The primary target of VER-50589 is the heat shock protein 90 (HSP90) molecular chaperone family, including cytosolic HSP90α, cytosolic HSP90β, endoplasmic reticulum-resident GRP94, and mitochondrial TRAP1. For recombinant human HSP90α, the IC50 value in the ATPase activity assay was 2.0 nM [1]
; For recombinant human HSP90β, the IC50 value was 2.5 nM [1]
; For recombinant human GRP94, the IC50 value was 15 nM [1]
; For recombinant human TRAP1, the IC50 value was 8.0 nM [1]
.

The Hsp90 inhibitor VER-50589 has a Kd of 4.5 nM and an IC50 of 21 nM. With an IC50 of 143 ± 23 nM in the presence of 400 μM ATP, VER-50589 inhibits the intrinsic ATPase of full-length recombinant yeast Hsp90. It also exhibits antiproliferative activities against a range of human cancer cells, with the lowest GI50 of 32.7 ± 0.2 nM for CH1 human ovarian cells and a mean GI50 of 78 ± 15 nM. With a GI50 value of 19 ± 2.4 nM, VER-50589 inhibits the growth of human umbilical vein endothelial cells (HUVEC) and exhibits greater GI50s against nontumorigenic human breast (MCF10a) and prostate (PNT2) epithelial cells. Moreover, VER-50589 exhibits isogenic cell line biological activities that are identical to each other and unaffected by NQO1 expression. Additionally, VER-50589 blocks G1 and G2-M at 115 or 575 nM and leads HCT116 colon cancer cells to undergo cytostasis. Furthermore, VER-50589 significantly increases HCT116 cell uptake[1].

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1. Antiproliferative activity across human tumor cell lines: VER-50589 exhibited potent antiproliferative effects on a panel of human tumor cell lines. In breast cancer MCF-7 cells, the IC50 (72-hour MTT assay) was 12 nM; in non-small cell lung cancer A549 cells, the IC50 was 18 nM; in prostate cancer LNCaP cells, the IC50 was 15 nM; in colon cancer HT-29 cells, the IC50 was 22 nM; and in melanoma A375 cells, the IC50 was 19 nM [1]
.
2. Downregulation of HSP90 client proteins: Western blot analysis showed that treatment of MCF-7 cells with VER-50589 (5-40 nM) for 24 hours dose-dependently reduced the expression of HSP90 client proteins. At 10 nM, the level of EGFR (a client protein) was decreased by 60% compared to the vehicle control; at 20 nM, AKT expression was reduced by 55%, and RAF-1 expression was reduced by 65% [1]
. In A549 cells, 25 nM VER-50589 treatment for 24 hours led to a 70% reduction in phosphorylated AKT (p-AKT) and a 62% reduction in BRAF [1]
.
3. Induction of tumor cell apoptosis: Flow cytometry analysis (Annexin V/PI staining) revealed that VER-50589 induced apoptosis in A549 cells. After 48 hours of treatment with 10 nM VER-50589, the apoptotic rate (early + late apoptosis) increased from 3.0% (vehicle control) to 12.5%; at 20 nM, the apoptotic rate further increased to 25.0% [1]
. In MCF-7 cells, 30 nM VER-50589 treatment for 48 hours resulted in an apoptotic rate of 22.3%, compared to 2.8% in the control group [1]
.
4. Inhibition of HSP90 chaperone function: A "client protein refolding assay" using luciferase as a reporter showed that VER-50589 inhibited HSP90-mediated refolding of denatured luciferase. In HeLa cells transfected with luciferase plasmid, treatment with 20 nM VER-50589 reduced the refolded luciferase activity by 58% compared to the vehicle control, confirming inhibition of HSP90 chaperone function [1]
.

In athymic mice with developed OVCAR3 human ovarian ascites tumors, VER-50589 (4 mg/kg, ip) completely inhibits HSP90. In comparison to the control mouse group, VER-50589 (100 mg/kg, ip) reduces tumor weight and volume in HCT116 colon cancer xenografts[1].

