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Enzastaurin (formerly known as LY317615; DB-102; DB102 and LY-317615) is a novel, potent, and selective inhibitor of PKCβ (Protein kinase C) with potential antitumor activity. It inhibits PKCβ with an IC50 of 6 nM in cell-free assays, and exhibits 6- to 20-fold selectivity against PKCα, PKCγ and PKCε. Enzastaurin is a synthetic macrocyclic bisindolemaleimide with potential antineoplastic activity. Enzastaurin binds to the ATP-binding site and selectively inhibits protein kinase C beta, an enzyme involved in the induction of vascular endothelial growth factor (VEGF)-stimulated neo-angiogenesis. It can decrease tumor blood supply and so tumor burden.
Physicochemical Properties
| Molecular Formula | C32H29N5O2 | |
| Molecular Weight | 515.61 | |
| Exact Mass | 515.232 | |
| Elemental Analysis | C, 74.54; H, 5.67; N, 13.58; O, 6.21 | |
| CAS # | 170364-57-5 | |
| Related CAS # | 170364-57-5; 359017-79-1 (HCl);365253-37-8 (2HCl); | |
| PubChem CID | 176167 | |
| Appearance | Typically exists as light orange to dark orange-red solids at room temperature | |
| Density | 1.34 | |
| Boiling Point | 767.2±60.0 °C at 760 mmHg | |
| Melting Point | 249-261℃ | |
| Flash Point | 417.8±32.9 °C | |
| Vapour Pressure | 0.0±2.6 mmHg at 25°C | |
| Index of Refraction | 1.723 | |
| LogP | 4.43 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 5 | |
| Heavy Atom Count | 39 | |
| Complexity | 974 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(C(C1=CN(C)C2=C1C=CC=C2)=C3C4=CN(C5CCN(CC6=NC=CC=C6)CC5)C7=C4C=CC=C7)NC3=O |
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| InChi Key | AXRCEOKUDYDWLF-UHFFFAOYSA-N | |
| InChi Code | 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) | |
| Chemical Name | 3-(1-methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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] |
| ln Vitro |
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]. 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] |
| ln Vivo |
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]. 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] |
| Enzyme Assay |
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. 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] |
| Cell Assay |
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). 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. 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. 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] |
| Animal Protocol |
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. 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. 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. 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] |
| ADME/Pharmacokinetics |
Oral bioavailability: ~30% in humans; ~45% in rats after oral administration of 100 mg/kg [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) = 2.6 L/kg in rats, with extensive distribution to tumor tissues, brain, and colon [1][3] - Metabolism: Primarily metabolized in the liver by CYP3A4 and CYP2C9 to inactive metabolites [1] - Excretion: 70% of dose excreted as metabolites in feces; 25% in urine; <3% excreted unchanged [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: Oral LD50 > 1500 mg/kg in rats; >1200 mg/kg in mice [1] - Subchronic toxicity (28-day oral administration in rats): No significant hepatotoxicity or nephrotoxicity at doses up to 100 mg/kg/day; mild anemia (≤8% reduction in RBC count) at 200 mg/kg/day [1] - In xenograft mice, no significant changes in serum creatinine, BUN, ALT/AST levels at therapeutic doses (50-100 mg/kg/day) [1][3] - Drug-drug interactions: Inhibited by strong CYP3A4 inhibitors (e.g., ketoconazole) which increased AUC by 2.3 fold; no interaction with chemotherapeutic agents (e.g., doxorubicin) [3] |
| References |
[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. |
| Additional Infomation |
3-(1-methyl-3-indolyl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-3-indolyl]pyrrole-2,5-dione is a member of indoles and a member of maleimides. Enzastaurin, an investigational, targeted, oral agent, will be evaluated at more than 100 sites worldwide for the treatment of relapsed glioblastoma multiforme (GBM), an aggressive and malignant form of brain cancer. Drug Indication Investigated for use/treatment in brain cancer, lymphoma (non-hodgkin's), and lung cancer. Mechanism of Action Enzastaurin is an oral serine-threonine kinase inhibitor that is designed to suppress tumor growth through multiple mechanisms. Preclinical data indicate it may reduce the cell's ability to reproduce (cell proliferation), increase the natural death of the tumor cells (apoptosis), and inhibit tumor- induced blood supply (angiogenesis). Enzastaurin has been shown to inhibit signaling through the PKC-B and PI3K/AKT pathways. These pathways have been shown to be activated in a wide variety of cancers. In addition to glioblastoma, enzastaurin is also being studied in multiple other tumor types, including non-Hodgkin's lymphoma, colorectal cancer, non-small cell lung cancer, pancreatic cancer, and mantle cell lymphoma. Enzastaurin (LY317615) is a potent, selective PKCβ inhibitor with oral bioavailability, developed for the treatment of solid tumors and hematological malignancies [1][3] - Its core mechanism involves direct inhibition of PKCβ and GSK-3, suppressing downstream AKT-mediated signaling pathways to induce tumor cell apoptosis, inhibit proliferation, and block angiogenesis [1][2][3] - Therapeutic applications include preclinical and clinical investigation for colon cancer, glioblastoma, and multiple myeloma, with demonstrated efficacy in xenograft models [1][3] - High selectivity for PKCβ over other PKC isoforms (e.g., PKCα, PKCγ) minimizes off-target effects on normal tissues [1][3] - Favorable oral bioavailability and tumor tissue penetration support its use as a single agent or in combination with other anticancer therapies [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. Solubility in Formulation 3: 15% Captisol: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9395 mL | 9.6973 mL | 19.3945 mL | |
| 5 mM | 0.3879 mL | 1.9395 mL | 3.8789 mL | |
| 10 mM | 0.1939 mL | 0.9697 mL | 1.9395 mL |