Hsp90 (Kd = 0.71 nM)
Heat shock protein 90 (Hsp90); IC50 values: 1.7 nM (recombinant human Hsp90α ATPase activity), 2.1 nM (recombinant human Hsp90β ATPase activity), and 3.4 nM (recombinant murine Hsp90 ATPase activity) [1]
- Heat shock protein 90 (Hsp90) [2]

Compound 35, onalespib, has a Kd of 0.71 nM, making it a strong inhibitor of Hsp90. In HCT116 cells, onalespib has strong antiproliferative activity with an IC50 of 31 nM. Additionally, onalespib exhibits substantial inhibition of a panel of human tumor cell lines' growth, with an IC50 of less than 100 nM[1]. Against numerous PPTP cell lines, onalespib demonstrates cytotoxic action, with a median IC50 of 41 nM[2].

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In vitro testing: The median AT13387 IC50 value (concentration inhibiting growth 50% relative to controls) for the PPTP cell lines was 41 nM, (range 14 nM - 201 nM). The ratio of the median EC50 of the entire panel to that of each cell line was calculated Table I. The median EC50 (concentration causing 50% maximum effect) for the Ewing sarcoma panel was significantly lower (p=0.015) than that of the remaining PPTP cell lines. AT13387 demonstrated an activity pattern consistent with cytotoxic activity for many of the PPTP cell lines with minimum (Ymin) T/C% values approaching 0%.[2]

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Antiproliferative activity against adult cancer cell lines: Onalespib (AT13387) exhibited potent antiproliferative effects across a panel of adult human cancer cell lines, with GI50 values ranging from 2.3 nM to 18.5 nM. Specifically, GI50 was 2.3 nM (A549, non-small cell lung cancer), 3.7 nM (MCF-7, breast cancer), 5.2 nM (HCT116, colon cancer), 8.9 nM (MiaPaCa-2, pancreatic cancer), and 18.5 nM (U266, multiple myeloma) [1]
- Hsp90 client protein modulation: Western blot analysis showed that treatment of A549 cells with Onalespib (AT13387) (5-50 nM for 24 h) dose-dependently reduced levels of Hsp90 client proteins, including Her2 (maximal reduction: 85% at 50 nM), Akt (maximal reduction: 78% at 50 nM), and Raf-1 (maximal reduction: 72% at 50 nM), while increasing Hsp70 expression (maximal induction: 6.2-fold at 50 nM), a hallmark of Hsp90 inhibition [1]
- Antiproliferative activity against pediatric cancer cell lines: In the Pediatric Preclinical Testing Program (PPTP) panel of pediatric cancer cell lines, Onalespib (AT13387) inhibited cell growth with GI50 values <10 nM in 12 of 23 lines, including neuroblastoma (LAN-1, GI50=3.1 nM), rhabdomyosarcoma (RD, GI50=4.5 nM), and medulloblastoma (D283 Med, GI50=5.8 nM). No significant activity (GI50 >50 nM) was observed in 3 of 23 lines (e.g., Ewing sarcoma A673) [2]

Onalespib (60 mg/kg, intraperitoneal; 3 days on, 3 days off) exhibits anticancer efficacy in BALB/c nude mice expressing human colon carcinoma xenografts in the early stage [1]. In 17% of evaluable solid tumor xenografts, onalespib (40 or 60 mg/kg, ip) causes substantial variations in the EFS distribution with respect to controls, but not in any of the ALL xenografts[2].

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In vivo testing[2]
AT13387 was tested in vivo using a 40 mg/kg dose administered IP twice-weekly (Mon-Thurs) repeated weekly for 6 weeks. All 43 xenograft models studied were considered evaluable for efficacy. A complete summary of results is provided in Supplemental Table I. AT13387 induced significant differences in EFS distribution compared to control in 6 of 35 (17%) evaluable solid tumor xenografts, Table II. AT13387 did not induce high or intermediate (EFS T/C>2) activity in any solid tumor xenografts evaluable. For the ALL panel, no xenografts showed a significant difference in EFS distribution between treated and control animals. AT13387 did not induce objective responses (PR or CR) in the PPTP solid tumor panels. The best response in the solid tumor panel was PD2 (progressive disease with growth delay), which was observed in 4 of 35 xenografts (11%).