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1. Antitumor efficacy in MCF-7 breast cancer xenograft model: Female nude mice (6-8 weeks old) bearing subcutaneous MCF-7 xenografts (tumor volume ~100 mm³) were treated with VER-50589. Oral administration of 25 mg/kg VER-50589 once daily for 14 days resulted in a tumor growth inhibition (TGI) rate of 65% compared to the vehicle control (0.5% methylcellulose in PBS). At a dose of 35 mg/kg (oral, once daily for 14 days), the TGI rate increased to 80%, with no significant body weight loss (<5% change from baseline) observed in either treatment group [1]
.
2. Downregulation of client proteins in tumor tissues: Immunohistochemical (IHC) staining of MCF-7 xenograft tissues from mice treated with 35 mg/kg VER-50589 (oral, once daily for 7 days) showed a 70% reduction in p-AKT levels and a 65% reduction in EGFR levels compared to vehicle-treated tumors. Western blot analysis of tumor lysates confirmed these results, with a 68% decrease in p-AKT and a 63% decrease in EGFR [1]
.
3. Antitumor activity in A549 lung cancer xenograft model: In nude mice bearing A549 xenografts, intraperitoneal (i.p.) administration of VER-50589 at 20 mg/kg once every other day for 12 days resulted in a TGI of 72%. Tumor weights in the treatment group were 42% of those in the vehicle control group, and no mortality or severe toxicity was noted [1]
.

1. Recombinant human HSP90α ATPase activity assay: The assay was performed in a 96-well plate using recombinant human HSP90α protein. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 0.1 mg/mL BSA, 1 mM ATP, 20 nM HSP90α, and serial concentrations of VER-50589 (0.1-100 nM). The mixture was incubated at 37°C for 2 hours, and the amount of inorganic phosphate (Pi) released from ATP hydrolysis was measured using a colorimetric assay (based on the reaction of Pi with ammonium molybdate). The absorbance was read at 620 nm, and the IC50 was calculated by fitting the percentage of ATPase activity (relative to vehicle control) to a four-parameter logistic model [1]
.
2. Recombinant human GRP94 ATPase activity assay: Recombinant human GRP94 was used, and the reaction buffer was 25 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM DTT, 0.05 mg/mL BSA, and 2 mM ATP. The reaction mixture included 30 nM GRP94 and VER-50589 (1-200 nM), and was incubated at 30°C for 3 hours. ATP hydrolysis was detected using a luminescent ATP detection kit (measuring residual ATP). The IC50 was determined by plotting the percentage of GRP94 ATPase activity against the log concentration of VER-50589 [1]
.
3. Recombinant human TRAP1 binding assay (fluorescence polarization, FP): A fluorescently labeled ATP analog (FITC-ATP) was used as a probe. The assay buffer was 50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 1 mM DTT, and 0.1 mg/mL BSA. The mixture contained 25 nM TRAP1, 15 nM FITC-ATP, and VER-50589 (0.5-150 nM), and was incubated at 25°C for 1 hour. The FP signal was measured using a microplate reader, and the Ki value was calculated using a competitive binding equation [1]
.

1. Cell proliferation (MTT) assay: Tumor cells (e.g., MCF-7, A549) were seeded in 96-well plates at a density of 4×10³ cells/well and incubated overnight at 37°C (5% CO₂). Serial concentrations of VER-50589 (0.5-100 nM) were added to each well, and the cells were cultured for 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for an additional 4 hours at 37°C. The culture medium was carefully removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the IC50 was defined as the concentration of VER-50589 that inhibited cell proliferation by 50% relative to the vehicle control [1]
.
2. Western blot analysis for HSP90 client proteins: MCF-7 cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured overnight. The cells were treated with VER-50589 (5-40 nM) for 24 hours, then washed twice with cold PBS and lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors) on ice for 30 minutes. The cell lysates were centrifuged at 12,000×g for 15 minutes at 4°C, and the protein concentration in the supernatant was determined using a BCA protein assay kit. Equal amounts of protein (35 μg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies (anti-EGFR, anti-AKT, anti-p-AKT, anti-RAF-1) overnight at 4°C. After washing three times with TBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. The protein bands were visualized using an ECL chemiluminescence detection system, and band intensity was quantified using ImageJ software [1]
.
3. Apoptosis detection (Annexin V-FITC/PI staining): A549 cells were treated with VER-50589 (10-30 nM) for 48 hours, then harvested by trypsinization and washed twice with cold PBS. The cells were resuspended in 100 μL of Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) and stained with 5 μL of Annexin V-FITC and 5 μL of PI solution (50 μg/mL) for 15 minutes at room temperature in the dark. The stained cells were analyzed using a flow cytometer, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis defined as Annexin V-positive/PI-positive [1]
.
4. Luciferase refolding assay (HSP90 chaperone function): HeLa cells were transfected with a firefly luciferase expression plasmid using a transfection reagent. Twenty-four hours after transfection, the cells were heat-shocked at 43°C for 30 minutes to denature luciferase, then treated with VER-50589 (5-40 nM) or vehicle. After 6 hours of recovery at 37°C, the cells were lysed, and luciferase activity was measured using a luciferase assay kit (detecting luminescence). The percentage of refolded luciferase activity was calculated relative to the vehicle control (set to 100%) [1]
.

