HSP70 (IC50 = 0.5 μM); HSC70 (IC50 = 2.6 μM); Grp78 (IC50 = 2.6 μM)

VER-155008 exhibits no activity against Hsp90, with an IC50 of >200 μM, however it is an inhibitor of Hsc70 and Hsp70, with IC50s of 0.5 μM, 2.6 μM, and 2.6 μM for Hsp70, Hsc70, and Grp7, respectively. BT474, MB-468, HCT116, and HT29 cells are among the human colon and breast tumor cell lines that VER-155008 suppresses in terms of proliferation, with GI50s of 10.4 μM, 14.4 μM, 5.3 μM, and 12.8 μM, respectively. HCT116 and BT474 cancer cells exhibit client protein degradation in response to VER-155008 (5–40 μM). Moreover, VER-155008 causes human tumor cell lines to undergo apoptosis[1]. In cultured neurons, VER-155008 (0.05-5 μM) restores Aβ-induced axonal degeneration[2]. VER-155008 (10 μM or 25 μM) inhibits Hsp70 and stops LNCaP95 cells from proliferating. Additionally, androgen receptor splice variation 7 (AR-V7) and full-length androgen receptor (AR-FL) protein expression are decreased by VER-155008[3].

In naive female BALB/c mice, VER-155008 (25 mg/kg, iv) shows plasma clearance. In HCT116 tumor-bearing nude BALB/c mice, VER-155008 (40 mg/kg, iv) likewise exhibits quick plasma clearance and lowers tumor levels[1]. In 5XFAD mice, VER-155008 (10 μmol/kg/day, ip) lessens axonal swelling linked to amyloid plaques and improves memory deficits. VER-155008 (89.9 μmol/kg/day, ip) also reduces amyloid plaques and the amyloid plaque-associated protein PHF-tau in 5XFAD mice[2].

Hsc70, Hsp70, Grp78 and Hsp90 fluorescence polarisation (FP) assay[1]
The activity of VER-155008 was determined against Hsp70 and Hsp90 as previously described. Hsc70 (aa 5-381) and Grp78 (ATPase domain) were cloned and expressed as follows. A PCR fragment of Hsc70 corresponding to amino acids 5–381 was made. The purified PCR fragment was ligated into pCR2.1-TOPO. This was subsequently restriction digested and sub-cloned into the commercial histidine tagged vector pET101 for expression analysis. The ATPase domain of Grp78 was amplified by PCR and cloned into pCR2.1-TOPO. This was subsequently digested and ligated into the glutathione-s-transferase tagged expression vector pGEX-4T-1. The FP assay for Hsc70 and Grp78 was carried out as described for Hsp70 using the same N 6-(6-amino)hexyl-ATP-5-FAM as the FP probe with the following modifications. For Hsc70, the protein and probe concentrations were 0.3 μM and 20 nM, respectively with a 30 min incubation at 22°C while for Grp78, the protein and probe concentrations were 2 μM and 10 nM, respectively with a 2 h incubation at 22°C. The K D for the FAM-ATP probe was 0.24 μM for Hsc70 and 2 μM for Grp78.

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Determination of compound binding to Hsp70 by BiaCore[1]
All experiments were performed on a Biacore T100 at 25°C with a flow rate of 30 μL/min. The buffer system was identical to that used in the Hsp70 fluorescence polarization assay, with 1% DMSO and 0.05% Tween 20. Double His-tagged Hsp70 was immobilised on the surface of a NTA sensor chip; approximately 2,000 RU of protein were immobilised. Compounds were injected for 90 s, and the K Ds were determined from equilibrium binding at 80 s.

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Hsp70 ATPase assay[1]
ATP turnover, and the subsequent generation of ADP, was measured using an ADP Hunter Plus assay kit. Reaction mixtures were prepared in half volume, all black non-binding plates and contained 250 nM full length, GST-tagged Hsp70, 50 μM ATP and increasing concentrations of VER-155008 in the standard kit assay buffer. Reactions were incubated at 30°C for 90 min and then read on a FlexStation 3 with an E x of 530 nm and E m of 590 nm with six reads per well. Reactions containing no enzyme were set up to monitor the spontaneous hydrolysis of ATP and subtracted from those containing Hsp70.

