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| Targets |
DC50: 2.56 μM (Hexokinase 2, HK2 in 4T1); 0.79 μM ((Hexokinase 2, HK2 in MDA-MB-231)
PROTAC HK2 Degrader-1 targets hexokinase 2 (HK2), a key enzyme in the glycolytic pathway that is frequently overexpressed in cancer cells. HK2 couples glycolysis to mitochondrial function and plays a role in protecting cells from apoptosis. The compound consists of the HK2 inhibitor lonidamine (target-binding moiety) and the CRBN ligand thalidomide (E3 ligase-recruiting moiety). It induces CRBN-dependent ubiquitination and proteasomal degradation of HK2. |
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| ln Vitro |
PROTAC HK2 Degrader-1 has an IC50 of 34.07 μM, 5.08 μM, 31.53 μM, 6.11 μM, and 21.65 μM for suppressing the growth of 786-O, 4T1, PANC-1, HGC-27, and MCF-1 cells, respectively [1]. PROTAC HK2 Degrader-1's DC50 against HK2 is 2.56 μM (4T1) and 0.79 μM (MDA-MB-231) [1]. Time- and concentration-dependent, PROTAC HK2 Degrader-1 (0.01-200 μM, 36 h) specifically suppresses the growth of breast cancer cells and promotes the ubiquitin-mediated proteasome pathway-mediated degradation of HK2 protein [1]. To degrade the HK2 protein, PROTAC HK2 Degrader-1 forms a ternary complex through the ubiquitin-proteasome system (10 μM for 4T1, 0.5 μM for MDA-MB-231, 24 h) [1]. This phrase can be included in the general product description and utilized as a mechanism. By degrading HK2, PROTAC HK2 Degrader-1 (20 μM, 36 h) activates caspase-3, releases cytochrome C, cleaves GSDME, induces a thermal coma, and causes cells to produce harmful signals like ATP, HMGB1, CRT, etc., ultimately resulting in caused cell death. immunity to demise [1]. The overall amount of PD-L1 protein can be decreased by inducing the internalization of the protein from the cell membrane to the cytoplasm using PROTAC HK2 Degrader-1 (20 μM, 36 h) [1].
In vitro, PROTAC HK2 Degrader-1 forms a ternary complex through the ubiquitin-proteasome system to degrade hexokinase 2 (HK2) protein, inducing mitochondrial damage and triggering pyroptosis. It selectively inhibits the proliferation of breast cancer cells. The compound induces mitochondrial damage, leading to programmed cell death. The degradation of HK2 disrupts glycolysis and mitochondrial function, which are critical for cancer cell survival. |
| ln Vivo |
In the 4T1 tumor model, PROTAC HK2 Degrader-1 (50 mg/kg, intraperitoneal injection, bid, for nine times, six-week-old female BALB/c mice) suppresses tumor growth [1]. PROTAC HK2 Degrader-1 (50 mg/kg, intraperitoneal injection, daily, nine times, in six-week-old female BALB/c mice) can successfully limit the growth of breast tumors and produce a GSDME-dependent heat reaction as well as elicit a tumor immunological response. [1]. Protac HK2 Degrader-1 (Cisplatin (HY-17394) 10mg/kg, iv, C-02 50mg/kg, ip, 25 days, six-week-old female BALB/c mice) has the potential to be clinically useful since it can sensitize cisplatin while lowering its intestinal adverse effects [1].
In vivo, PROTAC HK2 Degrader-1 can effectively suppress the growth of breast tumors and reduce the colonic side effects of cisplatin. The compound may be utilized in breast cancer research to study HK2 degradation as a therapeutic strategy. By degrading HK2, it induces mitochondrial damage and pyroptosis, leading to tumor growth inhibition. Specific dosing and tumor model data are not provided. |
| Enzyme Assay |
A cell-free binding assay for PROTAC HK2 Degrader-1 is not typical. To assess the binding of the lonidamine moiety to HK2, a biochemical assay using purified HK2 protein and a fluorescent glucose analog can be performed. Alternatively, SPR can measure the binding affinity of the PROTAC to HK2. For CRBN binding, a TR-FRET-based competition assay with recombinant CRBN and a fluorescent thalidomide probe can be used.
