MAPK-Erk Inhibitors (MAPK-Erk 抑制剂)

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MAPK-Erk Inhibitors

AZD8330 is an orally active, selective MEK inhibitor with an IC50 of 7 nM. AZD8330 has potential antineoplastic activity. AZD8330 specifically inhibits mitogen- activated protein kinase kinase 1 (MEK or MAP/ERK kinase1), resulting in inhibition of growth factor-mediated cell signaling and tumor cell proliferation. MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway that regulates cell growth. Constitutive activation of this pathway has been implicated in many cancers. [1][2]

References on AZD8330:

[1] http://www.cancer.gov/drugdictionary/?CdrID=543526

[2] Frémin C et al. J Hematol Oncol. 2010 Feb 11;3:8.

AZD6244 (Selumetinib) is highly potent to inhibit MEK1 with IC50 of 14nmol. AZD6244 is a not competitive with ATP and inactivates the ERK1/2 phosphorylation with IC50 concentrations below 40 nmol. AZD6244 also inhibits the growth of primary HCC cells through inhibition of ERK1/2 and p90RSK phosphorylation, accompanied with elevation of the cleavage of caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase. [1] AZD6244 is sensitive to raf mutations in breast cancer cell lines and ras mutations in NSCLC cell lines. [2] AZD6244 had little effects on the p38, c-Jun-NH2-kinase, phosphatidylinositol 3-kinase, and MEK5/ERK5 pathways. In vivo studies showed that AZD6244 significantly inhibits phosphorylation of ERK1/2 in 2-1318, 5-1318, and 26-1004 4-1318 xenografts and induces apoptosis in primary 2-1318 cells by activating the caspase pathway. [1] AZD6244 could inhibit the tumor growth in HT-29 xenograft, which is a colorectal tumor model carrying a B-Raf mutation, at a dose of 100mg/kg and the TGI of AZD6244 is better than it of gemcitabine. [3] Otherwise AZD6244 could inhibit HCC xenografts tumor growth, which associated with increased apoptosis and down-regulation of cell cycle regulators, including cyclin D1, cdc-2, cyclin-dependent kinases 2 and 4, cyclin B1, and c-Myc. [4] AZD6244 could be used to treat with many cancers including colorectal, NSCLC, pancreatic and breast. AZD6244 is current in Phase II clinical trial against NSCLC. AZD6244 is originally developed by Array BioPharma and latterly by Astra Zeneca.

Protocol:

Kinase Assay:
NH2-terminal hexahistidine tagged, constitutively active MEK1 is expressed in baculovirus-infected Hi5 insect cells and purified by immobilized metal affinity chromatography, ion exchange, and gel filtration. The activity of MEK1 is assessed by measuring the incorporation of [γ-33P]phosphate from [γ-33P]ATP onto ERK2. The assay is carried out in a 96-well polypropylene plate with an incubation mixture (100 μL) composed of 25 mmol/L HEPES (pH 7.4), 10 mmol/L MgCl2, 5 mmol/L β-glycerolphosphate, 100 μmol/L sodium orthovanadate, 5 mmol/L DTT, 5 nmol/L MEK1, 1 μmol/L ERK2, and AZD6244 (dissolved in 1% DMSO). The reactions are initiated by the addition of 10 μmol/L ATP (with 0.5 μC k[γ-33P]ATP/well) and incubated at room temperature for 45 min. An equal volume of 25% trichloracetic acid is added to stop the reaction and precipitate the proteins. Precipitated proteins are trapped onto glass fiber B filter plates, excess labeled ATP is washed off with 0.5% phosphoric acid, and radioactivity is counted in a liquid scintillation counter. ATP dependence is determined by varying the amount of ATP in the reaction mixture. [3]

Cell Assay:
Tumor cells are seeded at a density of 2.0 × 104. After 48h incubation, the cells are rinsed twice with culture media. Cells are treated with various concentrations of AZD6244 for 24 or 48 h. Cell viability is determined by the 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide (MTT) assay.
Cell proliferation is assayed using a bromodeoxyuridine kit. [1]

Animal Studies:
AZD6244 is suspended in water and administered to tumor bearing mice by p.o. with 50/100mg AZD6244 per kg of body weight after tumor implanting. Tumor size is measured by Vernier caliper. Tumor volume is calculated as (length × width2)/2. Animals are sacrificed 3 h after the last dose of ADZ6244, and body and tumor weights ae recorded, with the tumors harvested for analysis. [1]

References on AZD6244 (Selumetinib):

[1] Huynh H, et al, Mol Cancer Therapy, 2007, 6 (1), 138-146

[2] Garon EB, et al, Mol Cancer Thera, 9 (7), pp. 1985-1994.

