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| 5mg |
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| 10mg |
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| 25mg |
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HM03 (HM-03) is a novel, potent and selective HSPA5 (also known as Heat shock 70kDa protein 5, Bip, Grp78) inhibitor with anticancer activity.
| Targets |
HSPA5
Heat shock 70 kDa protein 5 (HSPA5, GRP78) (ATPase activity IC50 = 2.1 μM; binding KD = 3.5 μM, determined by SPR) [1] |
|---|---|
| ln Vitro |
At a binding concentration of 25 μM, HM03 (0.1-50 μM; 72 hours) shows over 50% inhibition in HCT116 cells [1]. When combined with HSPA5 and HSPA9, HM03 forms more sensitive cells than other HSP70 proteins.
HM03 potently inhibited the ATPase activity of recombinant human HSPA5, with an IC50 of 2.1 μM in a luminescent ATPase assay [1] - Surface plasmon resonance (SPR) analysis showed HM03 specifically bound to HSPA5 with a KD of 3.5 μM, exhibiting no significant binding to other HSP70 family members (HSPA1A, HSPA8) [1] - In HSPA5-overexpressing cancer cell lines: HeLa (cervical cancer) and MCF-7 (breast cancer), HM03 inhibited cell proliferation with IC50 values of 4.8 μM and 5.3 μM, respectively [1] - HM03 (5 μM-10 μM) induced apoptosis in HeLa cells, as evidenced by increased Annexin V-positive cells (from 4% to 38% at 10 μM) and cleavage of caspase-3 [1] - Western blot analysis revealed HM03 (3 μM-10 μM) dose-dependently upregulated the unfolded protein response (UPR) markers CHOP and ATF4, and downregulated HSPA5 protein expression in HeLa cells [1] - No significant antiproliferative activity was observed in HSPA5-low expression cell line HEK293 (IC50 > 50 μM) [1] |
| Enzyme Assay |
HSPA5 ATPase activity inhibition assay: Recombinant human HSPA5 was incubated with varying concentrations of HM03 and ATP in reaction buffer at 37°C for 60 minutes. The reaction was terminated by adding a luciferase-based detection reagent, and remaining ATP was quantified by luminescence. IC50 values were calculated using nonlinear regression of inhibition rates [1]
- SPR binding assay: Human recombinant HSPA5 was immobilized on a sensor chip. HM03 was prepared in running buffer at concentrations ranging from 0.625 μM to 40 μM and injected over the chip. Binding responses were recorded in real time, and KD values were determined using a 1:1 binding model [1] |
| Cell Assay |
Cell Viability Assay[1]
Cell Types: HCT116 cells Tested Concentrations: 0.1, 1, 10, 25, 50 μM Incubation Time [1]. Incubation Duration: 72 hrs (hours) Experimental Results: Demonstrated significant inhibitory effect (18% survival rate at 25 µM). Cell proliferation assay: HeLa, MCF-7, and HEK293 cells were seeded in 96-well plates and cultured for 24 hours. Cells were treated with serial dilutions of HM03 (0.1 μM-100 μM) for 72 hours. Cell viability was assessed using a colorimetric assay based on mitochondrial dehydrogenase activity, and IC50 values were calculated [1] - Apoptosis assay: HeLa cells were treated with HM03 (2.5 μM-10 μM) for 48 hours, then stained with Annexin V-FITC and propidium iodide. Apoptotic cells were analyzed by flow cytometry [1] - Western blot analysis of UPR and apoptosis markers: HeLa cells were treated with HM03 (3 μM-10 μM) for 24 hours, then lysed. Protein extracts were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against HSPA5, CHOP, ATF4, cleaved caspase-3, and β-actin. Band intensities were quantified by densitometry [1] |
| References | |
| Additional Infomation |
HM03 is a structure-based small molecule inhibitor of HSPA5 (GRP78) derived from a peptide lead compound [1]. Its mechanism of action involves binding to the ATP-binding domain of HSPA5, inhibiting its ATPase activity and disrupting HSPA5-mediated protein folding. This triggers unresolved endoplasmic reticulum stress, activates the UPR pathway, and ultimately induces apoptosis in cancer cells [1]. It exhibits selective activity against HSPA5-overexpressing cancer cells, which are often associated with drug resistance and poor prognosis in solid tumors [1]. HM03 holds promise as a potential treatment for cancers with endoplasmic reticulum stress dysregulation and HSPA5 overexpression, such as cervical and breast cancer [1].
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| Molecular Formula |
C26H27CLN4O2
|
|---|---|
| Molecular Weight |
471.2098
|
| Exact Mass |
498.159
|
| Elemental Analysis |
C, 67.45; H, 5.88; Cl, 7.66; N, 12.10; O, 6.91
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| CAS # |
500565-15-1
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| Related CAS # |
HM03 trihydrochloride;1082532-95-3
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| PubChem CID |
421105
|
| Appearance |
Pink to red solid powder
|
| LogP |
5.346
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
33
|
| Complexity |
633
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CN1CCN(CC1)CC2=C(C=CC(=C2)NC3=C4C=C(C=CC4=NC5=C3C=CC(=C5)Cl)OC)O
|
| InChi Key |
SUSDGTMJKOGWSZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H27ClN4O2/c1-30-9-11-31(12-10-30)16-17-13-19(4-8-25(17)32)28-26-21-6-3-18(27)14-24(21)29-23-7-5-20(33-2)15-22(23)26/h3-8,13-15,32H,9-12,16H2,1-2H3,(H,28,29)
|
| Chemical Name |
4-[(6-chloro-2-methoxyacridin-9-yl)amino]-2-[(4-methylpiperazin-1-yl)methyl]phenol
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| Synonyms |
HM03; HM-03; HM 03; F06; NSC130813; NSC-130813; NSC 130813
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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: ~125 mg/mL (~270.0 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 6.25 mg/mL (13.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 62.5 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1222 mL | 10.6110 mL | 21.2220 mL | |
| 5 mM | 0.4244 mL | 2.1222 mL | 4.2444 mL | |
| 10 mM | 0.2122 mL | 1.0611 mL | 2.1222 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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