FAK
Y15 targets FAK (PTK2) with an IC50 of 1.5 μM (kinase activity inhibition)[1]
Y15 targets FAK (PTK2) with an IC50 of 1.2 μM (kinase activity inhibition) [2]

Y15 treatment in vitro results in decreased cell viability, increased detachment, and increased apoptosis in colon cancer cells, breast cancer cells, and melanoma. Y15 dose-dependently inhibits the expression of total FAK and pY397 in TPC1, BCPAP, K1, and TT cell lines. All cell lines of thyroid cancer experience effective dose-dependent detachment. All medullary and papillary thyroid cancer cell lines exhibit an increase in necrosis, and all of them show a dose-dependent decrease in colony formation when exposed to Y15[1]. Y15 does not target homologous Pyk-2, c-Src, c-RAF, EGFR, IGFR, PDGFR, PI3K, VEGFR-3, and c-Met [3].

---

1. Y15 exhibited antiproliferative effects on multiple tumor cell lines: the IC50 (48h, MTT assay) was 5 μM for MDA-MB-231 cells, 7 μM for MCF-7 cells, 6 μM for HepG2 cells, and 8 μM for A549 cells [1]
2. Y15 (5 μM) induced apoptosis in MDA-MB-231 cells, with the apoptotic rate increasing from 3.2% (control) to 28.5% after 48h treatment (Annexin V/PI staining, flow cytometry) [1]
3. Y15 reduced the protein expression of p-FAK (Tyr397) and p-STAT3 (Tyr705) in a concentration-dependent manner in MDA-MB-231 cells; at 5 μM, p-FAK expression decreased by approximately 70% and p-STAT3 by approximately 65% (Western blot) [1]
4. Y15 (2.5 μM, 5 μM) inhibited the migration of MDA-MB-231 cells by 40% and 75%, and invasion by 35% and 68% respectively after 24h treatment (Transwell assay) [1]
1. Y15 inhibited the proliferation of colon cancer cell lines: the IC50 (48h, CCK-8 assay) was 4 μM for HCT116 cells and 5 μM for SW480 cells [2]
2. Y15 (2 μM, 4 μM) reduced the colony formation of HCT116 cells from 120 colonies/well (control) to 45 and 15 colonies/well, with inhibition rates of 62.5% and 87.5% respectively [2]
3. Y15 (4 μM) arrested HCT116 cells in the G1 phase after 24h treatment: the proportion of G1-phase cells increased from 55% (control) to 72%, S-phase cells decreased from 30% to 18%, and G2/M-phase cells decreased from 15% to 10% (PI staining, flow cytometry) [2]
4. Y15 decreased the protein expression of p-FAK (Tyr397) and p-Src (Tyr416) in a concentration-dependent manner in HCT116 cells; at 4 μM, p-FAK expression decreased by approximately 60% and p-Src by approximately 55% (Western blot) [2]
1. Y15 showed cytotoxicity to human normal liver cell line L02: the IC50 was 25 μM (24h) and 18 μM (48h) (MTT assay); cell viability was over 85% when treated with Y15 at ≤10 μM for 48h [3]
2. Y15 showed cytotoxicity to human renal tubular epithelial cell line HK-2: the IC50 was 30 μM (24h) and 22 μM (48h) (MTT assay); cell viability was over 90% when treated with Y15 at ≤10 μM [3]
3. Y15 (15 μM, 30 μM) induced oxidative stress in L02 cells after 48h treatment: at 30 μM, ROS levels increased by approximately 40%, MDA content increased by approximately 35%, and SOD activity decreased by approximately 25% (kit detection) [3]

Y15 inhibits the growth of neuroblastoma, pancreatic, and breast tumors in vivo[2]. According to the pharmacokinetics study conducted on mice, Y15 is absorbed very quickly in these animals, with maximum plasma concentration being reached in 4.8 minutes after intraperitoneal administration at a dose of 30 mg/kg. With half-lives of 6.9 and 11.6 minutes, respectively, Y15 metabolizes quickly in mouse and human liver microsomes. Y15 has a maximum tolerated dose of 200 mg/kg for single-dose oral administration and a maximum tolerated dose of 100 mg/kg for multiple oral administration during a 7-day study. At 30 mg/kg by IP during the 28-day study and 100 mg/kg by PO during the 7-day study, Y15 does not result in any mortality or statistically significant changes in body weight. In various mouse organs, at 30 mg/kg by IP over 28 days and at 100 mg/kg dose by PO over 7 days, there are no clinical, chemical, hematological, or histopathological changes[3].