[3] Yeh TC, et al, Clin Cancer Res, 2007, 13 (5), 1576-1583.

[4] Huynh H, et al, Mol Cancer Ther, 2007, 6 (9), 2468-2476.

GSK1120212 (JTP-74057) is a reversible, selective, allosteric MEK1/MEK2 kinase activity inhibitor with IC50 of 0.7 and 0.9 nM for MEK1 and MEK2. [1] GSK1120212 (JTP-74057) strongly prevented the activities of MEK1 and MEK2 kinases rather than activities of other 98 kinases. After treatment of GSK1120212 (JTP-74057), it led to the growth inhibition and upregulate p15INK4b and/or p27KIP1 in most of the colorectal cancer cell lines tested. In nude animal mice studies, JTP-74057 inhibited tumor growth of HT-29 and COLO205 xenografts when GSK1120212 (JTP-74057) was daily oral administered for 14 days. GSK1120212 (JTP-74057) showed an additive or a synergistic action in combination with the standard-of-care agents, 5-fluorouracil, oxaliplatin or SN-38. Sensitivity to JTP-74057-induced apoptosis may be an important factor for the estimation of in vivo efficacy, and sensitivity was enhanced by an Akt inhibitor. [2] GSK1120212 (JTP-74057) is originally developed by GlaxoSmithKline and is recruiting for phase I clinical trials for the treatment of solid tumors.

References on GSK1120212 (JTP-74057):

[1] Yamaguchi T et al. Int J Oncol. 2011 Jul;39(1):23-31.

[2] Gilmartin AG et al. Clin Cancer Res. 2011 Mar 1;17(5):989-1000.

PD0325901 is selective and non ATP-competitive mitogen activated protein kinase kinase (MEK or MAPKK) MEK inhibitor with an IC50 of 0.33 nM for inhibiton of MEK activity in mouse colon 26 cells. The dual specific threonine/tyrosine kinase MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway that is frequently activated in human tumors. PD0325901 is exquisitely specific and highly potent against purified MEK, revealing a Kiapp of 1 nM against activated MEK1 and MEK2. PD0325901 is roughly 500-fold more potent than CI-1040 with respect to its cellular effects on phosphorylation of ERK1 and ERK2, displaying subnanomolar activity. PD0325901 prevents the growth of melanoma cell lines in vitro and in vivo and causes G1-phase cell cycle arrest and apoptosis in a mouse xenograft model. A single oral dose of PD0325901 (25 mg/kg) suppressed phosphorylation of ERK by >50% at 24 hours post-dosing. Anticancer activity of PD 0325901 has been demonstrated for a broad spectrum of human tumor xenografts, significantly inhibiting the growth of 6 out of 7 human tumor models tested. [1] PD0325901 also blocks production of proangiogenic cytokines such as VEGF. PD0325901 is originally developed by Pfizer. A phase II clinical trial of PD0325901 has been terminated in the treatment of non-small-cell lung cancer.

Protocol:

Solubility:
Soluble to 25 mM in DMSO.

Biochemical assay:
PD0325901-and vehicle-treated tumors were excised after imaging, fixed in formalin, embedded in paraffin, and cut into 5.0-μm sections. The degree of tumor proliferation was assessed in tumor sections as previously described. For each tumor section, the total number of Ki67-positive brown-stained cells and hematoxylin-stained cells were counted in 16 randomly selected fields of view using an Olympus BX51 microscope at ×400 magnification. The Ki67 labeling index (LIKi67) was calculated using the equation LIKi67 = [Ki67-positive cells / (total cells)] × 100%. [2]