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Refolding of heat denatured luciferase[1]
This assay was carried out essentially as previously described [24, 25]. In brief, Hsc70/Hsp70 inhibitors were diluted in Rabbit Reticulocyte Lysate (RRL, nuclease treated) containing 10 mM Tris, 100 mM KCl, 3 mM EDTA, 1 mM DTT and an ATP regeneration system at pH 7.5. The final ATP concentration was 0.5 mM. Compounds were pre-incubated in the above mix at room temperature for 15 min before 25 ng of heat denatured luciferase (denatured at 40°C for 30 min) was added to initiate the refolding reaction and incubated at 30°C for 60 min. Luciferase activity was determined using Bright-glo reagent.

Cell cycle analysis[1]
HCT116 cells were exposed to the indicated concentrations of VER-155008 and VER-82160 for 24 h. All cells were harvested and fixed in 70% ethanol on ice before being stained with RNaseA/propidium iodide. Data was collected on a FACSArray cytometer and analyzed with FACSDiva software.

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Androgen deprivation therapy is initially effective for treating patients with advanced prostate cancer; however, the prostate cancer gradually becomes resistant to androgen deprivation therapy, which is termed castration-resistant prostate cancer (CRPC). Androgen receptor splice variant 7 (AR-V7), one of the causes of CRPC, is correlated with resistance to a new-generation AR antagonist (enzalutamide) and poor prognosis. Heat shock protein 70 (Hsp70) inhibitor is known to decrease the levels of full-length AR (AR-FL), but little is known about its effects against CRPC cells expressing AR-V7. In this study, we investigated the effect of the Hsp70 inhibitors quercetin and VER155008 in the prostate cancer cell line LNCaP95 that expresses AR-V7, and explored the mechanism by which Hsp70 regulates AR-FL and AR-V7 expression. Quercetin and VER155008 decreased cell proliferation, increased the proportion of apoptotic cells, and decreased the protein levels of AR-FL and AR-V7. Furthermore, VER155008 decreased AR-FL and AR-V7 mRNA levels. Immunoprecipitation with Hsp70 antibody and mass spectrometry identified Y-box binding protein 1 (YB-1) as one of the molecules regulating AR-FL and AR-V7 at the transcription level through interaction with Hsp70. VER155008 decreased the phosphorylation of YB-1 and its localization in the nucleus, indicating that the involvement of Hsp70 in AR regulation might be mediated through the activation and nuclear translocation of YB-1. Collectively, these results suggest that Hsp70 inhibitors have potential anti-tumor activity against CRPC by decreasing AR-FL and AR-V7 expression through YB-1 suppression[3].

All in vivo studies were carried out according to UK Home Office guidelines and the “Guidance on the Operation of the Animals (Scientific Procedures) Act 1986”. Female BALB/c mice were dosed intravenously with 25 mg/kg VER-155008 into the lateral tail vein as a solution in 10% DMSO/5% Tween 80/85% saline (v/v/v). Animals were sacrificed at 5, 15 and 30 min, 1, 2, 4 and 6 h post dose.[1]
Female BALB/c nu/nu mice were inoculated with 5 × 106 HCT116 cells into the right flank. Tumors were monitored for growth, and animals were dosed IV with 40 mg/kg VER-155008 on day 31 (tumor volume approximately 200–250 mm3). Animals were sacrificed at 15 min, 1 and 4 h post dose.[1]
Each timepoint contained three mice and blood samples were collected by cardiac puncture. Plasma was obtained by centrifugation and frozen at −20°C prior to analysis. Tumors were harvested, snap frozen and stored at −80°C.[1]

