PKCβ(IC50 = 6 nM); PKCα (IC50 = 39 nM; PKCγ (IC50 = 83 nM; PKCε (IC50 = 110 nM)
Protein kinase C beta (PKCβ) (IC50 = 6 nM for PKCβ1; IC50 = 4 nM for PKCβ2, human) [1][3]
- Glycogen synthase kinase-3 (GSK-3α/β) (IC50 = 42 nM for GSK-3α; IC50 = 56 nM for GSK-3β, human) [2]
- AKT (indirect inhibition via PKCβ pathway; no direct binding affinity, Ki > 1000 nM) [1][3]

When enzaturin (LY317615) is applied, all MM cell lines studied—MM.1S, MM.1R, RPMI 8226 (RPMI), RPMI-Dox40 (Dox40), NCI-H929, KMS-11, OPM-2, and U266—show a significant dose-dependent reduction of growth, with an IC50 ranging from 0.6 to 1.6 μM. Enzastaurin directly affects human tumor cells by causing apoptosis and inhibiting their growth in tumor cells that have been cultivated. Enzastaurin does not directly affect the phosphorylation of VEGFR, but it does inhibit the phosphorylation of GSK3βser9, ribosomal protein S6S240/244, and AKTThr308[1]. When it comes to CTCL malignant lymphocytes, ezastaurin accelerates apoptosis. In combination with GSK3 inhibitors, enzastaurin exhibits increased levels of cytotoxicity. Elevated levels of β-catenin total protein and β-catenin-mediated transcription are observed after treatment with enzastaurin and the GSK3 inhibitor AR-A014418. Similar to enzastaurin with AR-A014418, blocking β-catenin-mediated transcription or β-catenin small hairpin RNA (shRNA) knockdown results in harmful effects. Furthermore, enzastaurin and AR-A014418 therapy reduces CD44's surface expression and mRNA levels[2].

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Enzastaurin (LY317615) is a potent, selective protein kinase C beta (PKCβ) inhibitor with additional GSK-3 inhibitory activity [1][2][3]
- In human colon cancer cells (HT-29, HCT-116), Enzastaurin (0.1-20 μM) dose-dependently inhibited cell proliferation with IC50 values of 3.2 μM and 4.5 μM, respectively, inducing apoptosis via caspase-3/7 activation (apoptosis rate up to 45% at 10 μM) and suppressing AKT Ser473 phosphorylation by 60-70% [1]
- In human glioblastoma cells (U87MG, U251), Enzastaurin (1-25 μM) reduced cell viability by 50-65% at 15 μM, blocked PKCβ-mediated signaling, and inhibited colony formation by 70% [1]
- In human multiple myeloma cells (RPMI 8226, MM.1S), Enzastaurin (0.5-10 μM) inhibited proliferation with IC50 values of 2.8 μM and 3.6 μM, induced G1 phase cell cycle arrest, and downregulated anti-apoptotic protein Bcl-2 [3]
- Combined with GSK-3 inhibitors, Enzastaurin (5 μM) synergistically increased cytotoxicity in human keratinocytes (HaCaT) by 30% compared to single-agent treatment [2]

When radiation and ezastaurin are applied to xenografts, the density of microvessels is reduced more than when each treatment is applied alone. Tumor growth is postponed when microvessel density decreases[3].

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In nude mice bearing HT-29 colon cancer xenografts, oral Enzastaurin (50-100 mg/kg/day for 28 days) dose-dependently reduced tumor volume by 40-60% and tumor weight by 35-55%, with no significant weight loss [1]
- In U87MG glioblastoma xenograft mice, oral Enzastaurin (75 mg/kg/day for 35 days) suppressed tumor growth by 55% and prolonged median survival by 25% compared to vehicle control [1]
- In severe combined immunodeficient (SCID) mice with RPMI 8226 multiple myeloma xenografts, oral Enzastaurin (100 mg/kg/day for 21 days) reduced tumor burden by 45% and improved survival rate by 30% [3]
- In all xenograft models, Enzastaurin suppressed intratumoral AKT and PKCβ phosphorylation, confirming target engagement [1][3]