Cell assay:
SKMEL-28 and HCT116 cells were cultured in RPMI 1640 growth medium containing 10% (v/v) fetal bovine serum, 2 mM of l-glutamine, 100 units/ml of penicillin, and 100 g/ml of streptomycin and grown in a 5% CO2 incubator at 37°C. Cells in exponential growth were used for subsequent studies. To examine the effect of PD0325901 in vitro on SKMEL-28 cells, the growth medium was replaced with medium containing 1 μM of PD0325901 or DMSO (the final concentration of DMSO in both cases was 0.1%). The plates were then incubated for 0.5, 2, 4, 24, or 48 hours and harvested. [2]

Animal study:
Two xenograft models to used to assess the biological activity of PD0325901, i.e., human melanoma SKMEL-28 and human colon carcinoma HCT116 xenografts. Six- to 8-week-old BALB/c nu/nu mice were obtained and tumors were induced by s.c. inoculation of 5 × 106 SKMEL-28 cells (BRAF mutant) or HCT116 cells (K-RAS mutant) on the back of the mice. SKMEL-28 cells were prepared as a suspension in 25% of growth medium in basement membrane matrix; HCT116 cells were prepared in PBS. Tumor dimensions were measured continuously using a caliper and tumor volumes were calculated by the equation: volume = (π/6) × a × b × c, where a, b, and c represent three orthogonal axes of the tumor. Mice were used when their tumors reached 100 mm3. Size-matched tumor-bearing mice were randomized to receive 25 mg/kg (0.005 mL/g mouse) of PD0325901 or vehicle (0.5% hydroxypropyl methylcellulose plus 0.2% Tween 80) daily by oral gavage. [18F]FLT-PET scanning was done at 25 hours after commencing treatment (i.e., after two doses with the second dose given 1 hour before scanning) or after 10 daily treatments (again with the last dose given 1 hour before scanning). After imaging, one part of the tumor was snap-frozen in liquid nitrogen and stored at −80°C; the other part was fixed in formal saline. [2]

References on PD0325901:

[1] Judith S. Sebolt-Leopold et al. Proc Amer Assoc Cancer Res.2004,45

[2] Barrett SD et al. Bioorg Med Chem Lett. 2008 Dec 15;18(24):6501-4.

TAK-733 is highly potent and selective novel MEK allosteric site inhibitor with an IC50 of 3.2 nM. TAK-733 selectively binds to and inhibits the activity of MEK1/2. Subseqently, TAK-733 prevents the activation of MEK1/2-dependent effector proteins and transcription factors, which may result in the inhibition of growth factor-mediated cell signaling and tumor cell proliferation. MEK1/2 (MAP2K1/K2) are dual-specificity threonine/tyrosine kinases that play key roles in the activation of the RAS/RAF/MEK/ERK pathway and are often up-regulated in a variety of tumor cell types. TAK-733 has potent anticancer activity in various mouse xenograft models. TAK-733 showed potent enzymatic and cell activity with an EC50 of 1.9 nM against ERK phosphorylation in cells. [1][2]

References on TAK-733:

[1] Dong Q et al. Bioorg Med Chem Lett. 2011 Mar 1;21(5):1315-9

[2] http://www.termwiki.com/EN:MEK_inhibitor_TAK-733

RDEA119 is a highly potent and selective inhibitor of mitogen activated ERK kinase (MEK), a key component of the RAS/RAF/MEK/ERK pathway that is commonly defective in human tumors . [1,2]

Rdea119 has favorable properties, including oral dosing, excellent selectivity and low central nervous system (CNS) penetration. [3]

In mouse xenograft studies, when rdea119 was dosed orally once daily for 14 days, there are significant inhibition and delay of tumor growth. We observed inhibition of pERK in tumors (the target of therapy) at significantly lower concentrations (EC50 = 73 nM), compared to those required to inhibit brain pERK (EC50 > 5,000 nM). [3,4]

References on RDEA119(BAY 869766):

[1] New Oral MEK Inhibitor, RDEA119, Shows Favorable Anti-Tumor Properties

[2] Ardea Biosciences to Present Data on Lead MEK Inhibitor, RDEA119, at Digestive Disease Week 2008.

[3] Ardea Biosciences to Present Data on Lead MEK inhibitor, RDEA119, at EORTC-NCI-AACR Symposium.

[4] Cory Iverson. The effect of MEK inhibitor RDEA119 on biomarkers in advanced cancer patients in a phase I clinical trial. 2008 Molecular Markers meeting.

