Sıra | DOSYA ADI | Format | Bağlantı |
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01. | Conference Conference Kinase Cells | pptx | Sunumu İndir |
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About OMICS GroupOMICS Group is an amalgamation of Open Access Publications and worldwide international science conferences and events. Established in the year 2007 with the sole aim of making the information on Sciences and technology ‘Open Access’, OMICS Group publishes 500 online open access scholarly journals in all aspects of Science, Engineering, Management and Technology journals. OMICS Group has been instrumental in taking the knowledge on Science & technology to the doorsteps of ordinary men and women. Research Scholars, Students, Libraries, Educational Institutions, Research centers and the industry are main stakeholders that benefitted greatly from this knowledge dissemination. OMICS Group also organizes 500 International conferences annually across the globe, where knowledge transfer takes place through debates, round table discussions, poster presentations, workshops, symposia and exhibitions.
OMICS International ConferencesOMICS International is a pioneer and leading science event organizer, which publishes around 500 open access journals and conducts over 500 Medical, Clinical, Engineering, Life Sciences, Pharma scientific conferences all over the globe annually with the support of more than 1000 scientific associations and 30,000 editorial board members and 3.5 million followers to its credit.OMICS Group has organized 500 conferences, workshops and national symposiums across the major cities including San Francisco, Las Vegas, San Antonio, Omaha, Orlando, Raleigh, Santa Clara, Chicago, Philadelphia, Baltimore, United Kingdom, Valencia, Dubai, Beijing, Hyderabad, Bengaluru and Mumbai.
Towards pharmacological validation of MNKs as anti-cancer drug targetsDr Hugo AlbrechtSchool of Pharmacy and Medical SciencesUniversity of South Australia
4Kinases as Cancer Drug Targets“Hard Targets”KO leads to strong or lethal pheno-type (e.g. PLK1)Adverse effects are expected. Use of advanced formulation to target cancer cells (e.g. nanoparticles, liposomes etc.)http://www.cellsignal.com/reference/kinase/overview.html “Soft Targets” KO with no obvious or mild pheno-type (e.g. Mnk1/2)
5Anti-cancer strategy•Targeting cancer cells through block of several pathwaysTranscriptionCancerCellCell cycle TranslationMitosisR-point
Targeting Translation: Mnk1/2StressGrowthFactors IntegrinsRasP38a/bPDK1Mek1/2RafAktSOSGrb2PI3KSrcFAKErk1/2PTENmTORTSC1/2RhebS6K14E-BPeIF4ETranslationS6KsMKK3/6MEKKCancermut
7Phosphorylation of eIF4E by MnksAdapted from Hay N., 2010, PNAS, Vol.107, No. 32, 13975-13976 and Pyronnet, S.; Imataka, H.; Gingras, A. C.; Fukunaga, R.; Hunter, T.; Sonenberg, N. The EMBO journal 1999, 18, 270_P4E 4 A40SMnk1/23AAAAAAUGErk1/2, p38m7GCcl2, Ccl7, MMP-3, MMP-9, Mcl1,Bcl2, Myc, PDGF, FGF2, VEGF,IL-1B, IL-15, TNF-a
8Elevated eIF-4E phosphorylation promotes tumorigenesisDiab, S.; Kumarasiri, M.; Yu, M.; Teo, T. H. S.; Milne, R.; Proud, C.; Wang, S. Chem. Biol. 2014, 21, 441Graff, J. R.; Konicek, B. W.; Carter, J. H.; Marcusson, E. G. Cancer research 2008, 68, 631.
9Rationale• eIF4E is frequently overexpressed in cancers and is associated with cellular transformation, tumorigenesis and metastatic progression.• In Mnk1/2 DKO mice:-eIF4E phosphorylation is completely abolished-Cells are resistant to transformation by Ras and tumour formation is significantly delayed in PTEN-/- x Mnk DKO mice lymphoma model2)• Knock-in mice with a eIF4E S209A mutation are resistant to: -Oncogene induced transformation1)-PTEN loss-induced prostate cancer1)• Normal growth of Mnk DKO and eIF4E S209A KI mice.Mnk1 and Mnk2 inhibitors may be effective and non-toxic anticancer drugsReferences: 1. Luc Furic et al PNAS 2010, 107 (32), 14134-14139 /.2. Takeshi Ueda et al PNAS 2010, 107 (32), 13894-139908
10Aim: Design and characterization of Mnk1/2 inhibitorsCompetition effectIMAPADP-GloCompound libraryDualMnk1 Mnk2- + + -- - + +ATPeIF4E peptidePrimary hitsPrimary assayQuick, robust and cost effectiveSecondary assayDose response and competition assayCytotoxicity Mnk structureMigration/Metastasis3D structure of staurosporine-bound Mnk2-KR obtained from the Protein Databank (PDB: 2HW7)Default active site created by FRED receptor programDefault active site detected by FRED receptor program as shown in the blue box
