Ezrin (KD = 5.85 μM ± 3.85 μM from SPR binding assay)
Protein Kinase C iota (PKCI) (IC50 = 8.3 μM for inhibition of PKCI-mediated ezrin phosphorylation in vitro kinase assay; KD = 172.4 μM from SPR binding assay)
Moesin (IC50 = 9.4 μM for inhibition of PKCI phosphorylation in vitro)
Radixin (IC50 = 55 μM for inhibition of PKCI phosphorylation in vitro) [1]

The ezrin inhibitor NSC305787 hydrochloride has anti-tumor action and a Kd of 5.85 μM. Ezrin, Moesin, Radixin, and MBP's PKCI phosphorylation is inhibited by NSC305787, with IC50 values of 8.3, 9.4, 55, and 58.9 μM, respectively. With a Kd value of 172.4 μM, NSC305787 binds to PKCI and primarily blocks ezrin T567 phosphorylation by binding to ezrin instead of PKCI kinase activity. NSC305787 (1, 10 μM) has inhibitory effect against K7M2 osteosarcoma (OS) cell invasion mediated by ezrin. Moreover, NSC305787 (10 μM) inhibits the growth of OS metastases in lung organ preparations and decreases the cell motility phenotype of zebrafish [1].

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NSC305787 directly binds to recombinant wild-type ezrin protein with an average KD of 5.85 μM as determined by surface plasmon resonance (SPR). It inhibits the phosphorylation of recombinant ezrin at threonine 567 (T567) by protein kinase C iota (PKCI) in an in vitro kinase assay with an IC50 of 8.3 μM. This inhibition is primarily due to its binding to ezrin rather than direct inhibition of PKCI kinase activity, as its IC50 for PKCI activity on a non-specific substrate (myelin basic protein) is higher, and its binding affinity for PKCI (KD = 172.4 μM) is significantly weaker than for ezrin. [1]
NSC305787 (10 μM) inhibits T567 phosphorylation and actin binding of endogenous ezrin in highly metastatic K7M2 osteosarcoma (OS) cells without altering cellular ezrin levels. [1]
NSC305787 (10 μM) inhibits the invasion of high-ezrin-expressing K7M2 OS cells through a human umbilical vein endothelial cell (HUVEC) monolayer in an electric cell impedance sensing assay. This inhibition is statistically significant compared to the DMSO control at 5 hours post-treatment. It does not inhibit the invasion of low-ezrin-expressing K12 cells and shows no cytotoxicity to K7M2, K12, or HUVEC cells at this concentration. [1]
NSC305787 (10-20 μM) treatment of zebrafish embryos during gastrulation mimics the phenotype induced by ezrin morpholino knockdown, characterized by epiboly defects (cells piling up at the animal pole instead of spreading over the yolk cell). At 20 μM, 100% of embryos exhibited this phenotype. [1]
NSC305787 (10 μM) treatment of Xenopus embryos causes gastrulation defects at stage 12.5, and by stage 37 leads to developmental delays and ventral defects. By stage 50, embryos show short and bent tails and reduced eyes. [1]
In an ex vivo mouse lung organ culture assay, NSC305787 (10 μM) significantly inhibits the growth of metastatic foci formed by GFP-expressing K7M2 OS cells in lung slices. [1]

In mouse lungs, NSC305787 (0.240 mg/kg/day, intraperitoneal injection) prevents ezrin-dependent osteosarcoma from spreading [1]. In a transgenic mouse model of osteosarcoma (Osx-Cre+p53fl/flpRBfl/fl), NSC305787 (240 μg/kg, i.p.) significantly inhibits lung metastasis and seems to be more beneficial than NSC668394 in the mouse model. dynamic qualities [2].

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In a murine experimental metastasis model, female BALB/c mice were injected with GFP-expressing K7M2 OS cells via tail vein. Treatment with NSC305787 at 0.240 mg/kg/day, administered intraperitoneally 5 days a week starting one day after tumor cell injection, significantly extended median survival compared to the vehicle-treated group (50 days vs. 28.5 days). The overall survival of the treated group was statistically different from the control group. Analysis of lung tissues showed a significant decrease in the number of GFP-expressing metastatic foci in the treated animals. [1]
In a control experiment using MNNG-HOS OS cells (which have an ezrin-independent metastatic phenotype), treatment with NSC305787 at the same regimen showed no difference in survival or the number of lung metastatic foci compared to the vehicle group, indicating the in vivo effect is specific to ezrin-dependent metastasis. [1]

For surface plasmon resonance (SPR) binding assays, recombinant wild-type ezrin protein was immobilized on a sensor chip. NSC305787 was injected over the surface at varying concentrations (1-64 μM). Binding interactions were recorded in real-time, and steady-state affinity analysis was performed to calculate the dissociation constant (KD). [1]
For in vitro kinase assays to measure inhibition of ezrin phosphorylation, recombinant ezrin protein was incubated with varying concentrations of NSC305787 (1-100 μM) on ice for 15 minutes. Recombinant PKCI was then added, and the kinase reaction was allowed to proceed at 30°C for 30 minutes. The reaction was stopped, and phosphorylation levels were analyzed. Inhibition of PKCI activity on a non-specific substrate (myelin basic protein) was also tested using a radioactive in vitro kinase assay. [1]