U0126 is an inhibitor of both MEK1(IC50 of 72 nM) and MEK2(IC50 of 58 nM).
U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with IC50 of 72 nM for MEK1 and 58 nM for MEK2. [1] U0126 inhibited anchorage-independent growth of Ki-ras-transformed rat fibroblasts by simultaneously blocking both extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR)-p70(S6K) pathways. [2] The effects of U0126 on the growth of eight human breast cancer cell lines shown that U0126 selectively repressed anchorage-independent growth of MDA-MB231 and HBC4 cells, two lines with constitutively activated ERK. Loss of contact with substratum triggers apoptosis in many normal cell types, a phenomenon termed anoikis. U0126 sensitized MDA-MB231 and HBC4 to anoikis, i.e., upon treatment with U0126, cells deprived of anchorage entered apoptosis. [3]

References on U0126:

[1] Duncia JV et al. Bioorg Med Chem Lett. 1998 Oct 20;8(20):2839-44

[2] Hidesuke Fukazawa, and Yoshimasa Uehara.Cancer Res. 2000 April 4;60: 2104

[3] Hidesuke Fukazawa et al. Mol Cancer Ther . 2002 Mar;1(5):303-9

PD 98059 is a highly selective inhibitor of MEK1 and MEK2 with IC50 values of 4 µM and 50 µM respectively[1–3] . PD98059 does not inhibit activation of other highly related dual-specificity protein kinases or the activity of over 18 other Ser/Thr protein kinases. At concentrations up to 100 µM, PD98059 does not inhibit activation of MKK3 or SEK (MKK4) as determined by measuring phosphorylation at its activation site. [1–3]

References on PD98059:

[1] Rundén E et al. J Neurosci. 1998 Sep 15;18(18):7296-305

[2] Veeranna et al. J Neurosci. 1998 Jun 1;18(11):4008-21

[3] Xing J et al. Mol Cell Biol. 1998 Apr;18(4):1946-55

PD318088 is a non-ATP competitive allosteric MEK1/2 inhibitor. It is a small-molecule inhibitor bound within the allosteric site. The binding modes of two kinase inhibitors are shown in relation to the binding site of ATP in the kinase active site. The MEK1 inhibitor PD318088 binds simultaneously with ATP in a region of the kinase active site that is adjacent to the ATP-binding site. Birb796 binding to p38 extends into the ATP site, but also accesses this back pocket, partly overlapping the region where PD318088 binds in MEK1. [1][2]

References on PD318088:

[1] Ohren JF et al. Nat Struct Mol Biol. 2004 Dec;11(12):1192-7.

[2] Sebolt-Leopold JS et al. Nature. 2006 May 25;441(7092):457-62.

D-87503 is a potent PI3K/Akt/mTOR signaling pathway inhibitor with IC50 of 62nM and 0.76μM for PI3K and Erk2, respectively. In cellular assays, D-87503 also effectively suppressed the target downstream substrates Akt and Rsk1 kinase of the PI3K/Akt/mTOR signaling pathway. D-87503 inhibited several carcinoma cell lines, including BxPC3, Hct116, MDA-MB 468 and 231 with EC50 of median 5µM. [1]

References on D-87503:

[1] Maira SM et al. Biochem Soc Trans. 2009 Feb;37(Pt 1):265-72.

References on D-87503:

[1] Maira SM et al. Biochem Soc Trans. 2009 Feb;37(Pt 1):265-72.

CI-1040 is a non-competitive inhibitor of MEK1/2 with a Ki of 300nM in vitro[1] CI-1040 inhibited MEK (as measured by phosphorylated ERK (p-ERK) levels) with a half-maximal inhibitory concentration (IC50) of 100–500nM in all cell lines tested[2] .

References on CI-1040 (PD184352):

[1] Judith S. Sebolt-Leopold et al. Nature Medicine.1999,5:810-816

[2] David B. Solit et al. Nature. 2006 January 19;439:358-362

AS703026 is a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells (cell IC50 ranging from 0.005 to 2 µM) and cytokine-induced osteoclast differentiation more potently (9- to 10-fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression.

Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. [1]

References on AS703026:

[1] Kim K et al. Br J Haematol. 2010 May; 149(4):537-49