11Primary compound profiling with IMAP assaysIMAP: Immobilised Metal Ion Affinity Particle Progressive Binding SystemMnkATPfluorescent peptide substratephospho-peptidehighTR-FRETPO4MIIIMIIITb-donorIMAPbindingreagentlinkerTbsensitizerPO4PO40 25 50 75 100 1250255075100125Mnk1 100ng% Activity1M% Activity (10 M)0 25 50 75 100 1250255075100125Mnk2 30ng% Activity1M% Activity (10 M)
12ADP-Glo AssayMnkeIF4E202-214ATPADP-GloreagentADPKinasedetectionreagentADP/ATPmixLuciferaseATP AMPBeetleluciferin+ Light0 1 2 3 40100020003000eIF4E202-214 conc (mM)Luminescence (RLU) 1000Mnk1Mnk2 0.0 0.2 0.4 0.6 0.8 1.003006009001200ATP conc (mM)Luminescence (RLU) 1000Mnk1Mnk20 1 2 3 4 5 605001000150020002500ATP conc (mM)Luminescence (RLU) 1000 Mnk1Mnk2MNKI-6MNKI-7MNKI-5MNKI-57MNKI-8MNKI-19MNKI-83MNKI-67MNKI-37MNKI-15MNKI-2-22MNKI-2-30MNKI-2-25MNKI-7-50MNKI-28MNKI-85MNKI-4MNKI-5-17MNKI-12MNKI-4-61012345678910IC50 (M)IC50 Mnk2IC50 Mnk17 dual inhibitors13 Mnk2 specific (Mnk1 > 10x Mnk2)
13Mnk1/2 Crystal Structure DiscrepancyCrystal structure of Mnk2 (PDB entry 2AC3)Crystal structure of Mnk1 (PDB entry 2HW6)
14Identification of Type I/II Mnk inhibitorseIF4E fixed at 600 µM (KM)-4 -3 -2 -1 0 1 2020406080100120SD4-61, Mnk2 30ngLog SD4-61 (M)% Kinase Activity50 M ATP200 M ATP400 M ATP800 M ATP1200 M ATP2000 M ATP-4 -3 -2 -1 0 1 2020406080100120SD4-61, Mnk2 30ngLog SD4-61 (M)% Kinase Activity150 M eIF4E600 M eIF4E1200 M eIF4E1800 M eIF4E2400 M eIF4E3000 M eIF4EATP fixed at 200 µM (KM)Data Mean ± SEM, n=2MNKI-4-61NSNNNHOOO
15Identification of Type I/II Mnk inhibitors-4 -3 -2 -1 0 1 2020406080100120SD5-17, Mnk2 30ngLog SD5-17 (M)% Kinase Activity50 M ATP200 M ATP400 M ATP800 M ATP1200 M ATP2000 M ATPeIF4E fixed at 600 µM (KM)-4 -3 -2 -1 0 1 2020406080100120SD5-17, Mnk2 30ngLog SD5-17 (M)% Kinase Activity150 M eIF4E600 M eIF4E1200 M eIF4E1800 M eIF4E2400 M eIF4E3000 M eIF4EATP fixed at 200 µM (KM)Data Mean ± SEM, n=2MNKI-5-17NSNNNHOOFF
16Identification of Type III Mnk inhibitorsATP fixed at 200 µM (Km)-4 -3 -2 -1 0 1 2020406080100120TAG-6, Mnk2 30ngLog TAG-6 (M)% Kinase Activity150 M eIF4E600 M eIF4E1200 M eIF4E1800 M eIF4E2400 M eIF4E3000 M eIF4EeIF4E fixed at 600 µM (Km)-4 -3 -2 -1 0 1 2020406080100120TAG-6, Mnk2 30ngLog TAG-6 (M)% Kinase Activity50 M ATP200 M ATP400 M ATP800 M ATP1200 M ATP2000 M ATPData Mean ± SEM, n=2MNKI-6Allosteric inhibitorsNN SONHF OFFNH2
17Type I/II vs. Type III inhibitorsMNKI-5MNKI-15-3 -2 -1 0 1 2020406080100120Log TAG-5 (M)% Kinase Activity2000 M50 M200 MATP ConcentrationATP Concentration-3 -2 -1 0 1 2020406080100120Log TAG-15 (M)% Kinase Activity50 M2000 M200 M< 6 foldDFD in with MNKI-15 DFD in with MNKI-5 NN SONHF OFFOHNN SONHFOH
18MV4-11 Leukemia cells0 1 2020406080100IC50 Mnk2GI50 MV4-110 1 2 3 4 5 6 7 8020406080100IC50 Mnk1GI50 MV4-11r = 0.7276p = 0.0005r = 0.4006p = 0.0780
19SummarySummaryCompetition effectIMAPinhibitionADP-Glo IC50 < 2 µMCompound library66 compoundsDualMnk135- + + -- - + +ATPeIF4E peptide10 µM < 10% or 1 µM < 75%No11No0 13 78 4 1 0Compounds 1 5 1 08 3 1 0CDK2/9 Ki > 15 µM 1 4 0 0- + + -- - + +31Yes20Mnk2MV4-11cytotoxicity6 0 0 1 1 GI50 < 10 µM
20Prostate cancer cellsMnk1Mnk2eIF4E WBMV4-11PC-0PC-3A8MNKI-7 83 44 35 0.83 >10 8.65MNKI-19 279 136 135 7.30 >10 >10MNKI-67 1400 36 X 8.84 >10 >10IC50 (nM) GI50 (µM)PC-0 PC-3A8PTEN - -P-Akt + +Androgen dependent + -Metastatic - +
21Prostate cancer cells
22ConclusionsPotent Mnk1/2 and selective Mnk2 inhibitors have been identified.Detailed kinetic studies revealed three types of binding modes.Our studies confirm different structural properties of Mnk1 and Mnk2 at the ATP-binding pocket.But they share a common allosteric binding site. Mnk inhibitors are cytotoxic against some leukemia cells.No cytotoxicity has been observed against prostate cancer cells.Mnk inhibitors blocked LPS induced migration of prostate cancer cells.
23AcknowledgementProf Shudong WangDr Matt SykesA/Prof Bob MilneDr Tom PeatBiologyRaffaella SchmidTheodosia TeoDr Peng LiDr Frankie LamBen NollTracy LuChemistrySarah DiabDr Mingfeng YuYuchao YangTodd Alexander Gillam
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25Subheading when requiredMajor Heading Here
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