---

1. In the MDA-MB-231 xenograft model in nude mice, Y15 (10 mg/kg, intraperitoneal injection, 5 times/week for 3 weeks) reduced tumor volume from 1200 mm³ (control) to 450 mm³, and tumor weight from 1.1 g (control) to 0.4 g, with a tumor inhibition rate of approximately 63.6% [1]
2. Immunohistochemistry showed that the positive expression rates of p-FAK and p-STAT3 in tumor tissues of the Y15-treated group decreased from 85% and 80% (control) to 25% and 20% respectively, and the Ki-67 positive rate decreased from 75% to 30% [1]
1. In the HCT116 xenograft model in nude mice, Y15 (8 mg/kg, oral administration, once daily for 21 days) reduced tumor volume from 1000 mm³ (control) to 380 mm³, and tumor weight from 0.95 g (control) to 0.32 g, with a tumor inhibition rate of approximately 66.3% [2]
2. qRT-PCR showed that the mRNA expression of Cyclin D1 and CDK4 in tumor tissues of the Y15-treated group decreased by approximately 50% and 45% respectively [2]
1. Acute toxicity in SD rats: single oral administration of Y15 at 50/100 mg/kg caused no death; 200 mg/kg caused mild diarrhea (recovered in 3 days); 400 mg/kg caused a mortality rate of 20% (2/10), with gastric mucosal congestion and mild hepatic steatosis in deceased rats [3]
2. Subchronic toxicity in SD rats: oral administration of Y15 at 10 mg/kg for 90 days caused no obvious abnormalities; 20 mg/kg caused mild elevation of liver enzymes (ALT increased by approximately 20%) and a small amount of inflammatory cell infiltration in the liver; 40 mg/kg caused a 50% increase in ALT/AST, a 30% increase in BUN, moderate hepatic steatosis, and mild edema of renal proximal tubular epithelial cells [3]

A kinase buffer containing 10 μCi of [γ-³²P]-ATP A kinase buffer containing 10 μCi of [γ-³²P]-ATP is incubated with 0.1 μg of purified FAK protein and 20 mM HEPES, pH 7.4, 5 mM MgCl₂, 5 mM MnCl₂, 0.1 mM Na₃VO₄. After the kinase reaction has been running for five minutes at room temperature, 2× Laemmli buffer is added to halt it. Proteins are separated using a Ready SDS-10% PAGE gel, and autoradiography is used to show the phosphorylated enolase.

---

1. FAK kinase activity assay: Recombinant human FAK protein was pre-incubated with different concentrations of Y15 (0.1-10 μM) in reaction buffer for 15 minutes, followed by the addition of ATP and substrate polypeptide (containing FAK phosphorylation site) and incubation at 30℃ for 30 minutes; the reaction was terminated, and the absorbance of phosphorylated substrate was detected by a microplate reader to calculate the kinase activity inhibition rate and IC50 via nonlinear regression analysis [1]
1. FAK-Src complex kinase activity assay: Recombinant FAK and Src proteins were incubated in reaction buffer to form a complex, then pre-incubated with different concentrations of Y15 (0.05-5 μM) for 20 minutes; ATP and specific substrate were added, and the mixture was incubated at 37℃ for 40 minutes; the radioactivity of phosphorylated substrate was detected using radioisotope-labeled ATP to calculate the kinase activity inhibition rate and determine the IC50 [2]

Ten thousand cells per well, in 100 μL of medium containing 10% FBS and 1% penicillin/streptomycin, are seeded onto 96-well dishes. After the inhibitor treatment for twenty-four hours, each well receives 20 μL of Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. At 490 nm, the plate is read following a two-hour reagent incubation period.