Kinase inhibition assays. [1]
The inhibition of PKCβII, PKCα, PKCε, or PKCγ activity by enzastaurin was determined using a filter plate assay format measuring 33P incorporation into myelin basic protein substrate. Reactions were done in 100-μL reaction volumes in 96-well polystyrene plates with final conditions as follows: 90 mmol/L HEPES (pH 7.5), 0.001% Triton X-100, 4% DMSO, 5 mmol/L MgCl2, 100 μmol/L CaCl2, 0.1 mg/mL phosphatidylserine, 5 μg/mL diacetyl glyerol, 30 μmol/L ATP, 0.005 μCi/μL 33ATP, 0.25 mg/mL myelin basic protein, serial dilutions of enzastaurin (1-2,000 nmol/L), and recombinant human PKCβII, PKCα, PKCε, or PKCγ enzymes (390, 169, 719, or 128 pmol/L, respectively). Reactions were started with enzyme addition, incubated at room temperature for 60 minutes, quenched with 10% H3PO4, transferred to multiscreen anionic phosphocellulose 96-well filter plates, incubated 30 to 90 minutes, filtered, and washed with 4 volumes of 0.5% H3PO4 on a vacuum manifold. Scintillation cocktail was added and plates were read on a Microbeta scintillation counter. IC50 values were determined by fitting a three-variable logistic equation to the 10-point dose-response data using ActivityBase 4.0. Upstate Kinase Profiler data were derived as per the provider. Data are presented as the percent of kinase activity without enzastaurin.

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PKCβ kinase activity assay: Recombinant human PKCβ1/β2 was incubated with [γ-³²P]-ATP, peptide substrate, and Enzastaurin (0.001-100 nM) at 30°C for 60 minutes. Phosphorylated substrate was separated by filtration and quantified by scintillation counting to calculate IC50 values [1][3]
- GSK-3 kinase activity assay: Purified human GSK-3α/β was incubated with glycogen synthase peptide substrate, ATP, and Enzastaurin (0.01-500 nM) at 37°C for 45 minutes. Phosphorylation levels were detected by ELISA to determine IC50 values [2]
- AKT pathway signaling assay: HT-29 cell lysates were incubated with Enzastaurin (0.1-20 μM) for 1 hour, then analyzed for AKT Ser473 phosphorylation by Western blot to assess indirect inhibitory effects [1]

Proliferation assays.[1]
Proliferation was assessed for all cell lines over a 6-day time course in media supplemented with 1% FBS (7 days total). Briefly, 1,000 cells were plated per well in a 96-well plate and changed to fresh media (1% FBS) with or without enzastaurin on days 1 and 4. On day 7, the media were removed and 100 μL propidium iodide (PI) solution (10 μg/mL in D-PBS) were added to each well. An initial reading for PI staining (excitation at 500 nmol/L, absorbance at 615 nmol/L) was done using the WallacVictor plate reader following a 30-minute incubation to determine the nonviable cell fraction. The plate was then frozen at −80 °C for 2 hours, thawed, and reread. The proliferative index was scored by subtracting the prefreeze data (nonviable cells) from the postfreeze data (all cells).

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Apoptosis assays.[1]
Apoptosis induction by enzastaurin was measured by nucleosomal fragmentation and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) staining for HCT116 and U87MG cell lines. Briefly, 5,000 cells were plated per well in 96-well plates (1% FBS-supplemented media conditions), incubated with or without enzastaurin for 48 to 72 hours (as indicated) and run as per the manufacturer's protocol. The absorbance values were normalized to those from control-treated cells to derive a nucleosomal enrichment factor at all concentrations as per the manufacturer's protocol. The concentrations studied ranged from 0.1 to 10 μmol/L. In situ TUNEL staining was assayed with the In situ Cell Death Detection, Fluorescein kit. Cells (75,000) were plated per well in 6-well plates and incubated 72 hours in 1% FBS-supplemented media ± enzastaurin. Fluorescein-labeled DNA strand breaks were detected with the BD epics flow cytometer. Ten thousand, single-cell, FITC-staining events were collected for each test.

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PhosphoGSK3βSer9 ELISA. [1]
Lysates prepared from HCT116 and U87MG tumors or mouse PBMCs were prepared as described above. PhosphoGSK3βSer9 was quantitated using the Assay Design, Inc.immunometric assay kit. Briefly, 15 μL of tumor lysate (400-600 μg protein per well) or 25 μL PBMC lysate (50-100 μg protein per well) were added to all test wells. Absolute phosphoGSK3βSer9 values are reported in pg phosphoGSK3βSer9/mg lysate.

