- OSU-T315 (ILK-IN-1) targets integrin-linked kinase (ILK) with an IC₅₀ value of 0.6 μM [1]
- OSU-T315 (ILK-IN-1) targets protein kinase B (AKT) by inhibiting its membrane translocation and activation, independent of integrin-linked kinase [2]

Compound 22, OSU-T315 (IC50 ranging from 1-2.5 μM), demonstrates excellent potency in vitro against a panel of prostate and breast cancer cell lines [1]. The expression of YB-1, HER2, and EGFR can be decreased by OSU-T315 (0-2.5 μM; 24 hours); it also exhibits a dose-dependent influence on phosphorylated p38 and ERK1/2 and a modest inhibitory effect on phosphorylated S6 levels. sexual inhibition, whereas PC-3 cells show no change in phosphorylated JNK [1]. ILK inhibition is used by OSU-T315 (0–4 μM; 24 hours) to promote autophagy [1].

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- OSU-T315 (ILK-IN-1) exhibited potent antiproliferative activity against prostate cancer cell lines (PC-3, LNCaP) and breast cancer cell lines (MDA-MB-231, MDA-MB-468, SKBR3, MCF-7) with IC₅₀ values ranging from 1 to 2.5 μM, while normal epithelial cells (PrECs, MECs) were unaffected. It induced dephosphorylation of Akt at Ser-473, glycogen synthase kinase-3β (GSK3β), and myosin light chain (MLC), and suppressed the expression of YB-1, HER2, and EGFR at both protein and mRNA levels in PC-3 and SKBR3 cells. These effects could be rescued by stable expression of constitutively active ILK (CA-ILK). Additionally, it induced cell cycle arrest, caspase-dependent apoptosis (detected by annexin V/PI staining and PARP cleavage), and autophagy (evidenced by LC3-II conversion), which were integral to its antiproliferative activity. SiRNA-mediated knockdown of Atg5 inhibited autophagy and partially protected PC-3 cells from OSU-T315 (ILK-IN-1)-mediated viability suppression [1]
- OSU-T315 (ILK-IN-1) showed dose-dependent selective cytotoxicity in chronic lymphocytic leukemia (CLL)-derived cell lines (Mec-1, OSU-CLL) and primary CLL cells, with minimal effects on normal B cells (CD19⁺) and T cells (CD3⁺). It directly abrogated AKT activation by preventing its translocation into lipid rafts without altering receptor-associated kinase activation. It suppressed AKT activation mediated by B-cell receptor (BCR), CD49d, CD40, and Toll-like receptor 9, and reduced Mcl-1 expression. It triggered caspase-dependent apoptosis (confirmed by Caspase 3/7 activity assay and annexin V/PI staining), which could be blocked by z-VAD-FMK. It also exhibited cytotoxicity against ibrutinib-resistant CLL samples. In vitro kinase assay showed it did not inhibit class I PI3K activity or RAS activity [2]

PC-3 xenograft tumor growth is inhibited by OSU-T315 (oral gavage; 25 mg/kg, 50 mg/kg; once daily; 35 days) [1]. ?In mice, no additional noteworthy harm was noted [1].

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- In athymic nude mice bearing subcutaneous PC-3 xenograft tumors, oral administration of OSU-T315 (ILK-IN-1) at doses of 25 mg/kg and 50 mg/kg once daily for 35 days significantly suppressed tumor growth. Western blot analysis of intratumoral tissues confirmed modulation of biomarkers related to ILK signaling [1]
- In C57BL/6 mice engrafted with TCL1 leukemia cells, OSU-T315 (ILK-IN-1) prolonged overall survival. Two administration regimens were effective: oral gavage of 50 mg/kg daily after 10% leukemia cells were detected in peripheral blood; or intraperitoneal injection of 25 mg/kg daily for 2 weeks followed by every-other-day administration after 5% CD19⁺CD5⁺ leukemia cells were detected in peripheral blood mononuclear cells. Pharmacologically active drug levels were achieved in vivo [2]