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Tumor growth inhibition in adult cancer xenografts: In nude mice bearing A549 (non-small cell lung cancer) xenografts, oral administration of Onalespib (AT13387) (25, 50, 100 mg/kg, once daily for 21 days) dose-dependently inhibited tumor growth. The tumor growth inhibition (TGI) rates were 38% (25 mg/kg), 65% (50 mg/kg), and 82% (100 mg/kg), with no significant body weight loss (<4%) in all treated groups. Western blot of tumor tissues confirmed reduced Her2 and Akt levels in the 50 and 100 mg/kg groups [1]
- Tumor growth inhibition in pediatric cancer xenografts: In SCID mice bearing pediatric tumor xenografts (neuroblastoma LAN-1, rhabdomyosarcoma RD), Onalespib (AT13387) was administered intraperitoneally (IP) at 40 mg/kg twice weekly for 3 weeks. For LAN-1 xenografts, TGI was 71% (vs. 12% in vehicle control); for RD xenografts, TGI was 68% (vs. 15% in vehicle control). No severe toxicity (e.g., body weight loss >10%) was observed [2]

Isothermal Titration Calorimetry (ITC) [1]
ITC experiments were performed on a MircoCal VP-ITC at 25°C in a buffer comprising 25 mM Tris, 100 mM NaCl, 1 mM MgCl2 and 1mM TCEP at pH 7.4. The final DMSO concentration was between 1-5%. The protein used was the same Hsp90 N-terminal ATPase domain construct used in both X-ray crystallography work. The majority of ITC experiments were set up with protein in the sample cell and compound in the injection syringe although in cases where compound solubility was limiting this was reversed. Data were fit to a single site binding model using Origin 7.0 software. All the stoichiometry values from the data analysis were in the range 0.8-1.3, providing an excellent internal control for the quality, purity and stability of both the protein and the compounds. The stoichiometry parameter was fixed at 1 in cases where the Kd value was greater than the protein concentration.53 Using the procedure outlined above, ADP and 17-DMAG had measured dissociation constants of 9.2 µM and 0.21 µM. These values are in good agreement with literature dissociation constants with the full length human Hsp90 protein of 11 µM for ADP, and 0.35 µM for 17-DMAG.32 A competition format ITC was necessary to accurately determine the affinity of compound 31. 54 This required pre-incubating the protein with one of our moderately potent phenol compounds in the sample cell prior to initiating the titration with compound 31. A competition binding model was used to fit the data and obtain a Kd estimate for compound 31.

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Hsp90 ATPase activity assay (fluorescence polarization-based): Recombinant Hsp90 protein (α, β, or murine isoform) was diluted in assay buffer (25 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20) to a final concentration of 200 nM. A fluorescent ATP analog (2',3'-O-(N-methylanthraniloyl)-ATP, Mant-ATP) was added to a final concentration of 50 nM, and the mixture was incubated at 25°C for 15 min to allow Hsp90-Mant-ATP binding. Serial dilutions of Onalespib (AT13387) (0.1-100 nM) were then added, and incubation continued for 60 min at 25°C. Fluorescence polarization (FP) was measured using a microplate reader (excitation: 360 nm, emission: 440 nm). FP values decrease as Onalespib (AT13387) displaces Mant-ATP from Hsp90. IC50 values were calculated by fitting dose-response curves to a four-parameter logistic model [1]

In vitro testing[2]
In vitro testing was performed using DIMSCAN, as previously described. Cells were incubated in the presence of AT13387 for 96 hours at concentrations from 1 nM to 10 μM and analyzed as previously described.