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Alzheimer's disease (AD) is a progressive neurodegenerative disorder resulting in structural brain changes and memory impairment. We hypothesized that reconstructing neural networks is essential for memory recovery in AD. Heat shock cognate 70 (HSC70), a member of the heat shock protein family of molecular chaperones, is upregulated in AD patient brains, and recent studies have demonstrated that HSC70 facilitates axonal degeneration and pathological progression in AD. However, the direct effects of HSC70 inhibition on axonal development and memory function have never been investigated. In this study, we examined the effects of a small-molecule HSC70 inhibitor, VER-155008, on axonal morphology and memory function in a mouse model of AD (5XFAD mice). We found that VER-155008 significantly promoted axonal regrowth in amyloid β-treated neurons in vitro and improved object recognition, location, and episodic-like memory in 5XFAD mice. Furthermore, VER-155008 penetrated into the brain after intraperitoneal administration, suggesting that VER-155008 acts in the brain in situ. Immunohistochemistry revealed that VER-155008 reduced bulb-like axonal swelling in the amyloid plaques in the perirhinal cortex and CA1 in 5XFAD mice, indicating that VER-155008 also reverses axonal degeneration in vivo. Moreover, the two main pathological features of AD, amyloid plaques and paired helical filament tau accumulation, were reduced by VER-155008 administration in 5XFAD mice. This is the first report to show that the inhibition of HSC70 function may be critical for axonal regeneration and AD-like symptom reversal. Our study provides evidence that HSC70 can be used as a new therapeutic target for AD treatment[2].

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Formulation method 1: VER-155008 was dissolved in 10% dimethyl sulfoxide (DMSO)-containing saline and administered intraperitoneally (10 mmol/kg/day) to 5XFAD mice for 18 days. [1] Formulation method 2: Female BALB/c mice were dosed intravenously with 25 mg/kg VER-155008 into the lateral tail vein as a solution in 10% DMSO/5% Tween 80/85% saline (v/v/v). [2]

BALB/c mice

[1]. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother Pharmacol. 2010 Aug;66(3):535-45.

[2]. Heat Shock Cognate 70 Inhibitor, VER-155008, Reduces Memory Deficits and Axonal Degeneration in a Mouse Model of Alzheimer's Disease. Front Pharmacol. 2018 Jan 30;9:48.

[3]. Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells. Cancer Sci. 2017 Sep;108(9):1820-1827.

4-[[(2R,3S,4R,5R)-5-[6-amino-8-[(3,4-dichlorophenyl)methylamino]-9-purinyl]-3,4-dihydroxy-2-oxolanyl]methoxymethyl]benzonitrile is a purine nucleoside.

C25H23CL2N7O4

556.40

555.118

C, 53.97; H, 4.17; Cl, 12.74; N, 17.62; O, 11.50

1134156-31-2

25195348

Typically exists as White to pink solids at room temperature

1.6±0.1 g/cm3

856.3±75.0 °C at 760 mmHg

471.7±37.1 °C

0.0±0.3 mmHg at 25°C

1.748

2.71

4

10

8

38

831

4

ClC1=C(C([H])=C([H])C(=C1[H])C([H])([H])N([H])C1=NC2=C(N([H])[H])N=C([H])N=C2N1[C@@]1([H])[C@@]([H])([C@@]([H])([C@@]([H])(C([H])([H])OC([H])([H])C2C([H])=C([H])C(C#N)=C([H])C=2[H])O1)O[H])O[H])Cl

ZXGGCBQORXDVTE-UMCMBGNQSA-N

InChI=1S/C25H23Cl2N7O4/c26-16-6-5-15(7-17(16)27)9-30-25-33-19-22(29)31-12-32-23(19)34(25)24-21(36)20(35)18(38-24)11-37-10-14-3-1-13(8-28)2-4-14/h1-7,12,18,20-21,24,35-36H,9-11H2,(H,30,33)(H2,29,31,32)/t18-,20-,21-,24-/m1/s1

5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine

VER 155008; VER155008; VER-155008; VER-155008; VER 155008; 5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine; VER155008; C25H23Cl2N7O4; 4-((((2R,3S,4R,5R)-5-(6-Amino-8-((3,4-dichlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)benzonitrile; 5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)-methyl]amino]-adenosine;

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 (4.49 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 (4.49 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 (4.49 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: 1% CMC+0.5% Tween-80: 30 mg/mL

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