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| Targets |
Caspase-3/7 ( IC50 = 1.9 μM ); HSP70
The primary target of HSP70-IN-1 is the heat shock protein 70 (Hsp70), a molecular chaperone that plays a critical role in protein homeostasis (proteostasis). Hsp70 assists in the folding of newly synthesized proteins, prevents protein aggregation, and facilitates the refolding of denatured proteins under stress conditions. It is often overexpressed in cancer cells, where it promotes cell survival and contributes to resistance to chemotherapy. HSP70-IN-1 inhibits the chaperone function of Hsp70 by binding to its ATPase domain or allosteric sites, preventing its interaction with client proteins and co-chaperones. This inhibition disrupts the Hsp70-HOP-Hsp90 multi-chaperone complex, leading to the destabilization and degradation of client proteins such as Raf-1. The compound also inhibits caspase-3/7 with an IC₅₀ of 1.9 μM, contributing to its pro-apoptotic effects. |
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| ln Vitro |
Heat shock protein 70 (Hsp70) is a molecular chaperone involved in cell survival and signaling, as well as protein homeostasis. In cancer, Hsp70 is often overexpressed. It is also thought that this increased expression contributes to or causes resistance to chemotherapy and other treatments. Because HSP70-IN-1 can destabilize Raf-1, a client of Hsp70-Hsp90 machinery, and change the megacomplex components in a dose-dependent manner, it obstructs the formation of functional Hsp70-HOP-Hsp90 machinery. In cells, HSP70-IN-1 inhibits both endogenous and transfected Hsp70's ability to refold heat-denatured luciferase. Moreover, HSP70-IN-1 causes cancer cells to undergo programmed cell death. The formation of the Hsp70-HOP complex is altered in a dose-dependent manner when HSP70-IN-1 is added to cancer cells; this phenomenon is linked to their destabilization and reduction in half-life[1].
HSP70-IN-1 demonstrates potent in vitro activity against Hsp70. It inhibits the growth of Kasumi-1 cells with an IC₅₀ of 2.3 μM. The compound disrupts the formation of the Hsp70-HOP complex in a dose-dependent manner in cancer cells, leading to the destabilization and reduction in half-life of client proteins. It inhibits the refolding of heat-denatured luciferase by both endogenous and transfected Hsp70 in cells, directly demonstrating its inhibition of Hsp70 chaperone activity. The compound also induces programmed cell death in cancer cells. Its physicochemical properties, including a LogP of 3.27 and a density of 1.3 g/cm³, are consistent with a drug-like molecule. HSP70-IN-1 is a valuable tool for studying the role of Hsp70 in cancer and other diseases. |
| ln Vivo |
In vivo activity data for HSP70-IN-1 are not extensively documented in the literature. However, based on its potent in vitro activity, the compound is expected to exhibit anti-tumor efficacy in animal models. Hsp70 inhibitors have been shown to have therapeutic potential in various cancers, and HSP70-IN-1 is a promising candidate for further development. Its ability to destabilize the Hsp70-HOP-Hsp90 complex and induce apoptosis makes it a potential anti-cancer agent. Further in vivo studies are needed to evaluate its pharmacokinetics, efficacy, and safety profile.
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| Enzyme Assay |
In vitro enzyme assays for HSP70-IN-1 are focused on measuring its inhibitory activity against Hsp70. A common assay is the measurement of Hsp70 ATPase activity. The enzyme is incubated with ATP and varying concentrations of the compound, and the production of ADP is measured using a colorimetric or fluorometric method. The IC₅₀ for ATPase inhibition is calculated. Another assay measures the compound's ability to inhibit Hsp70-mediated protein refolding. In this assay, a denatured protein (e.g., luciferase) is incubated with Hsp70, its co-chaperones, and ATP in the presence or absence of the compound. The refolding of the protein is measured by its activity (e.g., luciferase activity). The compound's ability to inhibit refolding is a measure of its chaperone inhibitory activity.
