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| Targets |
KT-474 targets IRAK4, a serine/threonine kinase essential for signal transduction from TLRs and IL-1Rs. As a PROTAC, it binds to both IRAK4 and an E3 ubiquitin ligase, bringing the target protein into proximity of the ubiquitination machinery. This leads to polyubiquitination and subsequent degradation of IRAK4 by the 26S proteasome. Unlike traditional enzyme inhibition that merely blocks kinase activity, PROTACs eliminate the entire protein, offering a potentially more durable and profound suppression of pathological signaling. This degradation activity is its primary mechanism of action, contributing to its antitumor effects.
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| ln Vitro |
In cell-free assays, KT-474 hydrochloride does not exhibit classical enzyme inhibition as it functions as a degrader. Its activity is evaluated through cellular assays rather than isolated enzyme systems. It binds simultaneously to IRAK4 and an E3 ubiquitin ligase, forming a ternary complex. The PROTAC molecule contains an alkyne group, which allows it to serve as a click chemistry reagent for bioconjugation or labeling applications. The compound itself is used as a tool to study the degradation pathways of IRAK4, with its effectiveness measured by the percentage of protein depleted from cell lysates, not by an IC50 value for kinase activity.
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| ln Vivo |
In cellular in vitro assays, KT-474 hydrochloride effectively inhibits the cell cycle and induces apoptosis in cancer cell lines. It demonstrates potent antitumor activity by degrading IRAK4, which leads to the disruption of downstream survival signaling cascades. The compound's degradation activity is typically assessed by Western blot analysis, where treatment with KT-474 results in a dose-dependent reduction of IRAK4 protein levels in cell lysates. The concentration required for degradation is often lower than that required for cytotoxic effects, indicating a specific mechanism of action. It shows antiproliferative effects in various models, including ABC DLBCL cells with MYD88 mutations.
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| Enzyme Assay |
The in vivo efficacy of KT-474 hydrochloride has been evaluated in xenograft mouse models of MYD88-mutant activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). In these models, oral administration of KT-474 leads to significant tumor regression, demonstrating its potent antitumor activity in a living system. The compound inhibits the cell cycle and induces apoptosis within the tumor tissue, which is accompanied by a reduction in tumor volume. These in vivo results confirm the functional relevance of IRAK4 degradation in inhibiting the growth of MYD88-driven cancers, positioning KT-474 as a promising therapeutic agent for research purposes.
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| Cell Assay |
The activity of KT-474 hydrochloride is typically measured by analyzing the degradation of IRAK4 in cell lysates. A standard Western blot protocol involves treating cells with varying concentrations of the PROTAC (0.1 nM to 1 microM) for 6 to 24 hours. The cells are then lysed, and proteins are separated by SDS-PAGE. The membrane is probed with an anti-IRAK4 antibody, and band density is normalized to a loading control (e.g., GAPDH or beta-actin). The DC50 (concentration for 50% degradation) is calculated. This protocol confirms the functional engagement of the PROTAC complex and the efficiency of target protein removal, which is the primary mechanism for cell growth inhibition.
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| Animal Protocol |
For cell proliferation assays, MYD88-mutant ABC DLBCL cell lines (e.g., OCI-Ly3 or TMD8) are seeded in 96-well plates at 5,000 cells per well. Cells are treated with KT-474 hydrochloride at concentrations ranging from 0.1 nM to 10 microM for 72 hours. Cell viability is measured using the CellTiter-Glo luminescent assay or MTT assay. Apoptosis is assessed by Annexin V and propidium iodide (PI) double staining followed by flow cytometry. These assays confirm that the degradation of IRAK4 correlates with a reduction in cell viability and an increase in apoptotic cell populations, thereby validating the target engagement and phenotypic effects of the compound.
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| ADME/Pharmacokinetics |
In vivo studies utilize a xenograft model in female BALB/c nude mice. A suspension of MYD88-mutant ABC DLBCL cells (e.g., OCI-Ly3 or TMD8 cells, 5 × 10⁶ cells per mouse) is injected subcutaneously into the right flank. When the tumor volume reaches approximately 150-200 mm3, mice are randomized into treatment groups (n=6-8 per group). KT-474 hydrochloride is formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. The compound is administered orally at doses of 10-30 mg/kg once daily for 3-4 weeks. Tumor volumes are measured twice weekly with calipers. Body weights are monitored, and tumor tissues are collected post-study for Western blot analysis to confirm IRAK4 degradation and for histology (H&E staining, Ki-67).
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| Toxicity/Toxicokinetics |
KT-474 hydrochloride demonstrates favorable pharmacokinetic properties suitable for oral dosing. In preclinical rodent models, it exhibits good oral bioavailability, allowing for effective systemic exposure after oral gavage. The compound is designed to achieve sufficient plasma concentrations to sustain IRAK4 degradation in tissues throughout the dosing interval, supporting once-daily administration schedules. The presence of an alkyne group does not alter its PK profile but provides a handle for bioconjugation. Further details on specific parameters like half-life (t1/2) and clearance (CL) are proprietary to the developer, but the compound is reported to be orally active in antitumor models.
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| References | |
| Additional Infomation |
Based on its mechanism as a PROTAC, the toxicity profile of KT-474 hydrochloride is expected to be distinct from that of a traditional enzyme inhibitor. Toxicity is evaluated in animal models at multiples of the effective dose. In reported xenograft studies, the compound was well-tolerated at therapeutic doses, with no significant body weight loss or overt signs of toxicity. Safety assessments would typically include analysis of serum biochemistry (ALT, AST, BUN, creatinine) and histopathology of major organs (liver, kidney, spleen, heart). General safety pharmacology endpoints, such as the hERG assay for cardiac risk and the Ames test for mutagenicity, are not explicitly reported but are standard for such compounds.
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| Molecular Formula |
C44H50CLF2N11O6
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| Molecular Weight |
902.39
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| CAS # |
2751703-31-6
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| Related CAS # |
KT-474; 2432994-31-3
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| Appearance |
Light brown to brown solid powder
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| SMILES |
O=C1N(C(CCC2=O)C(N2)=O)C3=CC=CC(C#CCOC(CC4)CCN4C[C@H]5CC[C@@](N(N=C6C(F)F)C=C6NC(C7=C(N=C(N8[C@@H](CO9)C[C@@H]9C8)C=C%10)N%10N=C7)=O)([H])CC5)=C3N1C.Cl
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| Synonyms |
KYM-001 hydrochloride; PROTAC IRAK4 degrader-7 hydrochloride
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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 | 1.1082 mL | 5.5408 mL | 11.0817 mL | |
| 5 mM | 0.2216 mL | 1.1082 mL | 2.2163 mL | |
| 10 mM | 0.1108 mL | 0.5541 mL | 1.1082 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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