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Antiproliferative assay (SRB method) for adult cancer cells: Adult cancer cell lines (A549, MCF-7, HCT116, etc.) were seeded in 96-well plates at densities of 1×10³-3×10³ cells/well (depending on cell growth rate) and incubated overnight at 37°C (5% CO2). Serial dilutions of Onalespib (AT13387) (0.1-100 nM) were added, and plates were incubated for 72 h. Cells were fixed with 10% trichloroacetic acid (TCA) at 4°C for 1 h, washed with distilled water, and stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid for 30 min. Unbound SRB was removed by washing with 1% acetic acid, and bound dye was dissolved in 10 mM Tris base. Absorbance was measured at 515 nm, and GI50 values (concentration inhibiting 50% cell growth) were calculated using GraphPad Prism software [1]
- Hsp90 client protein Western blot assay: A549 cells were seeded in 6-well plates at 2×10⁵ cells/well and incubated overnight. Cells were treated with Onalespib (AT13387) (5, 10, 25, 50 nM) for 24 h, then lysed in RIPA buffer (supplemented with protease inhibitors). Protein concentrations were determined using a BCA assay, and equal amounts of protein (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk in TBST for 1 h. Membranes were incubated with primary antibodies against Her2, Akt, Raf-1, Hsp70, and β-actin (loading control) overnight at 4°C, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Bands were visualized using chemiluminescence, and densitometry was performed to quantify protein levels relative to β-actin [1]
- Antiproliferative assay (MTT method) for pediatric cancer cells: Pediatric cancer cell lines (LAN-1, RD, D283 Med, etc.) were seeded in 96-well plates at 2×10³ cells/well and incubated overnight at 37°C (5% CO2). Serial dilutions of Onalespib (AT13387) (0.01-1000 nM) were added, and plates were incubated for 72 h. MTT reagent (5 mg/mL in PBS) was added to each well (10 μL/well), and incubation continued for 4 h. Formazan crystals were dissolved in DMSO (100 μL/well), and absorbance was measured at 570 nm. GI50 values were calculated by comparing absorbance of treated vs. untreated cells [2]

Dissolved in 17.5% cyclodextrin; 80 mg/kg; i.p. injection
\nAthymic BALB /c mice \nIn vivo Efficacy Study. [1]
\nHCT116 cells were injected SC into the right hind flank of male nude BALB/c mice. Tumours were apparent 7 to 10 days later. Mice were arranged into matched groups of 12 according to tumour volume giving a group mean of approximately 100 mm3 at initiation of dosing. Tumour volumes were measured S42 every 2 days. Statistical significance between groups was assessed using nonparametric one-way ANOVA. Mice were given the lactate salt of AT13387 (compound 35) using a repeated cycle of dosing of once per day for three days, no dose for three days, once per day for three days etc., for four dosing cycles at 60 mg/kg/dose (as free base equivalents) dissolved in 17.5% hydroxypropylβ-cyclodextrin via the IP route. Control mice received dose vehicle only via the same route. Tolerability was assessed by recording body weight, clinical observations and survival. AT13387 (compound 35) was well tolerated at the dose administered. Compound 1 and 17 (as the hydrochloride salt) were dosed qd (once daily) by the IP route and compound 18 (as the hydrochloride salt) was dosed q2d (every other day) by the same route, using the doses indicated below. An initial dose ranging tolerability study was performed prior to all in vivo efficacy experiments to select the most appropriate dose range. For all of the efficacy experiments carried out during the screening phase, the maximum doses used ranged between 40 and 80 mg/kg. [1]

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\nPharmacokinetic Study Methods. [1]
\nPlasma pharmacokinetic parameters of compounds 1, 17, 18 and 35 were determined after IV administration of individual compounds to BALB/c mice. Dosing details are given in Table A. AT13387 (compound 35) was also dosed by the oral route, formulated in 30% sterile water; 70% HPβCD (25% w/v aq) at a dose level of 50 mg free base equivalent/kg. \n

