,(H2,44,55)(H,48,54)(H,49,57)(H,50,60)(H,51,61)(H,52,58)(H,53,59)(H4,45,46,47);3*(H,6,7)/t28-,29-,30-,31-,32-;;;/m0.../s1
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| Chemical Name |
(2S)-2-[[(2S)-2-acetamido-6-aminohexanoyl]amino]-N-[(2S)-6-amino-1-[[(2S)-1-[[(2S)-5-(diaminomethylideneamino)-1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]pentanediamide;tris(2,2,2-trifluoroacetic acid)
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| Synonyms |
Cathepsin S substrate TFA
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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) |
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 | 0.8243 mL | 4.1216 mL | 8.2433 mL | |
| 5 mM | 0.1649 mL | 0.8243 mL | 1.6487 mL | |
| 10 mM | 0.0824 mL | 0.4122 mL | 0.8243 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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