HSP90

Conglobatin 6.25-100 μM; 48 h) significantly inhibits the proliferation of MCF-7 and SKBR3 cells with IC50s of 12.11 and 39.44 μM, respectively[2]. EC109, KYSE70, KYSE450, KYSE150, KYSE180, and KYSE510 cells exhibit inhibited cell proliferation in response to conglobatin, with IC50 values of 16.43, 15.89, 10.94, 10.50, 10.28, and 9.31 μM, respectively[3]. SKBR3 and MCF-7 cells exhibit a clear G2/M phase arrest when exposed to conglobatin (10–40 μM) for a 24-hour period. Conglobatin causes SKBR3 and MCF-7 cells to undergo apoptosis via caspase-dependent mechanisms[2]. Hsp90 client protein levels are decreased and proteasome-dependent degradation is induced by conglobatin (10–40 μM; 3–24 h)[2]. binds to Hsp90's N-terminal, inhibiting Hsp90/Cdc37 chaperone/co-chaperone interactions but having no effect on Hsp90's ability to bind ATP[2].

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FW-04-806 binds to the N-terminal of Hsp90. FW-04-806 does not affect ATP-binding capability of Hsp90, but inhibits Hsp90/Cdc37 chaperone/co-chaperone interactions. FW-04-806 decreases Hsp90 client protein levels and induces proteasome-dependent degradation. FW-04-806 inhibits growth, induces cell cycle arrest, induces apoptosis, and downregulates the expression of anti-apoptotic proteins.[2]

In SKBR3 and MCF-7 human breast cancer xenograft models, conglobatin (50-200 mg/kg; ig q3d for 24 d) dose-dependently suppresses the growth of tumors[2]. In low-toxicity tumor xenograft models, EC109 and KYSE510, conglobatin (4–8 mg/kg; intraperitoneally every day for 21 days) suppresses the growth of tumors.[3]

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FW-04-806 inhibits the tumor growth of SKBR3 and MCF-7 tumor xenograft models [2]
SKBR3 and MCF-7 human breast cancer xenografts were established to assess the chemotherapeutic potential of FW-04-806. The antitumor activity of FW-04-806 at three doses (50, 100, and 200 mg/kg per dose i.g., q3d) were determined. ADM (4 mg/kg per dose i.p., q3d) was used as a positive control. The results demonstrated that FW-04-806 inhibited tumor growth in the SKBR3 and MCF-7 xenograft models in a dose-dependent manner (Figure 5A and B). Compared with the vehicle group, the three increasing doses of FW-04-806 showed, respectively, inhibition of tumor growth at a rate of 39.1% (P = 0.009), 52.7% (P = 0.003), and 67.5% (P = 0.0007) in the SKBR3 cell line groups and 27.3% (P = 0.021), 39.8% (P = 0.004), 54.3% (P = 0.001) in the MCF-7 cell line groups. Notably, the antitumor activity of high-dose FW-04-806 (0.37 ± 0.04 g, 67.5%) was better than positive control group(0.39 ± 0.04 g, 64.9%, P = 0.0008).All animals survived FW-04-806 treatment without appreciable adverse effects in terms of body weight loss or other signs of toxicity during the treatment (Figure 5C and D). Liver and renal function was similar between FW-04-806-treated and control mice. Additionally, lung, liver, heart, and kidneys of mice showed no histological abnormalities at the end of drug treatment (data not shown). This outcome demonstrates that FW-04-806 was well tolerated.

ATP-Sepharose binding assay [2]
ATP-Sepharose binding assay was modified base on previous protocol. Different concentrations of FW-04-806 or 17AAG were added into recombinant NBD Hsp90 protein (10 μg), and then mixtures were incubated with 25 µL preequilibrated γ-phosphate-linked ATP-Sepharose in 200 µL incubation buffer (10 mM Tris–HCl, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4 , 0.01% NP-40, pH 7.5) for 4 h at 4°C. The protein bound to Sepharose beads was separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed with protein immunoblotting.