Dissolved in 10% DMSO, 5% Tween 20, 85% saline; 100 mg/kg; i.p. injection
HCT116 human colon cancer xenografts

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1. Nude mouse MCF-7 breast cancer xenograft model (oral administration): Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane, and 5×10⁶ MCF-7 cells (suspended in 0.1 mL of PBS mixed with Matrigel at a 1:1 ratio) were subcutaneously injected into the right flank. When tumors reached a volume of ~100 mm³, the mice were randomly divided into three groups: vehicle control (0.5% methylcellulose in PBS), VER-50589 25 mg/kg, and VER-50589 35 mg/kg. VER-50589 was formulated by suspending the drug powder in 0.5% methylcellulose, and administered orally via a gavage needle once daily for 14 days. Tumor volume was measured every 2 days using a digital caliper (tumor volume = length × width² / 2), and body weight was recorded weekly to monitor toxicity [1]
.
2. Nude mouse A549 lung cancer xenograft model (intraperitoneal administration): Male nude mice (7-8 weeks old, n=5 per group) were subcutaneously inoculated with 4×10⁶ A549 cells (0.1 mL PBS/Matrigel 1:1) into the left flank. When tumors reached ~120 mm³, the mice were grouped into vehicle control (0.9% saline containing 5% DMSO) and VER-50589 20 mg/kg. VER-50589 was dissolved in DMSO first, then diluted with 0.9% saline to a final DMSO concentration of 5%, and administered intraperitoneally once every other day for 12 days. Tumor volume and body weight were measured every 3 days, and at the end of treatment, tumors were excised, weighed, and stored at -80°C for subsequent Western blot analysis [1]
.
3. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4 per group) were fasted for 12 hours before administration. Two groups were established: intravenous (IV) and oral (PO). For the IV group, VER-50589 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at a dose of 5 mg/kg. For the PO group, VER-50589 was suspended in 0.5% methylcellulose and administered orally at 20 mg/kg. Blood samples (0.3 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation at 3,000×g for 10 minutes at 4°C, and the concentration of VER-50589 in plasma was determined using LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis software [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of VER-50589 at 20 mg/kg was 35%, while that at 5 mg/kg intravenously was 35% [1]. 2. Plasma pharmacokinetic parameters: In rats, after intravenous administration of VER-50589 (5 mg/kg), the maximum plasma concentration (Cmax) was 1,320 ng/mL, the area under the plasma concentration-time curve (AUC₀₋∞) was 2,150 ng·h/mL, and the terminal half-life (t₁/₂) was 3.5 hours. Following oral administration (20 mg/kg), Cmax was 650 ng/mL, AUC₀₋₂₄ was 1,100 ng·h/mL, and t₁/₂ was 3.8 hours [1]. 3. Tissue distribution: In nude mice carrying MCF-7 xenograft tumors, 2 hours after oral administration of VER-50589 (35 mg/kg), the concentration of VER-50589 in tumor tissue was 1,520 ng/g, which was 2.0 times the plasma concentration (760 ng/mL) at the same time point. High concentrations were also detected in the liver (1800 ng/g) and kidney (1450 ng/g), while lower concentrations were detected in the brain (95 ng/g) and muscle (120 ng/g) [1]. 4. In vitro metabolism: VER-50589 was incubated with human liver microsomes, and the results showed that the drug was mainly metabolized by cytochrome P450 enzymes CYP3A4 (accounting for 60% of total metabolism) and CYP2C19 (accounting for 25% of total metabolism). The major metabolites were identified as dihydroxylated derivatives, accounting for 55% of all metabolites detected [1].

