GSK-3β (IC50 = 0.9 nM); PI3Kγ (IC50 = 282 nM)

LY2090314 selectively inhibits the activity of GSK-3 by interrupting ATP binding. LY2090314 has the ability to keep β-catenin stable. As a monotherapy, LY2090314 exhibits a limited efficacy. Cisplatin and carboplatin are more effective in vitro against solid tumor cancer cell lines when combined with LY3090314. [1]

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LY2090314 is a potent, selective GSK3 inhibitor which activates the Wnt pathway in melanoma cell lines. LY2090314 potently induces apoptotic cell death in a panel of melanoma cell lines irrespective of BRAF mutation status. Cell death induced by LY2090314 is dependent upon β-catenin and Wnt signaling.LY2090314 remains active in cell lines resistant to Vemurafenib and has an independent mechanism of action. [2]

LY2090314 improves the effectiveness of cisplatin and carboplatin in solid tumor cancer xenografts. [1]

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LY2090314 demonstrates single agent activity in the A375 melanoma model and synergizes with DTIC in vivo [2]
We sought to assess the ability of LY2090314 to activate the Wnt pathway in vivo and subsequently question if pathway elevation could lead to antitumor efficacy in melanoma. In mouse, LY2090314 is rapidly cleared and has a plasma half-life of 36 minutes (Fig 5A). In studies assessing the in vivo gene expression of Axin2, a Wnt responsive gene, we observed a significant induction of Axin2 mRNA at 2 and 4 hours post dose of LY2090314 in A375 xenograft tumor tissue (Fig 5B). This finding is in agreement with our in vitro experiments which also reveal Axin2 elevation 2–4 hours after initial drug exposure (Fig 1E). The rapid decline in Axin2 gene expression after 4 hours is consistent with the short half-life and pharmacokinetic properties of the compound in vivo (Fig 5A and 5B). Despite the transient elevation of the Wnt pathway with LY2090314 treatment, we were able to observe single agent antitumor efficacy in subcutaneous A375 xenografts dosed every 3 days (Fig 5C, p<0.003). In addition, we explored the ability of LY2090314 to synergize with DTIC in vivo and observed that the combination treatment displayed statistically significant greater than additive effects relative to control and single treatment groups (Fig 5D, p<0.02). In these studies we did not detect significant animal weight loss or other clinical signs. It is important to note that caution should be adopted when exploring the potential use of Wnt activators in cancer therapy due to their ability to increase the proliferation of normal tissues. Optimization of compound dosing and scheduling will be of vital importance when determining if compounds such as these have a sufficient therapeutic window for the treatment of melanoma. The studies presented here provide proof-of-concept data supporting the use of Wnt activators in the treatment of melanoma and support further investigation of GSK3 inhibitors for melanoma therapy with particular attention given to the effects on healthy tissues.

GSK3 biochemical assay [2]
The inhibitory enzyme activity of LY2090314 was assessed by incubating human recombinant GSK3α or GSK3β in the presence of the peptide substrate YRRAAVPPSPSLSRHSSPHQ(Ps)EDEEE as previously conducted

Cell chemosensitivity and caspase activation assay [1]
Cells were seeded in 96 well plates at a density of 2,000 cells/well and allowed to adhere overnight in regular growth media. Following 24 hours, media was removed and replaced with growth media containing 2% fetal bovine serum and the cells exposed to test agents (0.5nmol/L to 10μmol/L) for 72 hours. In vitro chemosensitivity of melanoma cells to LY2090314, BIA and Vemurafenib was determined using the CellTiter-Glo assay according to manufacturer’s instructions. Caspase 3/7 activation was determined using the Caspase-Glo assay according to manufacturer’s instructions. Nonlinear regression and sigmoidal dose-response curves were used to calculate the half maximal inhibitory concentration (IC50) using GraphPad Prism 6 software.