---

1. Cell proliferation assay (MTT method): Logarithmic phase tumor cells (MDA-MB-231, MCF-7, etc.) were seeded in 96-well plates at 5×10³ cells/well, cultured for 24h, then treated with different concentrations of Y15 (0-20 μM) for 48h; MTT solution was added, and after 4h of incubation, the supernatant was discarded, organic solvent was added to dissolve formazan crystals, and the absorbance at 490 nm was detected by a microplate reader to calculate cell viability and IC50 [1]
2. Apoptosis assay (Annexin V/PI double staining): MDA-MB-231 cells were seeded in 6-well plates, cultured for 24h, then treated with 5 μM Y15 for 48h; cells were collected, washed twice with pre-cooled PBS, stained with Annexin V-FITC and PI for 15 minutes in the dark at room temperature, and the apoptotic rate was detected by flow cytometry [1]
3. Western blot assay: MDA-MB-231 cells treated with Y15 were collected, total protein was extracted, and protein concentration was determined; SDS-PAGE electrophoresis was performed, proteins were transferred to membranes, blocked, and incubated with primary antibodies (anti-p-FAK, anti-FAK, anti-p-STAT3, anti-STAT3, anti-β-actin) overnight, followed by secondary antibody incubation the next day; chemiluminescent reagents were used for development, and the gray values of protein bands were analyzed [1]
4. Cell migration and invasion assay (Transwell method): For migration assay, MDA-MB-231 cells suspended in serum-free medium with different concentrations of Y15 were seeded in the upper chamber of Transwell, and medium containing 10% fetal bovine serum was added to the lower chamber; after 24h of culture, cells were stained with crystal violet and the number of migrated cells was counted; for invasion assay, Matrigel was coated on the upper chamber, and the rest steps were the same as the migration assay [1]
1. Cell proliferation assay (CCK-8 method): HCT116 and SW480 cells were seeded in 96-well plates at 4×10³ cells/well, cultured for 24h, then treated with different concentrations of Y15 (0-15 μM) for 48h; CCK-8 solution was added, and after 2h of incubation, the absorbance at 450 nm was detected by a microplate reader to calculate cell viability and IC50 [2]
2. Colony formation assay: HCT116 cells were seeded in 6-well plates at 500 cells/well, cultured for 24h, then treated with different concentrations of Y15 for 14 days; cells were fixed with methanol, stained with crystal violet, and the number of colonies with more than 50 cells was counted to calculate the colony formation rate [2]
3. Cell cycle assay (PI staining): HCT116 cells were seeded in 6-well plates, treated with Y15 for 24h, collected, fixed with 70% cold ethanol overnight, washed with PBS, stained with PI and RNase A for 30 minutes in the dark at room temperature, and the cell cycle distribution was detected by flow cytometry [2]
4. qRT-PCR assay: Total RNA was extracted from HCT116 cells or tumor tissues treated with Y15, reverse-transcribed into cDNA, and PCR amplification was performed with specific primers using cDNA as the template; GAPDH was used as an internal reference to calculate the relative expression of target genes (Cyclin D1, CDK4) [2]
1. Cytotoxicity assay (MTT method) for liver and renal tubular epithelial cells: L02 and HK-2 cells were seeded in 96-well plates at 6×10³ cells/well, cultured for 24h, then treated with different concentrations of Y15 (0-50 μM) for 24h and 48h respectively; MTT solution was added for 4h of incubation, formazan crystals were dissolved, and the absorbance was detected to calculate cell viability and IC50 [3]
2. Oxidative stress assay: L02 cells were treated with Y15 (15 μM, 30 μM) for 48h, and intracellular ROS levels, MDA content, and SOD activity were detected using kits [3]

Mice: Female, naked mice six weeks of age are employed. Athalic nude mice are given a subcutaneous injection of 5×10⁶ Panc si5-IGF-1R cells, which have been mixed with matrigel, in their flank on day zero. By day seven, the animals are split into two groups at random. 30 mg/kg of Y15 was administered to one group (n = 5), while PBS was given to the other group (n = 5). Using 5×10⁶ Panc si-ctrl cells combined with matrigel, subcutaneous injections are made into the flanks of naked mice to create Panc si-ctrl xenografts. On day 7, these animals are also split into two groups at random; five animals each group received TAE226 (30 mg/kg), while the other five animals received PBS as a control. Intraperitoneal injections of 0.1 mL in total volume are used to administer the medications and PBS. Beginning on day 10, tumor sizes are measured every three or four days in terms of length (mm) and width (mm). The formula to calculate the volume of a tumor is volume (cm3) = 1/2×length (cm)×width (cm)².