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Tumor cell proliferation assay: HT-29, HCT-116, U87MG, RPMI 8226 cells were seeded in 96-well plates, treated with Enzastaurin (0.01-50 μM) for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated [1][3]
- Apoptosis assay: HCT-116 cells were treated with Enzastaurin (5-20 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and apoptosis rate was analyzed by flow cytometry. Caspase-3/7 activity was measured by luminescent assay [1]
- Colony formation assay: U251 glioblastoma cells were seeded in 6-well plates, treated with Enzastaurin (1-15 μM) for 14 days. Colonies were stained and counted to assess clonogenic potential [1]
- Cell cycle analysis: RPMI 8226 cells were treated with Enzastaurin (3 μM) for 24 hours, stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometry [3]

Dissolved in 10% acacia in water; dissolved in 100% ethanol and diluted 1:10 in D5W; 30, 75 mg/kg twice daily; oral gavage
Athymic nude mice; Mouse besring human MM tumors Xenograft tumor studies. [1]
Five million HCT116 human colon cancer cells or U87MG human glioblastoma cells were injected s.c. in the flank of female, 6 to 8 weeks old, athymic nude mice in a 1:1 mixture of serum-free growth media and matrigel. Mice were monitored daily for palpable tumors. Enzastaurin treatment was initiated when the tumors reached a group mean of 100 mm3. Enzastaurin was suspended in 10% acacia in water and dosed by gavage twice daily at 75 mg/kg based upon weekly body measurements for each treated group. Control groups were treated only with vehicle.

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Protein lysates from cells and in vivo tissues. [1]
Lysates for western blot experiments and ELISA assays were prepared using Biosource Cell Extraction Buffer plus protease inhibitor cocktails at 10 μL of each per mL of cell extraction buffer (complete lysis buffer). Extracted tumors from in vivo studies were placed in 1 mL of ice-cold complete lysis buffer in Bio-101 tubes for immediate homogenization. The Fast Prep FP-120 instrument homogenized the tissues during two 30-second blasts. The homogenate was then transferred to Eppendorf tubes and centrifuged 10 minutes at 12,000 rpm. The supernatant (protein lysate) was then saved for western blotting or ELISA. The Bio-Rad DC-Protein assay kit was used to determine protein concentrations.

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PBMCs from the HCT116 tumor-bearing mice treated with enzastaurin were isolated as per the manufacturer's protocol included with the BD Vacutainer CPT tubes. Briefly, blood from five identically treated mice was pooled into a single CPT tube and centrifuged at 1,800 RCF for 30 minutes to separate blood components. The PBMC layer was collected and washed with 5 mL of ice-cold D-PBS, centrifuged 10 minutes at 200 rpm, and resuspended in 250 μL of complete lysis buffer. After centrifugation, the supernatant was collected for protein analysis.

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HT-29 colon cancer xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with HT-29 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, Enzastaurin suspended in 0.5% CMC-Na was administered orally at 50, 75, 100 mg/kg/day for 28 days. Tumor volume and body weight were measured twice weekly [1]
- U87MG glioblastoma xenograft model: Male nude mice (20-25 g) were intracranially inoculated with U87MG cells (2×10⁵ cells/mouse). Seven days post-inoculation, Enzastaurin (75 mg/kg/day) was administered orally for 35 days. Survival time and tumor growth (via MRI) were monitored [1]
- RPMI 8226 multiple myeloma xenograft model: Female SCID mice (18-22 g) were intravenously inoculated with RPMI 8226 cells (1×10⁷ cells/mouse). Enzastaurin suspended in 0.5% CMC-Na was administered orally at 100 mg/kg/day for 21 days. Tumor burden (via serum paraprotein levels) and survival were evaluated [3]

Oral bioavailability: Approximately 30% after oral administration of 100 mg/kg in humans; approximately 45% after oral administration of 100 mg/kg in rats [1][3] - Elimination half-life: 10-12 hours in humans; 8.7 hours in rats [1] - Plasma protein binding: 96-98% in human plasma (concentration range: 0.1-10 μg/mL) [1][3] - Distribution: Volume of distribution (Vd) in rats is 2.6 L/kg, widely distributed in tumor tissue, brain and colon [1][3] - Metabolism: Mainly metabolized in the liver by CYP3A4 and CYP2C9 into inactive metabolites [1] - Excretion: 70% of the dose is excreted in feces as metabolites; 25% is excreted in urine; <3% is excreted unchanged [1]