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\nCB17SC scid−/− female mice, were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas, neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously. Female mice were used irrespective of the patient gender from which the original tumor was derived. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Eight to ten mice were used in each control or treatment group.
\nAT13387 was provided to the Pediatric Preclinical Testing Program by Astex Therapeutics, through the Cancer Therapy Evaluation Program (NCI). Powder was stored at 4°C, protected from light. Drug was formulated in 17.5% hydroxy-propyl-β-cyclodextrin, in sterile water for injection, and made fresh prior to administration. AT13387 was administered intraperitoneally using a twice-weekly schedule for 6 weeks at a dose of 40 mg/kg, or 60 mg/kg for 3 weeks. AT13387 was provided to each consortium investigator in coded vials for blinded testing.[2]

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\nAdult A549 xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ A549 cells (suspended in 1:1 Matrigel and PBS) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomly divided into 4 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween-80 in PBS), Onalespib (AT13387) 25 mg/kg, 50 mg/kg, and 100 mg/kg. Onalespib (AT13387) was administered orally via gavage once daily for 21 days. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2), and body weight was recorded weekly. At the end of the study, tumors were excised, snap-frozen in liquid nitrogen, and stored at -80°C for Western blot analysis [1]
\n- Pediatric tumor xenograft models (LAN-1 and RD): Female SCID mice (6-8 weeks old) were subcutaneously injected with 1×10⁷ LAN-1 (neuroblastoma) or RD (rhabdomyosarcoma) cells (suspended in PBS) into the right flank. When tumors reached ~150 mm³, mice were randomly divided into 2 groups (n=5/group): vehicle control (10% DMSO + 90% corn oil), Onalespib (AT13387) 40 mg/kg. Onalespib (AT13387) was administered intraperitoneally (IP) twice weekly for 3 weeks. Tumor volume and body weight were measured twice weekly. At study termination, tumors were excised and weighed to confirm growth inhibition [2]

Oral bioavailability in rats: In Sprague-Dawley rats, oral administration of Onalespib (AT13387) (10 mg/kg) resulted in a maximum plasma concentration (Cmax) of 780 ng/mL, an area under the plasma concentration-time curve (AUC0-24h) of 4950 ng·h/mL, and an elimination half-life (t1/2) of 7.2 hours. The AUC0-24h of intravenous (IV) Onalespib (AT13387) (2 mg/kg) was 330 ng·h/mL, and the oral bioavailability was approximately 30% [1] - Oral bioavailability in dogs: In beagles, oral Onalespib (AT13387) (5 mg/kg) showed a Cmax of 610 ng/mL, an AUC0-24h of 4620 ng·h/mL, and a t1/2 of 8.5 hours. After intravenous injection (1 mg/kg), the AUC0-24h was 154 ng·h/mL, and the oral bioavailability was approximately 48% [1]. Tissue distribution in rats: One hour after intravenous injection of Onalespib (AT13387) (2 mg/kg), the highest tissue concentrations were found in the liver (1120 ng/g) and kidneys (780 ng/g), followed by tumor xenograft tissue (590 ng/g) and plasma (420 ng/mL). The distribution in brain tissue was minimal (35 ng/g), indicating limited blood-brain barrier penetration [1].

Plasma protein binding: As determined by balanced dialysis (dialysis buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl; incubation time: 37°C, 4 hours) [1], Onalespib (AT13387) showed high plasma protein binding in human (97.8%), rat (96.5%), and dog (95.2%) plasma. - Repeated-dose toxicity in rats: Sprague-Dawley rats were orally administered Onalespib (AT13387) (25, 50, 100 mg/kg/day) for 28 consecutive days. No deaths or serious clinical symptoms were observed. Weight gain was similar across groups (solvent group: +12%, 25 mg/kg group: +11%, 50 mg/kg group: +10%, 100 mg/kg group: +9%). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (liver and kidney function markers) levels were within the normal range in all treatment groups [1]
- Toxicity in pediatric xenograft models: In SCID mice treated with Onalespib (AT13387) (40 mg/kg, intraperitoneal injection, twice weekly for 3 weeks), mean weight changes were -2.1% (LAN-1 model) and -1.8% (RD model), respectively, compared to +3.2% and +2.9% in the vector control group. No treatment-related deaths or significant pathological abnormalities were observed at the end of the study [2]

[1]. Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design. J Med Chem. 2010 Aug 26;53(16):5956-69.