- ILK kinase activity inhibition assay: ILK was immunoprecipitated from cells, and its kinase activity was measured in the presence of different concentrations of OSU-T315 (ILK-IN-1). The phosphorylation of the ILK substrate myelin basic protein (MBP) was detected using ³²P labeling to evaluate the inhibitory effect of OSU-T315 (ILK-IN-1) on ILK activity, with an IC₅₀ of 0.6 μM calculated [1]
- Class I PI3K kinase activity assay: The kinase activity of class I PI3K was evaluated using a specific assay kit. OSU-T315 (ILK-IN-1) was added to the kinase reaction system, and the relative percentage of inhibition was determined by referencing the biotinylated-PIP3 signal, with wortmannin as a positive control [2]

Western Blot Analysis[1]
Cell Types: PC-3 cells; MDA-MB-231 cells
Tested Concentrations: 1μM, 2μM, 3μM, 4μM; 0.5μM, 1μM, 1.5μM, 2μM, 2.5μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Exhibits a dose-dependent reduction in the phosphorylation of pS6, ERK and p38 in PC-3 cells and MDA-MB-231 cells.

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Cell viability assay[1]
Cell Types: Prostate cancer cells: LNCaP, PC-3; Breast cancer cells: MDA-MB-231, MDA-MB-468, SKBR3, MCF-7; PrEC and MEC cell
Tested Concentrations: 0-5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of cancer cell viability in breast and prostate cancer cells (IC (50), 1-2.5 μM).

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Apoptosis analysis[1]
Cell Types: PC-3 Cell
Tested Concentrations: 1 μM, 2 μM, 3 μM, 4 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induction of accumulation of LC3-II and PARP cleavage.

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- Antiproliferative activity and signaling pathway assay: Prostate, breast cancer cells, and normal epithelial cells were seeded and treated with different concentrations of OSU-T315 (ILK-IN-1) for 24 hours. Cell viability was detected by MTT assay. Western blot analysis was performed to measure the phosphorylation levels of Akt, GSK3β, MLC, and the expression of YB-1, HER2, EGFR. Stable transfectants expressing CA-ILK were used to verify the specificity of the drug [1]
- Apoptosis and autophagy assay: PC-3 cells were treated with OSU-T315 (ILK-IN-1) at various concentrations for 24 hours. Cell cycle distribution was analyzed by flow cytometry. Apoptosis was detected by annexin V/PI staining and PARP cleavage via Western blot. Autophagy was evaluated by LC3-II conversion using Western blot, and siRNA-mediated knockdown of Atg5 was used to confirm the role of autophagy [1]
- CLL cell cytotoxicity and signaling assay: Primary CLL cells, CLL-derived cell lines, and normal lymphocytes were treated with increasing doses of OSU-T315 (ILK-IN-1) for 24 hours. Cell viability was analyzed by flow cytometry. Western blot was used to detect the expression of Mcl-1 and phosphorylation of signaling molecules. Caspase 3/7 activity was measured by a specific assay kit. Ibrutinib-resistant CLL samples were also tested for sensitivity to OSU-T315 (ILK-IN-1) [2]
- Lipid raft translocation assay: Mec-1 cells expressing Myr-flag-AKT were treated with OSU-T315 (ILK-IN-1). Lipid raft fractions were purified by ultracentrifugation, and AKT content in raft and non-raft compartments was analyzed by Western blot. Immunofluorescence staining with AKT and cholera toxin subunit B (CT-B, a lipid raft marker) was performed, and the colocalization index was measured by confocal microscopy [2]

Animal/Disease Models: Male NCr athymic nude mice with PC-3 tumor xenografts
Doses: 25 mg/kg; 50 mg/kg
Route of Administration: po (oral gavage); daily single; 35 days
Experimental Results: After 35 days of treatment, relative Tumor growth was inhibited compared to vehicle control (48% and 62% inhibition at 25 mg/kg and 50 mg/kg, respectively).