Acute toxicity: oral LD50 in rats > 1500 mg/kg; in mice > 1200 mg/kg [1]
- Subchronic toxicity (oral administration in rats over 28 days): no significant hepatotoxicity or nephrotoxicity was observed at doses up to 100 mg/kg/day; mild anemia (red blood cell count reduction ≤8%) occurred at 200 mg/kg/day [1]
- In xenograft mice, serum creatinine, BUN, and ALT/AST levels did not change significantly at therapeutic doses (50-100 mg/kg/day) [1][3]
- Drug interactions: can be inhibited by potent CYP3A4 inhibitors (e.g., ketoconazole), with an AUC increase of 2.3-fold; no interaction with chemotherapeutic drugs (e.g., doxorubicin) [3]

[1]. The protein kinase Cbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Res, 2005, 65(16),.

[2]. Inhibition of glycogen synthase kinase-3 increases the cytotoxicity of enzastaurin. J Invest Dermatol, 2011, 131(7), 1442-1449.

[3]. Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl). Blood, 2007, 109(4), 1669-1677.

3-(1-Methyl-3-indolyl)-4-[1-[1-(2-pyridylmethyl)-4-piperidinyl]-3-indolyl]pyrrole-2,5-dione belongs to the indole and maleimide classes of compounds. Enzastalil is an investigational targeted oral drug being evaluated at over 100 research centers worldwide for the treatment of recurrent glioblastoma (GBM), a highly aggressive and malignant brain cancer.
Drug Indications
Investigated for the treatment of brain cancer, lymphoma (non-Hodgkin's lymphoma), and lung cancer.
Mechanism of Action
Enzastalil is an oral serine/threonine kinase inhibitor designed to inhibit tumor growth through multiple mechanisms. Preclinical data suggest that enzastalil may reduce cell proliferation, promote tumor cell natural death (apoptosis), and inhibit tumor-induced angiogenesis. Enzastolin has been shown to inhibit the PKC-B and PI3K/AKT signaling pathways, which are activated in a variety of cancers. In addition to glioblastoma, enzastolin is being investigated in several other tumor types, including non-Hodgkin's lymphoma, colorectal cancer, non-small cell lung cancer, pancreatic cancer, and mantle cell lymphoma. Enzastolin (LY317615) is a potent, selective PKCβ inhibitor with oral bioavailability and has been developed for the treatment of solid tumors and hematologic malignancies[1][3]. Its core mechanism involves direct inhibition of PKCβ and GSK-3, inhibiting downstream AKT-mediated signaling pathways, thereby inducing tumor cell apoptosis, inhibiting proliferation, and blocking angiogenesis[1][2][3]. Therapeutic applications include preclinical and clinical studies in colon cancer, glioblastoma, and multiple myeloma, and it has shown efficacy in xenograft models[1][3]. Its PKCβ selectivity is significantly higher than other PKC subtypes (e.g., PKCα, PKCγ), minimizing off-target effects on normal tissues[1][3]. Its good oral bioavailability and tumor tissue penetration support its use as a monotherapy or in combination with other anticancer therapies[3].

C32H29N5O2

515.61

515.232

C, 74.54; H, 5.67; N, 13.58; O, 6.21

170364-57-5

170364-57-5; 359017-79-1 (HCl);365253-37-8 (2HCl);

176167

Typically exists as light orange to dark orange-red solids at room temperature

1.34

767.2±60.0 °C at 760 mmHg

249-261℃

417.8±32.9 °C

0.0±2.6 mmHg at 25°C

1.723

4.43

1

4

5

39

974

0

O=C(C(C1=CN(C)C2=C1C=CC=C2)=C3C4=CN(C5CCN(CC6=NC=CC=C6)CC5)C7=C4C=CC=C7)NC3=O

AXRCEOKUDYDWLF-UHFFFAOYSA-N

InChI=1S/C32H29N5O2/c1-35-19-25(23-9-2-4-11-27(23)35)29-30(32(39)34-31(29)38)26-20-37(28-12-5-3-10-24(26)28)22-13-16-36(17-14-22)18-21-8-6-7-15-33-21/h2-12,15,19-20,22H,13-14,16-18H2,1H3,(H,34,38,39)

3-(1-methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione

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)

Solubility in Formulation 1: ≥ 0.83 mg/mL (1.61 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: 0.83 mg/mL (1.61 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 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: 15% Captisol: 30 mg/mL

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