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PARP cleavage assay [2]
Cells were seeded in 6 well plates at a density of 250,000 cells/well and allowed to adhere overnight in regular growth media. Following 24 hours, media was replaced with fresh growth medium containing LY2090314 (final concentration 5nM, 15nM) and the cells exposed to test agents for 72 hours. PARP cleavage was assessed per 50μg total protein using the Pathscan Cleaved PARP ELISA (Asp214) sandwich ELISA kit according to manufacturer’s instructions. Levels of cleaved PARP were calculated relative to DMSO control treated cells.

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Immunoprecipitation [2]
In order to assess ‘free’ β-catenin within cell lysates before and after treatment with LY2090314, we measured the amount of β-catenin available for binding to the C-terminal region of E-cadherin fused to a FLAG tag. This method has been described previously. Briefly, lysates representing 300μg total protein were incubated with 10μg His-Flag-E-cadherin. Anti-FLAG M2 Affinity gel was washed and incubated with the lysate/E-cadherin mix overnight at 4°C in order to bind E-cadherin/β-catenin complexes. Following the overnight incubation, the resin was washed 3 times with lysis buffer before being resuspended in gel loading buffer containing β-mercaptoethanol. Samples were centrifuged at 8000g and supernatants used for immunoblot analysis..

Five million A375 human melanoma cancer cells are injected S.C. in the flank of female 6 to 8 week old athymic nude mice in a 1:1 mixture with matrigel. Tumors that can be felt are checked for daily in mice. When tumors are approximately 100 mm2 in size, mice are divided into groups and given LY2090314 (25 mg/kg Q3D) or a vehicle (20% Captisol/0.01N HCl) intravenously. Animal body weight and tumor volume (calculated using calipers) are recorded twice weekly. The formula used to determine tumor volumes is (a2 b)/2, where a represents the tumor's smaller dimension and b its larger dimension. LY2090314 is dosed at 2.5 mg/kg Q3D for combination studies with DTIC (60 mg/kg QD), and tumor growth is tracked.

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LY2090314 pharmacokinetic studies [2]
To understand the plasma pharmacokinetics of LY2090314 in vivo following i.v. administration, CD1 nu/nu non-tumor bearing mice (Harlan, Indianapolis, IN) were injected i.v. with 5mg/kg LY2090314 and blood collected by cardiac puncture at the times indicated (5, 15, 30, 60 120 minutes post dose). Mice were sacrificed using isoflurane and cervical dislocation. Blood was centrifuged at 5,000 x g for 10 minutes, and the resulting plasma analyzed for drug concentration using HPLC and mass spectrometry.

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In vivo studies [2]
Five million A375 human melanoma cancer cells were injected S.C. in the flank of female 6 to 8 week old athymic nude mice (Harlan, Indianapolis, IN) in a 1:1 mixture with matrigel. Mice were monitored daily for palpable tumors. When tumors reached ~100mm2 mice were randomized into groups receiving either LY2090314 (25 mg/kg Q3D) or vehicle (20% Captisol/0.01N HCl) via i.v. administration. Tumor volume (measured by calipers) and animal body weight were recorded twice weekly. Tumor volumes were calculated using the formula: (a2 x b)/2 (a being the smaller and b being the larger dimension of the tumor). For combination studies with DTIC (60 mg/kg QD), LY2090314 was dosed at 2.5 mg/kg Q3D and tumor growth monitored. For in vivo target inhibition studies in xenograft tissue LY2090314 (25mg/kg) was administered to mice harboring A375 tumors approximately 200mm2 in volume and tumor tissue collected for RNA expression analysis at 1, 2, 4, 6, 8 and 24hours postdose.