For immunoprecipitation and immunoblotting to assess ezrin phosphorylation and actin binding, K7M2 cells were treated with NSC305787 (10 μM) for 6 hours. Cells were lysed, and endogenous ezrin was immunoprecipitated using an ezrin antibody. The immunoprecipitates were then subjected to immunoblotting with antibodies against phospho-ezrin (T567), actin, and total ezrin. [1]
For the invasion assay using an electric cell impedance sensing system, human umbilical vein endothelial cells (HUVECs) were seeded to form a confluent monolayer. A layer of K7M2 osteosarcoma cells was then added in media containing NSC305787 (10 μM). Invasion was monitored in real-time for 5 hours by measuring changes in electrical resistance at the cell-electrode interface. The cell index, representing the level of invasion, was calculated from the resistance data. [1]
For the cell migration assay, K7M2 cells were placed in the upper chamber of a migration plate in serum-free media containing NSC305787 at various concentrations. The lower chamber contained media with serum as a chemoattractant. Cell migration across the membrane was monitored in real-time over 24 hours using an electric cell impedance system. [1]

In the in vivo experimental metastasis model, GFP-expressing K7M2 osteosarcoma cells were injected into female BALB/c mice via the tail vein. Starting one day after tumor cell injection, mice were treated with vehicle (1% DMSO) or NSC305787 at a dose of 0.240 mg/kg/day. The compound was administered via intraperitoneal injection, 5 days a week. Mice were monitored for signs of morbidity (anorexia, dehydration, dyspnea, decreased activity). Animals were euthanized upon development of severe symptoms or at the end of the study (day 66). Lung tissues were harvested for analysis of metastatic burden. [1]

Following a single intravenous injection of NSC305787 (240 µg/kg) into female Balb/c mice, the plasma pharmacokinetic parameters were: Cmax = 195.1 ng/ml, AUCinf = 415.3 ng·h/ml, elimination half-life (T1/2) = 13.6 h, clearance (CI) = 9.6 ml/min/kg, and volume of distribution (V) = 11.3 L/kg. [2] Following a single intraperitoneal injection of NSC305787 (240 µg/kg), NSC305787 was detectable in plasma for at least 6 hours, and the exposure was relatively stable (approximately 16 ng/ml at 6 hours). In contrast, NSC668394 was undetectable 1 hour after intraperitoneal injection. [2]
The mean steady-state plasma concentration of NSC305787 was estimated to be 20.0–35.0 nM within 6–14 hours after intraperitoneal or intravenous injection. [2]

In cell viability assays, NSC305787 at a concentration of 10 μM did not show cytotoxicity against K7M2, K12 osteosarcoma cells, or human umbilical vein endothelial cells (HUVECs). [1] In zebrafish embryo experiments, compounds that cause embryonic death before 70% epiblast formation during gastrulation were excluded according to toxicity criteria; NSC305787 did not meet this criterion, but induced specific developmental phenotypes at concentrations of 10–20 μM. [1] In a mouse maximum tolerated dose study, all five animals survived after 5 days of intraperitoneal injection of NSC305787 at a dose of 2.4 mg/kg/day, indicating good tolerability at this dose and administration regimen. In an in vivo metastasis model, no significant acute toxicity was observed at a treatment dose of 0.240 mg/kg/day. [1]

[1]. Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells. Oncogene. 2012 Jan 19;31(3):269-81.

[2]. Ezrin Inhibition Up-regulates Stress Response Gene Expression. J Biol Chem. 2016 Jun 17;291(25):13257-70.

NSC305787 is a small molecule compound that was discovered by screening 3081 compounds using surface plasmon resonance (SPR) technology. This compound can directly bind to ezrin. It has a functionalized quinoline pharmacophore. Calculation analysis predicted its partition coefficient to be 5.8, its molecular weight to be 443 g/mol, and it conforms to the Lipinski five rules, indicating that it has drug-like properties. Its mechanism of action involves direct binding to ezrin, thereby inhibiting phosphorylation at its T567 site, which in turn inhibits subsequent functions related to cell motility and metastasis. It exhibits specific anti-metastatic activity in an ezrin-dependent cancer model. [1]

C25H31CL3N2O

481.8854

480.15

53868-26-1

(Rac)-NSC305787;785718-37-8

54607543

White to off-white solid powder

3

3

3

31

603

0

JQALIVSYOXLOBE-UHFFFAOYSA-N

InChI=1S/C25H30Cl2N2O.ClH/c26-17-8-18-19(24(30)21-3-1-2-4-28-21)10-22(29-23(18)20(27)9-17)25-11-14-5-15(12-25)7-16(6-14)13-25;/h8-10,14-16,21,24,28,30H,1-7,11-13H2;1H

(2-(adamantan-1-yl)-6,8-dichloroquinolin-4-yl)(piperidin-2-yl)methanol hydrochloride

NSC-305787HCl; NSC 305787 Hydrochloride; NSC305787 Hydrochloride; NSC-305787 HCl; NSC 305787 HCl; NSC305787 HCl;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~8 mg/mL (~16.60 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.59 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 12.5 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: ≥ 1.25 mg/mL (2.59 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 12.5 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: ≥ 1.25 mg/mL (2.59 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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