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| Cell Assay |
Cell Cytotoxicity Assay[1]
Cell Types: 4T1, MDA-MB-231, PUMC-HUVEC-T1 Tested Concentrations: 20 μM Incubation Duration: 72 h(MTT), 48 h(CCK-8) Experimental Results: demonstrated the greatest impact on 4T1 and HGC-27 cells, with IC50 dosages of 5.08 and 6.11 μM. Selectively suppressed breast cancer cell proliferation and stimulates HK2 protein degradation. Prevented 4T1 cells to form a colony and had little influence on HUVECT-1. Western Blot Analysis[1] Cell Types: 4T1, MDAMB-231 Tested Concentrations: 20 μM; 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 150, 200 μM; 10 μM; 0.5 μM. Incubation Duration: 36 h ; 24 h Experimental Results: Degraded 71.06% of HK2 at 20 μM in 4T1 and MDAMB-231 cells. DC50=2.56 μM(4T1) and 0.79 μM(MDA-MB-231), respectivley. Promoted the degradation of HK2 protein within 12 h, with the greatest degradation impact at 36 h in 4T1 cells and MDA-MB-231 cells. Degradation capacity was decreased, because pretreatment with Tha and LND occupy the protein pocket and disrupt the formation of the ternary complex of HK2, CRB HK2-expressing breast cancer cells (e.g., MCF-7 or MDA-MB-231) are seeded in 96-well plates. Cells are treated with PROTAC HK2 Degrader-1 at varying concentrations (0.1-1000 nM) for 24-72 hours. Cell viability is measured by MTT or CellTiter-Glo. HK2 protein levels are assessed by Western blot. Mitochondrial damage is evaluated by JC-1 staining (loss of mitochondrial membrane potential) or by measuring ATP levels. Pyroptosis is assessed by GSDMD cleavage and LDH release. |
| Animal Protocol |
Animal/Disease Models: xenograft models, into six-weekold female balb/c (Bagg ALBino) mouse[1]
Doses: 50 mg/kg Route of Administration: intraperitoneal (ip)injection, bid, for nine times. Experimental Results: decreased proliferation and damaged nuclei in mouse models. Increased the levels of Cytokines IL-1β, IFN-γ, and TNF-α Dramatically and diminished the level of TGF-β and IL-10. Elevated levels of cleaved-Casp-3 and GSDME-N in tumor tissues of mouse. Animal/Disease Models: breast tumor model in mice by injecting 4T1 cells subcutaneously (sc) into six-weekold female balb/c (Bagg ALBino) mouse[1] Doses: Cisplatin (HY-17394) 10mg/kg, 50mg/kg Route of Administration: Cisplatin (HY-17394) (10mg/kg, iv) , 50mg/kg, ip, 25 days Experimental Results: Inhibited tumor growth and tumor volume. diminished HK2 protein level, while co- treated with Cisplatin (HY-17394). Could alleviate Cisplatin (HY-17394) aggravated colon damage. PROTAC HK2 Degrader-1 can be studied in mouse breast cancer xenograft models. Female BALB/c nude mice are implanted subcutaneously with breast cancer cells (e.g., MCF-7 or 4T1). When tumors reach 100-150 mm3, mice are randomized and treated intraperitoneally with PROTAC HK2 Degrader-1 (10-50 mg/kg) dissolved in a vehicle (10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline) every 2-3 days. Tumor volume is measured. HK2 degradation in tumors is confirmed by Western blot. Mitochondrial damage and pyroptosis markers are assessed. |
| ADME/Pharmacokinetics |
PROTAC HK2 Degrader-1 has a molecular weight of 647.51 and the molecular formula C32H28Cl2N6O5. The product appears as a light yellow to yellow solid. It should be stored at -20degC for up to 3 years. In solvent, it is stable for 6 months at -80degC. Specific PK parameters have not been reported. The compound is soluble in DMSO. For in vivo use, it requires formulation with a suitable vehicle.
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| Toxicity/Toxicokinetics |
No detailed toxicity data for PROTAC HK2 Degrader-1 are available. In animal studies, the compound effectively suppresses breast tumor growth and reduces the colonic side effects of cisplatin, suggesting it may have a favorable safety profile. However, formal toxicology studies have not been reported. As a degrader of HK2, which is involved in normal glucose metabolism, there may be mechanism-based effects on non-cancerous tissues. Standard safety precautions for handling PROTAC molecules should be followed.
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| References | |
| Additional Infomation |
PROTAC HK2 Degrader-1 (C-02) is a research-grade PROTAC and is not approved for clinical use. It is a CRBN-recruiting HK2 degrader composed of lonidamine (HK2 inhibitor) and thalidomide (CRBN ligand). It degrades HK2 protein, induces mitochondrial damage, triggers pyroptosis, and suppresses breast tumor growth. This compound is a valuable research tool for studying HK2-targeted therapy in cancer. It is for research use only.
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| Molecular Formula |
C32H28CL2N6O5
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|---|---|
| Molecular Weight |
647.51
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| Exact Mass |
646.15
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| CAS # |
3033812-84-6
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| PubChem CID |
171361405
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.53±0.1 g/cm3(Temp: 20 °C; Press: 760 Torr)(predicted)
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| Boiling Point |
954.7±65.0 °C(predicted)
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| LogP |
0
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
45
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| Complexity |
1160
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(=O)NC(=O)C1N2C(=O)C3=C(C2=O)C(=CC=C3)NCCCCNC(=O)C4=NN(C5=CC=CC=C54)CC6=C(C=C(C=C6)Cl)Cl
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| InChi Key |
OFEUWOIETWIJID-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C32H28Cl2N6O5/c33-19-11-10-18(22(34)16-19)17-39-24-9-2-1-6-20(24)28(38-39)30(43)36-15-4-3-14-35-23-8-5-7-21-27(23)32(45)40(31(21)44)25-12-13-26(41)37-29(25)42/h1-2,5-11,16,25,35H,3-4,12-15,17H2,(H,36,43)(H,37,41,42)
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| Chemical Name |
1-[(2,4-dichlorophenyl)methyl]-N-[4-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]butyl]indazole-3-carboxamide
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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) |
DMSO :~100 mg/mL (~154.44 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 | 1.5444 mL | 7.7219 mL | 15.4438 mL | |
| 5 mM | 0.3089 mL | 1.5444 mL | 3.0888 mL | |
| 10 mM | 0.1544 mL | 0.7722 mL | 1.5444 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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