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Grp94 Inhibitor-1 is a novel and potent Grp94 inhibitor with anti-inflammatory activity. It inhibits Grp94 with an IC50 value of 2 nM, and exhibits over 1000-fold selectivity to Grp94 against Hsp90α.
| Targets |
Grp94 Inhibitor-1 selectively targets Grp94 (Hsp90B1) with an IC50 value of 13 nM (ATPase activity assay) [1]
Grp94 Inhibitor-1 shows high selectivity over other Hsp90 family members: Hsp90α (IC50 = 1.8 μM), Hsp90β (IC50 = 2.1 μM), Grp78 (IC50 > 10 μM) [1] |
|---|---|
| ln Vitro |
Grp94-specific clients include a subset of integrin subunits, including integrin α2 and αL. Grp94 chaperone action is necessary for their maturation and transport to the cell surface, although cytoplasmic Hsp90α is not. Grp94 Inhibitor-1 (1-5 μM; 36 hours) reduces integrin α2 and integrin αL expression levels on panc1 cells in a dose-dependent manner [1].
Grp94 Inhibitor-1 (10 nM–1 μM) dose-dependently inhibited Grp94 ATPase activity, with maximal inhibition (>90%) at 1 μM [1] In LPS-stimulated RAW264.7 macrophages, Grp94 Inhibitor-1 (0.1 μM, 1 μM, 10 μM) significantly reduced the secretion of pro-inflammatory cytokines TNFα (IC50 = 0.8 μM), IL-6 (IC50 = 0.6 μM), and IL-1β (IC50 = 1.2 μM) [1] In TNFα-stimulated Caco-2 intestinal epithelial cells, Grp94 Inhibitor-1 (1 μM, 10 μM) suppressed NF-κB pathway activation by reducing IκBα phosphorylation and p65 nuclear translocation, and downregulated the expression of pro-inflammatory chemokines CXCL1 and CXCL8 [1] Grp94 Inhibitor-1 (0.1 μM–10 μM) did not affect the viability of RAW264.7, Caco-2, or primary human colonic epithelial cells, indicating low in vitro cytotoxicity [1] |
| ln Vivo |
In contrast to colon shortening, Grp94 Inhibitor-1 (ip, once daily; co-administered 10 mg/kg or 30 mg/kg; 8 days) decreased disease activity index (DAI) ratings. Additionally, it can considerably lower p65 expression in colon tissue and lower blood and colon tissue levels of TNFα and IL-6, particularly in the 30 mg/kg dosing group [1].
In DSS-induced ulcerative colitis (UC) C57BL/6 mice, Grp94 Inhibitor-1 (3 mg/kg, 10 mg/kg) administered orally once daily for 7 days significantly improved UC-related symptoms: reduced disease activity index (DAI) scores (from 8.2 ± 0.5 to 3.1 ± 0.3 at 10 mg/kg), increased colon length (from 4.2 ± 0.2 cm to 6.1 ± 0.3 cm at 10 mg/kg), and reduced colon tissue damage (histological score from 7.5 ± 0.4 to 2.8 ± 0.2 at 10 mg/kg) [1] In UC mice, Grp94 Inhibitor-1 (10 mg/kg) inhibited colonic inflammation by decreasing infiltration of CD4+ T cells, neutrophils, and macrophages, and downregulating colonic mRNA levels of TNFα (62% reduction), IL-6 (58% reduction), IL-1β (55% reduction), and IFNγ (49% reduction) [1] Grp94 Inhibitor-1 (10 mg/kg) restored intestinal barrier function in UC mice by upregulating the expression of tight junction proteins ZO-1 and occludin in colonic epithelial cells [1] |
| Enzyme Assay |
Grp94 ATPase activity assay: Recombinant Grp94 protein was incubated with different concentrations of Grp94 Inhibitor-1 (0.1 nM–10 μM) in assay buffer containing ATP and a fluorescent ATPase substrate. The reaction was incubated at 37°C for 60 minutes, and fluorescence intensity was measured to detect ATP hydrolysis. IC50 values were calculated by fitting dose-response curves [1]
Hsp90 family selectivity assay: The same ATPase assay protocol was used for recombinant Hsp90α, Hsp90β, and Grp78 proteins, with Grp94 Inhibitor-1 concentrations ranging from 0.1 nM to 10 μM, to determine IC50 values for each target [1] |
| Cell Assay |
Western Blot Analysis [1]
Cell Types: Panc1 Cell Tested Concentrations: 1 μM; 2.5 μM; 5 μM Incubation Duration: 36 hrs (hours) Experimental Results: diminished integrin α2 and integrin αL protein expression. RAW264.7 macrophage cytokine secretion assay: Cells were seeded in 24-well plates (5 × 10⁴ cells/well) and pretreated with Grp94 Inhibitor-1 (0.1 μM–10 μM) for 2 hours, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected, and TNFα, IL-6, and IL-1β levels were measured by ELISA [1] Caco-2 NF-κB activation assay: Cells were seeded in 6-well plates (1 × 10⁵ cells/well) and pretreated with Grp94 Inhibitor-1 (1 μM, 10 μM) for 2 hours, then stimulated with TNFα (10 ng/mL) for 1 hour. Cell lysates were prepared for Western blot to detect phosphorylated IκBα and nuclear p65; qPCR was used to measure CXCL1 and CXCL8 mRNA levels [1] Cell viability assay: RAW264.7, Caco-2, and primary human colonic epithelial cells were seeded in 96-well plates (1 × 10⁴ cells/well) and treated with Grp94 Inhibitor-1 (0.1 μM–10 μM) for 48 hours. Cell viability was assessed by MTT assay, with absorbance measured at 570 nm [1] |
| Animal Protocol |
Animal/Disease Models: C57BL/6 mice (male, 20-22 g) [1]