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Colorimetric determination of ATPase activity [2]
Malachite green reagent was prepared on the day of use and contained malachite green (0.0812%, w/v), polyvinyl alcohol (2.32%, w/v, dissolves with difficulty and requires heating), ammonium molybdate (5.72%, w/v, in 6 M HCl), and argon water mixed in a ratio of 2:1:1:2 to a golden yellow solution. The assay buffer consisted of 100 mM Tris–HCl, 20 mM KCl, and 6 mM MgCl2, with a pH of 7.4. The experiments were performed in 100 μL of test solution containing 80 μL of malachite green reagent. The test solution contained 0.5 μM Hsp90 protein, 1 mM ATP, and 25, 50, 100, or 200 μM FW-04-806 or vehicle (DMSO).

Cell Proliferation Assay[2]
Cell Types: SKBR3 and MCF-7 cells
Tested Concentrations: 6.25, 12.5, 25, 50, 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Inhibited the proliferation of SKBR3 and MCF-7 cells in a dose-dependent manner.

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Cell Cycle Analysis[2]
Cell Types: SKBR3 and MCF-7 cells
Tested Concentrations: 10, 20, 40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased the G2/M cell population and diminished the population in the S and G0/G1 phases.

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Western Blot Analysis[2]
Cell Types: SKBR3 and MCF-7 cells
Tested Concentrations: 10, 20, 40 μM
Incubation Duration: 3, 6, 12, 24 hrs (hours)
Experimental Results: diminished the levels of the client proteins HER2, p-HER2, Raf-1, Akt, and p-Akt in a dose and time-dependent manner in SKBR3 cells. decreased the levels of the client proteins Raf-1, Akt, and p-Akt in a dose and time -dependent manner in MCF-7 cells.

Animal/Disease Models: BALB/c (nu/nu) athymic mice with SKBR3 and MCF-7 tumor xenograft[2]
Doses: 50, 100, 200 mg/kg
Route of Administration: po (oral gavage) every 3 days for 24 days
Experimental Results: demonstrated inhibition of tumor growth at a rate of 39.1%, 52.7%, and 67.5% in the SKBR3 cell line groups and 27.3%, 39.8%, 54.3% in the MCF-7 cell line groups at the three increasing doses, respectively. Was well tolerated.

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Animals, tumor xenografts, and test agents for in vivo studies and efficacy [2]
BALB/c (nu/nu) athymic mice were used. For SKBR3 and MCF-7 xenografts, 6-mm3 tumor fragments were implanted into the subcutaneous tissue of the axillary region using a trocar needle, and the animals were randomly divided into groups (n = 6) when the bearing tumor reached approximately 20 mm3. FW-04-806 was suspended at the desired concentration for each dose group in an aqueous vehicle containing 10% ethanol, 10% polyethylene glycol 400, and 10% Tween 80. The control group was given 0.4 mL/mouse vehicle solution i.g.; mice in other groups were given 50, 100, or 200 mg/kg of FW-04-806. Doxorubicin hydrochloride was purchased as 10 mg injections and diluted with saline as necessary to achieve the prescribed concentration.

[1]. Conglobatin, a novel macrolide dilactone from Streptomyces conglobatus ATCC 31005. J Antibiot (Tokyo). 1979 Sep;32(9):874-7.

[2]. FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation. Mol Cancer. 2014 Jun 14;13:150.

[3]. Macrolide analog F806 suppresses esophageal squamous cell carcinoma (ESCC) by blocking β1 integrin activation. Oncotarget. 2015 Jun 30;6(18):15940-52.

There have been reports of the presence of coccidialin in Streptomyces conglobatus, and relevant data are available for reference.

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A culture of Streptomyces conglobatus, a known producer of the polyether antibiotic ionomycin, was fermented to isolate and identify its second metabolite, coccidialin (C28H38N2O6). X-ray diffraction analysis showed that coccidialin has a dimeric macrocyclic lactone structure, similar to the structures of fungal metabolites helminthin and pyranomycin, thus inferring the absolute configuration of coccidialin. The dimer is composed of two 7-hydroxy-8-oxazolyl-2,4,6-trimethyl-2-octenic acid molecules linked by two ester bonds. [1]