LY2090314 (3-[9-fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydro[1,4]diazazo[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1H-pyrrole-2,5-dione) is an intravenously administered glycogen synthase kinase-3 inhibitor used in clinical trials for oncology. This study characterized the drug distribution following intravenous infusion of [(14)C]LY2090314 in rats and dogs and correlated it with existing clinical data. LY2090314 exhibits high clearance (close to hepatic blood flow) and moderate volume of distribution (approximately 1–2 L/kg), leading to rapid elimination (half-lives of approximately 0.4, 0.7, and 1.8–3.4 hours in rats, dogs, and humans, respectively). Standardized results for hepatic microsomal clearance accurately predicted perfusion-limited clearance in different species. LY2090314 is extensively metabolized and cleared, with its numerous metabolites rapidly excreted into feces via bile (69-97% of the dose; 62-93% excreted within 0-24 hours); urinary recovery of drug-related substances is low (≤3% of the dose). Despite extensive metabolism, the only identifiable drug-related component in plasma in rats and humans is the parent compound. Even in Mdr1a, Bcrp, and Mrp2 knockout rats, LY2090314 metabolites did not appear in the circulatory system, and urinary excretion was not increased, as the hypothetical impaired bile excretion of metabolites in the absence of these capillary bile transporters was not observed. The distribution of metabolites in dogs is generally similar, with the canine-specific LY2090314 glucuronide being a notable exception. This conjugate is generated in the canine liver and preferentially excreted into the bloodstream, accounting for the majority of late-circulating radioactivity, and is primarily recovered in urine (16% of the dose). In summary, LY2090314 is rapidly cleared through extensive metabolism, and since the metabolites are excreted into the feces via bile and there is no significant intestinal reabsorption, the exposure to circulating metabolites is negligible. [1] In mice, LY2090314 is rapidly cleared with a plasma half-life of 36 minutes (Fig. 5A). In a study evaluating the in vivo expression of the Wnt-responsive gene Axin2, we observed that Axin2 mRNA was significantly induced in A375 xenograft tissues 2 and 4 hours after LY2090314 administration (Fig. 5B). This finding is consistent with our in vitro results, which also showed an increase in Axin2 expression 2–4 hours after the first administration (Fig. 1E). The rapid decrease in Axin2 gene expression after 4 hours is consistent with the short half-life and pharmacokinetic properties of the compound in vivo (Fig. 5A and 5B). [2]

[1]. Pharmacokinetics, metabolism, and excretion of the glycogen synthase kinase-3 inhibitor LY2090314 in rats, dogs, and humans: a case study in rapid clearance by extensive metabolism with low circulating metabolite exposure. Dr

[2]. Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3. PLoS One. 2015 Apr 27;10(4):e0125028.

