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]diazepino[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1H-pyrrole-2,5-dione) is an intravenous glycogen synthase kinase-3 inhibitor in oncology trials. Drug disposition was characterized after intravenous infusion of [(14)C]LY2090314 to rats and dogs, and was related to available clinical data. LY2090314 exhibited high clearance (approximating hepatic blood flow) and a moderate volume of distribution (∼1-2 l/kg) resulting in rapid elimination (half-life ∼0.4, 0.7, and 1.8-3.4 hours in rats, dogs, and humans, respectively). Scaled clearance from liver microsomes accurately predicted perfusion-limited clearance across species. LY2090314 was cleared by extensive metabolism, and the numerous metabolites were rapidly excreted into feces via bile (69-97% of dose; 62-93% within 0-24 hours); urinary recovery of drug-related material was low (≤3% of dose). Despite extensive metabolism, in rats and humans the parent compound was the sole identifiable drug-related moiety in plasma. Even in Mdr1a-, Bcrp-, and Mrp2-knockout rats, LY2090314 metabolites did not appear in circulation, and their urinary excretion was not enhanced, because the hypothesized impaired biliary excretion of metabolites in the absence of these canalicular transporters was not observed. Canine metabolite disposition was generally similar, with the notable exception of dog-unique LY2090314 glucuronide. This conjugate was formed in the dog liver and was preferentially excreted into the blood, where it accounted for the majority of circulating radioactivity at later times, and was predominantly recovered in urine (16% of dose). In conclusion, LY2090314 was rapidly cleared by extensive metabolism with negligible circulating metabolite exposures due to biliary excretion of metabolites into feces with no apparent intestinal reabsorption. [1]

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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). [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 is a member of the class of diazepinoindoles that is 1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indole substituted by piperidin-1-ylcarbonyl, 4-(imidazo[1,2-a]pyridin-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl and fluoro groups at position 2, 7 and 9, respectively. It is a potent ATP-competitive inhibitor of glycogen synthase kinase-3 (GSK-3) with IC50 values of 1.5 nM and 0.9 nM for GSK-3alpha and GSK-3beta. The drug is in clinical development for the treatment of advanced/metastatic cancer. It has a role as an apoptosis inducer, an antineoplastic agent, a Wnt signalling activator and an EC 2.7.11.26 (tau-protein kinase) inhibitor. It is an imidazopyridine, a diazepinoindole, a member of monofluorobenzenes, a piperidinecarboxamide, a member of ureas and a member of maleimides.
Ly2090314 has been used in trials studying the treatment of Leukemia, Advanced Cancer, and Pancreatic Cancer.
GSK-3 Inhibitor LY2090314 is an inhibitor of glycogen synthase kinase-3 (GSK-3), with potential antineoplastic activity. Upon administration, LY2090314 binds to and inhibits GSK-3 in an ATP-competitive manner. This prevents GSK-3-mediated phosphorylation of beta-catenin, which inhibits the subsequent ubiquitination and proteasomal degradation of beta-catenin. This leads to the activation of the Wnt/beta-catenin pathway and the induction of apoptosis in susceptible tumor cells. GSK-3, a serine/threonine kinase, plays a key role in numerous pathways involved in protein synthesis, cellular proliferation, differentiation, and apoptosis. The Wnt/beta-catenin signaling pathway plays key roles in both cellular proliferation and differentiation. The increased expression of beta-catenin, a transcriptional activator, correlates with decreased cellular proliferation and improved prognosis in select cancers.

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It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10 uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma 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.)