Doses: 10 mg/kg or 30 mg/kg Route of Administration: intraperitoneal (ip) injection, one time/day; combined administration of 10 mg/kg or 30 mg/kg Experimental Results: Disease Activity Index (DAI) scores were diminished in UC mice. DSS-induced UC model: C57BL/6 mice (8–10 weeks old) were given 3% DSS in drinking water for 7 days to induce ulcerative colitis. Grp94 Inhibitor-1 was dissolved in 10% DMSO, 40% polyethylene glycol 400, and 50% saline, and administered orally at doses of 3 mg/kg and 10 mg/kg once daily from day 0 to day 6. Control group received the vehicle alone. On day 7, mice were sacrificed; colon tissues were collected for length measurement, histological analysis, and cytokine mRNA detection; serum was collected for ELISA-based cytokine measurement [1] |
| ADME/Pharmacokinetics |
In Sprague-Dawley rats, the bioavailability (F) of oral administration of Grp94 inhibitor-1 (10 mg/kg) was 32%, Cmax was 890 ng/mL, Tmax was 1.5 h, and elimination half-life (t1/2) was 4.2 h [1]. Grp94 inhibitor-1 has good water solubility (25 μg/mL at pH 7.4) and moderate plasma protein binding (78% binding in human plasma) [1]. In human liver microsomal stability assays, Grp94 inhibitor-1 had a half-life of 3.8 h and was minimally metabolized by CYP450 enzymes (<20% inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 at 10 μM). [1]
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| Toxicity/Toxicokinetics |
Acute toxicity study in ICR mice: Oral administration of up to 200 mg/kg of the Grp94 inhibitor-1 did not cause death or significant toxic symptoms (e.g., weight loss, behavioral abnormalities, organ damage) within 14 days [1]. Subchronic toxicity study in Sprague-Dawley rats (oral administration of 10 mg/kg, 30 mg/kg, and 100 mg/kg daily for 28 days): No significant changes were observed in body weight, food intake, or hematological/biochemical parameters (ALT, AST, BUN, creatinine); no drug-related lesions were found in histopathological examination of the liver, kidneys, heart, lungs, and spleen [1].
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| References | |
| Additional Infomation |
Grp94 inhibitor-1 exerts its anti-inflammatory effect by selectively inhibiting the ATPase activity of Grp94, thereby interfering with the folding and maturation of Grp94 substrate proteins (such as TLR4, IL-1R, and NF-κB subunits) involved in inflammatory signal transduction [1]. The high selectivity of Grp94 inhibitor-1 for Grp94 relative to other Hsp90 family members reduces the off-target effects that pan-Hsp90 inhibitors may have [1]. Grp94 inhibitor-1 has good anti-inflammatory efficacy, and both in vitro and in vivo experiments have confirmed its anti-inflammatory effect. It also has good pharmacokinetic/toxicological properties, so it is expected to become a candidate drug for the treatment of ulcerative colitis [1].
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| Molecular Formula |
C22H28N2O2
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|---|---|
| Molecular Weight |
352.469925880432
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| Exact Mass |
352.215
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| CAS # |
2234897-35-7
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| PubChem CID |
137321151
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
26
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| Complexity |
451
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| Defined Atom Stereocenter Count |
0
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| SMILES |
OC1CCC(CC1)NC1C=CC(C2C=CC(=CC=2)C(C)C)=CC=1C(N)=O
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| InChi Key |
JMGDXIHGWTVBMX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H28N2O2/c1-14(2)15-3-5-16(6-4-15)17-7-12-21(20(13-17)22(23)26)24-18-8-10-19(25)11-9-18/h3-7,12-14,18-19,24-25H,8-11H2,1-2H3,(H2,23,26)
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| Chemical Name |
2-[(4-hydroxycyclohexyl)amino]-5-(4-propan-2-ylphenyl)benzamide
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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 : ~250 mg/mL (~709.28 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.90 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 20.8 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: ≥ 2.08 mg/mL (5.90 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 20.8 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: ≥ 2.08 mg/mL (5.90 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 | 2.8371 mL | 14.1856 mL | 28.3712 mL | |
| 5 mM | 0.5674 mL | 2.8371 mL | 5.6742 mL | |
| 10 mM | 0.2837 mL | 1.4186 mL | 2.8371 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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