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| Cell Assay |
In vitro cell-based assays for HSP70-IN-1 are used to study its effects on cancer cell viability and Hsp70 function. A common assay is the MTT or CellTiter-Glo assay, where cancer cells are treated with the compound for 48-72 hours, and cell viability is measured. The IC₅₀ for cell growth inhibition is calculated (e.g., 2.3 μM for Kasumi-1 cells). Apoptosis is assessed by measuring caspase-3/7 activity (IC₅₀ of 1.9 μM) or by using Annexin V staining and flow cytometry. The effect of the compound on Hsp70 function is assessed by measuring the levels of Hsp70 client proteins (e.g., Raf-1) by western blotting and by analyzing the formation of the Hsp70-HOP complex by co-immunoprecipitation. These assays provide a comprehensive picture of the compound's cellular activity.
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| Animal Protocol |
In vivo animal studies for HSP70-IN-1 are likely conducted in xenograft models of cancer. Tumor-bearing mice are treated with the compound, and tumor growth is monitored. The compound's effect on Hsp70 function in the tumor tissue can be assessed by measuring the levels of Hsp70 client proteins and by analyzing the formation of the Hsp70-HOP complex. Pharmacodynamic endpoints, such as the induction of apoptosis, are also measured. These studies are essential for evaluating the compound's anti-tumor efficacy and its mechanism of action in vivo.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of HSP70-IN-1 are not extensively documented. As a small molecule with a molecular weight of 464.58 g/mol and a LogP of 3.27, it is expected to have reasonable cell permeability and oral bioavailability. It is soluble in DMSO and can be formulated for in vivo administration. For research use, it is typically stored as a powder at -20°C for up to three years. Detailed PK parameters, such as half-life, clearance, and volume of distribution, would need to be determined in standard preclinical studies.
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| Toxicity/Toxicokinetics |
The toxicological profile of HSP70-IN-1 is not extensively documented. As an inhibitor of a key cellular chaperone, its toxicity is likely related to its on-target effects on protein homeostasis. Hsp70 is essential for the survival of both normal and cancer cells, so its inhibition could lead to toxicity in normal tissues. For laboratory handling, standard safety precautions for research chemicals should be observed, including the use of personal protective equipment (gloves, lab coat, safety goggles). The compound is intended for research use only and is not for human therapeutic or diagnostic use.
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| References | |
| Additional Infomation |
HSP70-IN-1 is a heat shock protein 70 (Hsp70) inhibitor. It is also known as LUN-00465. The compound inhibits the growth of Kasumi-1 cells with an IC₅₀ of 2.3 μM and inhibits caspase-3/7 with an IC₅₀ of 1.9 μM. It disrupts the formation of the Hsp70-HOP complex and destabilizes client proteins such as Raf-1. It has a molecular formula of C₂₄H₂₈N₆O₂S and a molecular weight of 464.58 g/mol. HSP70-IN-1 is supplied as a light yellow to yellow solid powder with a purity of ≥98%. The compound is for research use only.
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| Molecular Formula |
C24H28N6O2S
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|---|---|
| Molecular Weight |
464.5831
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| Exact Mass |
464.199
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| Elemental Analysis |
C, 62.05; H, 6.08; N, 18.09; O, 6.89; S, 6.90
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| CAS # |
1268273-90-0
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| PubChem CID |
66973893
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.686
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| LogP |
3.27
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
33
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| Complexity |
600
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1=C([H])C([H])=C([H])C(=C1[H])N([H])C(C([H])([H])N([H])[H])=O)C1=C([H])N=C(N=C1OC([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]
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| InChi Key |
LBJCUDZMNPAWPX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H28N6O2S/c1-29-10-12-30(13-11-29)24-26-16-21(23(28-24)32-17-18-6-3-2-4-7-18)33-20-9-5-8-19(14-20)27-22(31)15-25/h2-9,14,16H,10-13,15,17,25H2,1H3,(H,27,31)
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| Chemical Name |
2-amino-N-[3-[2-(4-methylpiperazin-1-yl)-4-phenylmethoxypyrimidin-5-yl]sulfanylphenyl]acetamide
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| Synonyms |
LUN-00465; LUN 00465; LUN00465; HSP70-IN-27c; HSP70-IN-1
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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: ~50 mg/mL (~107.6 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1525 mL | 10.7624 mL | 21.5248 mL | |
| 5 mM | 0.4305 mL | 2.1525 mL | 4.3050 mL | |
| 10 mM | 0.2152 mL | 1.0762 mL | 2.1525 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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