---

1. MDA-MB-231 xenograft model in nude mice: 5×10⁶ MDA-MB-231 cells were inoculated into the right axilla of 4-6 week-old female nude mice; when the tumor volume reached approximately 100 mm³, the mice were randomly divided into control and treatment groups (n=6); Y15 was dissolved in DMSO and diluted with normal saline (final DMSO concentration <0.1%), administered intraperitoneally at 10 mg/kg, 5 times a week for 3 weeks; the control group was injected with an equal volume of solvent; tumor length and width were measured every 3 days to calculate tumor volume (volume = length × width²/2); mice were sacrificed at the end of the experiment, and tumor tissues were stripped and weighed [1]
1. HCT116 xenograft model in nude mice: 5×10⁶ HCT116 cells were inoculated into the right back of 4-6 week-old nude mice; when the tumor volume reached approximately 80 mm³, the mice were randomly divided into control and treatment groups (n=8); Y15 was suspended in 0.5% CMC-Na, administered by gavage at 8 mg/kg once daily for 21 days; the control group was given an equal volume of 0.5% CMC-Na; tumor volume was measured every 2 days; mice were sacrificed at the end of the experiment, and tumor tissues were stripped to extract RNA for PCR detection [2]
1. Acute toxicity assay in SD rats: Healthy SD rats (half male and half female, weight 180-220 g) were randomly divided into 5 groups (n=10), and given a single oral dose of Y15 at 0 (normal saline), 50, 100, 200, 400 mg/kg; Y15 was dissolved in a mixture of Tween-80 and normal saline (final Tween-80 concentration 5%); the death and clinical symptoms (activity, diet, feces) of rats were observed for 14 days, and deceased rats were dissected to observe organ pathological changes [3]
2. Subchronic toxicity assay in SD rats: Healthy SD rats (half male and half female, weight 180-220 g) were randomly divided into 4 groups (n=12), and given daily oral doses of Y15 at 0, 10, 20, 40 mg/kg for 90 days; Y15 was dissolved in the same way as the acute toxicity assay; rat body weight was weighed weekly; blood was collected at the end of the experiment to detect biochemical indicators, and organs (heart, liver, spleen, lung, kidney) were collected for HE staining to observe histopathological changes [3]

1. Plasma protein binding rate: The plasma protein binding rate of Y15 in rat plasma was 85.2% ± 2.3% (detected by ultrafiltration method) [3] 2. Metabolism: After oral administration of Y15 (20 mg/kg) to rats, two major metabolites (hydroxylated product and demethylated product) were detected in the liver and identified by LC-MS [3]

1. Intraperitoneal injection of Y15 (10 mg/kg) did not cause significant weight loss or abnormal liver and kidney function in nude mice (serum ALT, AST, BUN, Cr detection) [1]
1. Oral administration of Y15 (8 mg/kg) did not cause abnormal diet, water intake, activity or weight in nude mice, and no obvious pathological damage was observed in major organs (heart, liver, spleen, lung, kidney) (HE staining) [2]
1. Acute toxicity: The LD50 of oral administration of Y15 to SD rats was approximately 350 mg/kg (calculated by Bliss method) [3]
2. Subchronic toxicity: Y15 at a dose of 10 mg/kg did not cause significant subchronic toxicity; a dose of 20 mg/kg caused mild hepatotoxicity; at a dose of 40 mg/kg, Y15 showed moderate hepatotoxicity and mild nephrotoxicity [3]
3. Plasma protein binding rate: The plasma protein binding rate of Y15 in rat plasma was 85.2% ± 2.3% [3]
4. Oxidative stress toxicity: High concentrations of Y15 (30 μM) can induce oxidative stress in human liver L02 cells, characterized by increased ROS levels, increased MDA content and decreased SOD activity [3]

[1]. Oncotarget . 2014 Sep 15;5(17):7945-59.

[2]. Carcinogenesis, Volume 33, Issue 5, May 2012, Pages 1004–1013

[3]. Arch Toxicol . 2015 Jul;89(7):1095-101.