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- PC-3 xenograft tumor model in athymic nude mice: Mice with established subcutaneous PC-3 tumors were randomly divided into vehicle group and OSU-T315 (ILK-IN-1) treatment groups (25 mg/kg and 50 mg/kg). The drug was administered orally once daily for 35 days. Tumor growth was monitored regularly, and at the end of the experiment, tumor tissues were collected for Western blot analysis of relevant biomarkers [1]
- TCL1 leukemia mouse model: C57BL/6 mice were engrafted with TCL1 leukemia cells. In the first regimen, mice were treated with oral OSU-T315 (ILK-IN-1) at 50 mg/kg daily after 10% leukemia cells were detected in peripheral blood; peripheral blood white blood cell counts were monitored weekly. In the second regimen, mice were treated with intraperitoneal injection of OSU-T315 (ILK-IN-1) at 25 mg/kg daily for 2 weeks after 5% CD19⁺CD5⁺ leukemia cells were detected, followed by every-other-day administration to prevent weight loss. The drug was formulated in phosphate-buffered saline containing 10% Cremophor EL. Overall survival was analyzed, and pharmacokinetic studies were conducted after intravenous, intraperitoneal, or oral dosing [2]

In mice, after intravenous, intraperitoneal or oral administration, OSU-T315 (ILK-IN-1) reached pharmacologically active concentrations in plasma and detailed pharmacokinetic characteristics were established (specific parameters were not specified in the literature) [2] ## Supplementary Information - OSU-T315 (ILK-IN-1) is a novel integrin-linked kinase inhibitor with a wide range of anticancer mechanisms, including inhibition of ILK signaling, inhibition of oncogenic protein expression and induction of apoptosis and autophagy, showing therapeutic potential in the treatment of prostate cancer and breast cancer [1] - OSU-T315 (ILK-IN-1) disrupts the PI3K/AKT pathway through a novel ILK-independent mechanism, preventing AKT translocation to lipid rafts. It exhibits selective cytotoxicity against chronic lymphocytic leukemia (CLL) cells (including ibrutinib-resistant cells) and can prolong the survival of leukemia mouse models, making it a potential targeted therapy for CLL [2]

[1]. Identification and Characterization of a Novel Integrin-Linked Kinase Inhibitor.J Med Chem. 2011 Sep 22; 54(18): 6364–6374.

[2]. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells. Blood. 2015 Jan 8;125(2):284-95.

OSU-T315 (ILK-IN-1) is a novel integrin-linked kinase inhibitor with a broad range of anticancer mechanisms, including inhibition of the ILK signaling pathway, inhibition of oncogenic protein expression, and induction of apoptosis and autophagy, showing therapeutic potential in the treatment of prostate cancer and breast cancer [1]. OSU-T315 (ILK-IN-1) disrupts the PI3K/AKT pathway through a novel ILK-independent mechanism, preventing AKT translocation to lipid rafts. It exhibits selective cytotoxicity against chronic lymphocytic leukemia (CLL) cells (including ibrutinib-resistant cells) and can prolong the survival of leukemia mouse models, making it a potential targeted therapy for CLL [2].

C₃₀H₃₀F₃N₅O

533.59

533.24

2070015-22-2

118986645

White to off-white solid powder

1.3±0.1 g/cm3

739.2±60.0 °C at 760 mmHg

400.9±32.9 °C

0.0±2.4 mmHg at 25°C

1.615

4.65

2

7

7

39

767

0

AJLOJUFSIDSBNN-UHFFFAOYSA-N

InChI=1S/C30H30F3N5O/c1-34-29(39)15-14-27-20-28(36-38(27)26-12-10-25(11-13-26)37-18-16-35-17-19-37)23-4-2-21(3-5-23)22-6-8-24(9-7-22)30(31,32)33/h2-13,20,35H,14-19H2,1H3,(H,34,39)

N-methyl-3-[2-(4-piperazin-1-ylphenyl)-5-[4-[4-(trifluoromethyl)phenyl]phenyl]pyrazol-3-yl]propanamide

OSUT315 OSU T315 OSU-T315

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)

DMSO : ≥ 260 mg/mL (~487.27 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (4.07 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 21.7 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: ≥ 2.17 mg/mL (4.07 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 21.7 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.)