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Background: Heat shock protein 90 (Hsp90) is a promising therapeutic target, and inhibiting Hsp90 is expected to inhibit multiple signaling pathways. FW-04-806 is a bioxazolyl macrolide compound extracted from the Chinese native Streptomyces FIM-04-806, and its structure has been reported to be identical to the polyketide compound Conglobatin. Methods: We employed chemical proteomics, computer-aided docking, immunoprecipitation, siRNA gene knockdown, quantitative real-time PCR, and xenograft models to investigate the antitumor mechanism of FW-04-806 in the HER2-overexpressing breast cancer cell line SKBR3 and the HER2-low-expressing breast cancer cell line MCF-7. Results: We confirmed that FW-04-806 directly binds to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits the Hsp90/cyclin 37 (Cdc37) molecular chaperone/helper chaperone interaction, but does not affect the ATP-binding capacity of Hsp90, thereby leading to the degradation of various Hsp90 substrate proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibited cell proliferation in a dose- and time-dependent manner, leading to G2/M phase cell cycle arrest, inducing apoptosis, and downregulating the Hsp90 substrate proteins HER2, Akt, Raf-1, and their phosphorylated forms (p-HER2, p-Akt). Importantly, FW-04-806 exhibited better anti-tumor effects in the HER2-overexpressing SKBR3 tumor xenograft model than in the HER2-low-expressing MCF-7 model. This result is consistent with cell proliferation and in vitro apoptosis assays performed on SKBR-3 and MCF-7 cells. Furthermore, FW-04-806 exhibited favorable toxicity characteristics. Conclusion: As a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminus of Hsp90, inhibiting the interaction between Hsp90 and Cdc37, leading to the dissociation of the Hsp90/Cdc37/substrate complex and the degradation of the Hsp90 substrate protein. FW-04-806 showed good antitumor activity against breast cancer cells in vitro and in vivo, especially against HER2-overexpressing breast cancer cells. [2] The lack of new drugs for the treatment of esophageal squamous cell carcinoma (ESCC) limits treatment options. In this study, the therapeutic effects and mechanism of action of a novel natural macrolide compound, F806, were elucidated in human ESCC xenograft models and cell lines. F806 inhibited the growth of ESCC, and more importantly, it had fewer side effects on normal tissues in two human ESCC xenograft models. F806 inhibited the proliferation of six ESCC cell lines, with half-maximal inhibitory concentrations (IC50) ranging from 9.31 to 16.43 μM. In addition, F806 induced apoptosis in ESCC cells, thereby exerting its growth inhibitory effect. At the same time, F806 inhibited cell adhesion, leading to apoptosis. Mechanistic studies showed that F806 partially inhibited the activation of β1 integrin by binding to a novel site, Arg610, thereby inhibiting the formation of adhesion focals, reducing cell adhesion to the extracellular matrix, and ultimately inducing apoptosis. We conclude that F806 may be a well-tolerated anticancer drug that induces anodic apoptosis in esophageal squamous cell carcinoma (ESCC) cells by targeting β1 integrin. [3]

C28H38N2O6

498.273

C, 67.45; H, 7.68; N, 5.62; O, 19.25

72263-05-9

6440452

White to off-white solid powder

1.06 g/cm3

673.4ºC at 760 mmHg

361.1ºC

1.484

5.502

0

8

4

36

746

6

C[C@H]1/C=C(\C(=O)O[C@H]([C@H](C[C@H](/C=C(\C(=O)O[C@H]([C@H](C1)C)CC2=CN=CO2)/C)C)C)CC3=CN=CO3)/C

LAJRJVDLKYGLOO-NLISZJEWSA-N

InChI=1S/C28H38N2O6/c1-17-7-19(3)25(11-23-13-29-15-33-23)35-28(32)22(6)10-18(2)8-20(4)26(12-24-14-30-16-34-24)36-27(31)21(5)9-17/h9-10,13-20,25-26H,7-8,11-12H2,1-6H3/b21-9-,22-10-/t17-,18-,19+,20+,25+,26+/m1/s1

(3Z,5R,7S,8S,11Z,13R,15S,16S)-3,5,7,11,13,15-hexamethyl-8,16-bis(1,3-oxazol-5-ylmethyl)-1,9-dioxacyclohexadeca-3,11-diene-2,10-dione

72263-05-9; 3,5,7,11,13,15-Hexamethyl-8,16-bis(1,3-oxazol-5-ylmethyl)-1,9-dioxacyclohexadeca-3,11-diene-2,10-dione; (3E,5R,11E)-3,5,7S,11,13R,15S-hexamethyl-8S,16S-bis(5-oxazolylmethyl)-1,9-dioxacyclohexadeca-3,11-diene-2,10-dione

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)