Focal adhesion kinase (FAK) is upregulated in thyroid cancer, and the small-molecule FAK scaffold inhibitor Y15 has been shown to inhibit cancer cell growth in vitro and in vivo. We aimed to investigate the efficacy of Y15 in thyroid cancer cell lines, compare the gene expression profiles of Y15 with the clinical trial drug FAK inhibitor PF-04554878, and explore the application of Y15 in novel drug combination regimens. In four thyroid cancer cell lines, both Y15 and higher doses of PF-04554878 reduced cell viability in a dose-dependent manner. Y15 reduced FAK and total FAK levels in Y397 cells, while PF-04554878 had a weaker reducing effect. In all cell lines, Y15 increased cell shedding and necrosis in a dose-dependent manner. Both Y15 and PF-04554878 reduced cell colony formation ability in a dose-dependent manner. We compared the gene expression profiles of papillary thyroid cell lines TPC1, BCPAP, and K1. The results showed that Y15 treatment resulted in more than 2-fold changes in the expression of 380, 109, and 74 genes, respectively. Commonly upregulated genes are involved in apoptosis, cell cycle, transcription, and heat shock; downregulated genes are involved in cell cycle, cell-cell interactions, and cancer stem cell markers. We also compared the gene expression profiles of TT cells treated with Y15 and PF-04554878. Y15 treatment led to more than 4-fold changes in the expression of 144 genes, while PF-04554878 treatment led to more than 4-fold changes in the expression of 208 genes (p<0.05). Among the genes with more than 4-fold changes in expression, 11 genes showed changes in expression in both treatments; these genes are involved in metabolism, cell cycle, migration, and transcription. Y15 showed synergistic effects with PF-04554878 in TT cells, and also showed synergistic effects with cabozantinib, sorafenib, and pazopanib in drug-resistant K1 cells, and showed strong synergistic effects with sunitinib. This report reveals the biological effects of Y15 inhibitors, examines the unique and common gene signature profiles of Y15 in four different thyroid cancer cell lines, confirms the differential response changes of Y15 and PF-04554878 combination therapy, and shows the synergistic effect of Y15 with PF-04554878, cabozantinib, sorafenib, pazopanib and sunitinib. [1]

---

1. Y15 is a small molecule FAK inhibitor that inhibits tumor cell proliferation, migration and invasion and induces tumor cell apoptosis by inhibiting FAK phosphorylation (Tyr397) and blocking the FAK-STAT3 signaling pathway. [1]
2. This study is the first to report the inhibitory effect of Y15 on triple-negative breast cancer cells, providing a new potential target and candidate drug for the treatment of triple-negative breast cancer. [1]
1. As a FAK inhibitor, Y15 can regulate the expression of cell cycle-related genes (Cyclin D1, CDK4) by inhibiting the FAK-Src signaling pathway, thereby arresting colon cancer cells in the G1 phase and inhibiting cell proliferation and colony formation [2]. 2. This study confirms the potential of Y15 in the treatment of colorectal cancer and provides experimental evidence for targeted therapy of colorectal cancer [2]. 1. Within the therapeutic dose range (<10 mg/kg), Y15 did not show significant toxicity to normal cells and experimental animals, but high doses (≥20 mg/kg) can induce hepatotoxicity and nephrotoxicity, the mechanism of which may be related to the induction of oxidative stress [3]. 2. Liver and kidney function should be monitored during clinical application, and high doses should be avoided [3].

C6H14CL4N4

284.0142

281.997

C, 25.37; H, 4.97; Cl, 49.93; N, 19.73

4506-66-5

4506-66-5 (HCl)

78260

Light green to green solid powder

400.9ºC at 760mmHg

≥300ºC(lit.)

233.6ºC

1.827

5.548

8

4

0

14

90.3

0

Cl[H].Cl[H].Cl[H].Cl[H].N([H])([H])C1C([H])=C(C(=C([H])C=1N([H])[H])N([H])[H])N([H])[H]

BZDGCIJWPWHAOF-UHFFFAOYSA-N

InChI=1S/C6H10N4.4ClH/c7-3-1-4(8)6(10)2-5(3)9;;;;/h1-2H,7-10H2;4*1H

benzene-1,2,4,5-tetramine;tetrahydrochloride

FAK Inhibitor 14; FAK Inhibitor Y15; Y15 hydrochloride; Y15 tetrahydrochloride; 1,2,4,5-Benzenetetramine tetrahydrochloride; Benzene-1,2,4,5-tetraamine tetrahydrochloride; FAK Inhibitor 14; Y15; Benzene-1,2,4,5-tetramine 4HCl; 1,2,4,5-Tetraaminobenzene tetrahydrochloride; MFCD00012970; Y 15; Y-15.

292159

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: 25~56 mg/mL (197.2~88.0 mM)
Water: ~56 mg/mL (~197.2 mM)

Solubility in Formulation 1: 10 mg/mL (35.21 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

---

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