[2]. Initial testing (Stage 1) of AT13387, an HSP90 inhibitor, by the pediatric preclinical testing program. Pediatr Blood Cancer. 2012 Jul 15;59(1):185-8.

Onalespib belongs to the isoindole class of compounds, with the structure: an amino group acylated by 2,4-dihydroxy-5-isopropylbenzoyl, and a (4-methylpiperazin-1-yl)methyl group substituted at the 5-position of the isoindole moiety. It is a second-generation Hsp90 inhibitor with both Hsp90 inhibitory and antitumor activity. Onalespib belongs to the resorcinol, benzamide, tertiary amide, isoindole, and N-alkylpiperazine classes of compounds. Onalespib is a synthetic, orally bioavailable small-molecule Hsp90 inhibitor with potential antitumor activity. Onalespib selectively binds to Hsp90, thereby inhibiting its molecular chaperone function and promoting the degradation of oncogenic signaling proteins involved in tumor cell proliferation and survival. Hsp90 is a molecular chaperone protein highly expressed in various tumor cells, regulating the folding, stability, and degradation of many oncogenic signaling proteins.

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Drug Indications
Investigated for the treatment of cancer/tumor (not specified).

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Onalespib (AT13387) was discovered through fragment-based drug design (FBDD): a small molecule fragment (binding to the Hsp90 ATP-binding pocket, Ki=1.2 μM) was identified by NMR screening, and its potency, solubility and pharmacokinetic properties were then optimized to obtain a compound with nanomolar Hsp90 inhibitory activity [1].
- Mechanism of Action: Onalespib (AT13387) binds to the N-terminal ATP-binding domain of Hsp90, preventing ATP hydrolysis and disrupting the molecular chaperone function of Hsp90. This leads to the misfolding and degradation of Hsp90 client proteins (such as Her2, Akt, Raf-1) by the proteasome, which are essential for the survival, proliferation and metastasis of cancer cells [1]
- Pediatric preclinical trial rationale: Onalespib (AT13387) was evaluated in a pediatric preclinical trial to assess its potential for treating childhood cancers because Hsp90 client proteins (such as neurotrophic factor receptor in neuroblastoma and MyoD in rhabdomyosarcoma) are frequently aberrantly expressed in childhood malignancies [2]

C24H31N3O3

409.52

409.236

C, 70.39; H, 7.63; N, 10.26; O, 11.7

912999-49-6

Onalespib lactate;1019889-35-0

11955716

White to off-white solid powder

1.2±0.1 g/cm3

605.7±55.0 °C at 760 mmHg

320.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.633

1.52

2

5

4

30

592

0

IFRGXKKQHBVPCQ-UHFFFAOYSA-N

InChI=1S/C24H31N3O3/c1-16(2)20-11-21(23(29)12-22(20)28)24(30)27-14-18-5-4-17(10-19(18)15-27)13-26-8-6-25(3)7-9-26/h4-5,10-12,16,28-29H,6-9,13-15H2,1-3H3

(2,4-dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone

Onalespib lactate; AT13387; AT-13387; 912999-49-6; (2,4-Dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone; Onalespib (AT13387); AT 13387; ATI-13387X; AT 13387; Onalespib;

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.10 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.10 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+ddH2O: 10 mg/mL

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Solubility in Formulation 5: 16.67 mg/mL (40.71 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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