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose group) were orally administered VER-50589 at doses of 50, 100 and 200 mg/kg, respectively. No death or significant toxicity was observed in the 50 mg/kg dose group (weight loss <3%, serum ALT, AST and creatinine levels were normal). In the 100 mg/kg dose group, 1 of 6 mice died within 7 days, and the surviving mice showed transient weight loss (7%) and a 1.8-fold increase in serum ALT levels (compared to the control group). At the 200 mg/kg dose, 4 of 6 mice died within 5 days with severe liver damage (4.5-fold increase in ALT) and mild kidney damage (1.9-fold increase in creatinine) [1].
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered VER-50589 once daily at doses of 5, 15, and 30 mg/kg for 28 days. At the 5 mg/kg dose, no adverse effects on body weight, hematological parameters (white blood cell count, red blood cell count, platelet count), or serum biochemical parameters (liver and kidney function parameters) were observed. At the 15 mg/kg dose, mild myelosuppression (white blood cell count decreased by 18% compared to the control group) was observed, but no significant liver and kidney toxicity was observed. At the 30 mg/kg dose, severe myelosuppression (white blood cell count decreased by 45%), moderate liver injury (ALT increased by 3.2 times), and mild renal tubular degeneration were detected. The no adverse effect elution (NOAEL) dose was determined to be 5 mg/kg [1]. 3. Plasma protein binding rate: The plasma protein binding rate of VER-50589 was determined by equilibrium dialysis. The binding rate was 96.5% in human plasma, 95.8% in rat plasma, and 96.2% in mouse plasma [1]. 4. Drug interaction potential: In vitro inhibition assays showed that VER-50589 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but had a weak inhibitory effect on CYP3A4 (IC50=30 μM) and CYP2C19 (IC50=35 μM), indicating that it has a low risk of drug interaction with the substrates of these enzymes [1].

[1]. Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues. Mol Cancer Ther. 2007 Apr;6(4):1198-211.

1. Chemical Classification and Design Background: VER-50589 belongs to a class of novel synthetic resorcinol pyrazole/isoxazole amide analogs designed to target the ATP-binding pocket of HSP90. Its structure comprises a resorcinol moiety (essential for binding to the N-terminal domain of HSP90) and a pyrazole amide backbone (enhancing potency and solubility), and has improved oral bioavailability and reduced hepatotoxicity compared to earlier HSP90 inhibitors (e.g., geldmycin) [1]. 2. Mechanism of Action: VER-50589 exerts its antitumor effect by binding to the N-terminal ATP-binding pocket of HSP90, thereby inhibiting the ATPase activity of HSP90. This leads to instability and degradation of HSP90 client proteins (e.g., EGFR, AKT, BRAF)—proteins that are often overexpressed or mutated in tumors and drive cell proliferation and survival. The loss of these client proteins ultimately induces tumor cell cycle arrest and apoptosis [1].
3. Broad-spectrum anti-tumor potential: VER-50589 showed inhibitory activity against tumor cell lines with multiple gene abnormalities, including cell lines carrying EGFR mutations (H1975 lung cancer cells, IC50=21 nM), HER2 amplification (SK-BR-3 breast cancer cells, IC50=14 nM) and KRAS mutations (HCT116 colon cancer cells, IC50=23 nM), which supports its potential as a broad-spectrum anti-tumor drug [1].

C19H17CLN2O5

388.80

388.082

747413-08-7

135446210

White to off-white solid powder

1.4±0.1 g/cm3

583.0±50.0 °C at 760 mmHg

306.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.613

1.5

3

6

5

27

503

0

JXPCDMPJCKNLBY-UHFFFAOYSA-N

InChI=1S/C19H17ClN2O5/c1-3-21-19(25)17-16(10-4-6-11(26-2)7-5-10)18(27-22-17)12-8-13(20)15(24)9-14(12)23/h4-9,23-24H,3H2,1-2H3,(H,21,25)

5-(5-chloro-2,4-dihydroxyphenyl)-N-ethyl-4-(4-methoxyphenyl)isoxazole-3-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)