LY-2090314 belongs to the diazacyclic heptaphylindo class of compounds. Its structure is 1,2,3,4-tetrahydro[1,4]diazacyclic heptaphylindo[6,7,1-hi]indole, with piperidin-1-ylcarbonyl, 4-(imidazo[1,2-a]pyridin-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrole-3-yl, and fluorine substitutions at positions 2, 7, and 9, respectively. It is a potent competitive inhibitor of glycogen synthase kinase-3 (GSK-3) against ATP, with IC50 values of 1.5 nM and 0.9 nM for GSK-3α and GSK-3β, respectively. This drug is currently in clinical development for the treatment of advanced/metastatic cancer. It exhibits apoptosis-inducing, anti-tumor, Wnt signaling pathway activation, and EC 2.7.11.26 (tau protein kinase) inhibition effects. It is an imidazopyridine, diazacycloheptanindole, monofluorobenzene, piperidinecarboxamide, urea, and maleimide compound. Ly2090314 has been used in clinical trials for the treatment of leukemia, advanced cancer, and pancreatic cancer. The GSK-3 inhibitor LY2090314 is a glycogen synthase kinase-3 (GSK-3) inhibitor with potential antitumor activity. After administration, LY2090314 binds to GSK-3 in an ATP-competitive manner and inhibits its activity. This prevents GSK-3-mediated β-catenin phosphorylation, thereby inhibiting subsequent β-catenin ubiquitination and proteasome degradation. This leads to activation of the Wnt/β-catenin pathway and induces apoptosis in susceptible tumor cells. GSK-3 is a serine/threonine kinase that plays a crucial role in numerous pathways involved in protein synthesis, cell proliferation, differentiation, and apoptosis. The Wnt/β-catenin signaling pathway plays a crucial role in cell proliferation and differentiation. β-catenin is a transcriptional activator, and increased expression is associated with reduced cell proliferation and improved prognosis in some cancers. Previous studies have observed decreased β-catenin expression during melanoma progression, and a negative correlation between nuclear β-catenin levels and cell proliferation, suggesting that activation of the Wnt/β-catenin pathway may be beneficial for melanoma patients. To further explore this concept, we tested LY2090314, a potent and selective small molecule inhibitor active against both GSK3α and GSK3β subtypes. In a range of melanoma cell lines, nanomolar concentrations of LY2090314 stimulated the activity of the TCF/LEF TOPFlash reporter gene, stabilized β-catenin, and increased Axin2 expression. Axin2 is a Wnt-responsive gene and a marker of pathway activation. Cytotoxicity assays showed that melanoma cell lines were highly sensitive to LY2090314 in vitro (IC50 approximately 10 nM after 72 hours of treatment), while other solid tumor cell lines were not (IC50 > 10 μM), as confirmed by caspase activation and PARP cleavage. Cell lines carrying mutant B-RAF or N-RAS were as sensitive to LY2090314 as cell lines resistant to the BRAF inhibitor vemurafenib. shRNA studies showed that β-catenin stability is essential for apoptosis following GSK3 inhibitor treatment, as knockdown of β-catenin could overcome the sensitivity of melanoma cell lines to LY290314. We further demonstrated that in vivo, a single dose of LY290314 increased Axin2 gene expression, and repeated doses delayed the growth of A375 melanoma xenografts. The activity of LY290314 in preclinical models suggests that the role of Wnt activators in melanoma treatment should be further explored. [2]

C28H25FN6O3

512.5349

512.197

C, 65.61; H, 4.92; F, 3.71; N, 16.40; O, 9.36

603288-22-8

603288-22-8

10029385

Off-white to light yellow solid powder

1.6±0.1 g/cm3

1.776

3.43

1

5

2

38

1030

0

FC1C([H])=C2C(C3C(N([H])C(C=3C3=C([H])N=C4C([H])=C([H])C([H])=C([H])N34)=O)=O)=C([H])N3C([H])([H])C([H])([H])N(C(N4C([H])([H])C([H])([H])C([H])([H])C([H])([H])C4([H])[H])=O)C([H])([H])C(C=1[H])=C32

HRJWTAWVFDCTGO-UHFFFAOYSA-N

InChI=1S/C28H25FN6O3/c29-18-12-17-15-34(28(38)32-7-3-1-4-8-32)11-10-33-16-20(19(13-18)25(17)33)23-24(27(37)31-26(23)36)21-14-30-22-6-2-5-9-35(21)22/h2,5-6,9,12-14,16H,1,3-4,7-8,10-11,15H2,(H,31,36,37)

3-[6-fluoro-10-(piperidine-1-carbonyl)-1,10-diazatricyclo[6.4.1.04,13]trideca-2,4,6,8(13)-tetraen-3-yl]-4-imidazo[1,2-a]pyridin-3-ylpyrrole-2,5-dione

LY 2090314; LY2090314; 603288-22-8; LY-2090,314; LY 2090,314; Kinome_3681; 3-(9-Fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione; UNII-822M3GYM67; CHEMBL362558; LY-2090314

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: ~100 mg/mL (~195.1 mM)
Water: <1 mg/mL
Ethanol: ~2 mg/mL warmed (~3.9 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.44 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 (2.44 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: 5% DMSO+45% PEG 300+ddH2O: 17mg/mL

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Solubility in Formulation 4: 10 mg/mL (19.51 mM) in 20% HP-β-CD/10 mM citrate pH 2.0 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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