心力衰竭的细胞分子生物学基础：PKC激活和心力衰竭-英文课件PPT课件.ppt

心力衰竭的心力衰竭的细细胞胞分子生物学基分子生物学基础础：PKC激活和心力激活和心力衰竭衰竭-英文英文课课件件Heart Failure in AustralialThe prevalence of heart failure in Australia is 6.3%.lThe heart failure prevalence is 10%in people aged over 65 years(this population will double over the next 50 years in Australia).Stages of heart failure from Goldman:Cecil Medicine,23rd ed Effusion from congestive heart failure from Mason:Murray&Nadels Textbook of Respiratory Medicine,4th ed2 2Molecular Cell Biology underlying heart failure:PKC Activation and Heart FailureCirculation Research.2007;101:195 3 3J.Clin.Invest.,115:527,2005Activation of PKC and Heart FailurePKC is activated by Gq/G11-PLC -IP3-SR-released Ca+and DAGSarcoplasmic Reticulum)4 4wo5 5Nature Medicine,10:239,2004Nature Medicine,10:239,2004Protein Phosphatase-1Phospholamban P ca+uptake,increased Ca+release&contractile force)Sarcoplasmic reticular calcium uptake proteinPKCa Ca+cyclingReduced1-contraction strength2ralaxation3Cardiac reservePKCa is activated by Ca+,stress agonists,pressure load,myocardial infarction,angiotensin,Transition in failing hearts fromadequate compensation To advanced failureProtein Phosphatase Inhibitor-1Calcium handling proteins:PLB,SERCA-2a6 6Domain Structure of PKC7 7Most available kinase inhibitors are ATP-binding site analoguesMost available kinase inhibitors are ATP-binding site analoguesLack of specificity:Lack of specificity:Nat.BiotechnolNat.Biotechnol.23:329-336,200523:329-336,2005Biochem.JBiochem.J.351:95-105,2000.351:95-105,2000.ATPN-LobeC-LobeStaurosporine Staurosporine(originally developed(originally developed as a PKC inhibitor)as a PKC inhibitor)Is there such a thing as a“Is there such a thing as a“SpecificSpecific”kinase inhibitor?”kinase inhibitor?8 83-D structure of PKADIFFICULTIES in structural determination of C-tailRole of the very C-termini of PKCsN-LobeC-Lobe9 9Study of the function of C-terminus of PKC-alphaStudy of the function of C-terminus of PKC-alpha1010The last 10 amino acid residues are essential for the catalytic competence The last 10 amino acid residues are essential for the catalytic competence of PKC-alphaof PKC-alphaIntrinsic activity,protamine sulfate as substrate.DAG-activation Histone H1 as substrateS.S.Yeong,et al.,J.Biol.Chem.281:30768,20061111The critical role of the very C-terminus of PKC-a in the activation of MAPKThe critical role of the very C-terminus of PKC-a in the activation of MAPK S.S.Yeong,et al.,J.Biol.Chem.281:30768,20061212The critical role of the very C-terminus of PKC-a in Augmenting Melatonin-stimulated Neurite Outgrowth in Neuronal CellsS.S.Yeong,et al.,J.Biol.Chem.281:30768,20061313Targeting“intrinsically disordered segment”Targeting“intrinsically disordered segment”instead of“instead of“druggable pocketdruggable pocket”.”.1414Proof-of-principle dataS.S.Yeong,et al.,J.Biol.Chem.,20061515Aptamers(from Latin(from Latin aptusaptus,means“fitting”),means“fitting”)Synthetic RNA,DNA or peptide that binds to protein targets Synthetic RNA,DNA or peptide that binds to protein targets with high specificity and affinitywith high specificity and affinity-Fold into well-defined 3-D structure-Fold into well-defined 3-D structure-Bind by-Bind by complementary shapecomplementary shape interactions interactions-Not toxic or immunogenic-Not toxic or immunogenic-Effectively inhibit the function of target protein-Effectively inhibit the function of target protein-No prior knowledgeNo prior knowledge of the of the 3-D structure3-D structure of the target is required of the target is requiredthrombinCurrent Opinion in Chemical BiologyCurrent Opinion in Chemical Biology,9:336-342,2005,9:336-342,20051616Nucleic Acids Base PairingtRNA1717SELEX:SELEX:S Systemic ystemic E Evolution of volution of L Ligands by igands by ExExponential enrichment.ponential enrichment.1818Develop Aptamers Targeting the very C-terminus of PKC C-terminus of PKC is exposed(accessible)from IP experiments1919From Aptamer to Small Molecule Drugs:From Aptamer to Small Molecule Drugs:Aptamer-guided HTSAptamer-guided HTSFluorescence Polarization-based screening.Homogenous assay system.Fluorescence Intensity-based screeningProf.Michael FamulokLaboratory of Chemical Biology University of Bonn 2020Source of Chemical LibrarieslCommercial sources(free of royalty)l-NCI(http:/dtp.nci.nih.gov/docs/misc/available_samples/dtp_indsamples.html)lStructural Diversity Set,version 1(1,991 compounds)lStructural Diversity Set,version 2(1,986 compounds)lMechanistic Diversity Set(879 compounds)lOpen Collection 1(90,000 compounds)lOpen Collection 2(10,000 compounds)l-ChemDiv(http:/)l-ChemBridge Corporation(http:/ with pharmaslPartnership with academia MOLECULAR LIBRARIES SCREENING CENTERS NETWORK(Established in 2004)9 Centers Sources of Current or Off-patient medications-Prestwick Chemical Co.(1120 off-patient drugs)-Microsources:The Spectrum Collection(2000)-Sequoia(a large collection)-NIH Brain Bioactive Compound collection2121Aptamer-guided Heart-Specific Delivery2222Molecular Medicine for Heart DiseasesEngineering nanoparticlesDrug loadingTargetingControlled Release(heat,magnetic field,pH),engineering the nanoparticle system such that the intracellular drug-release kinetics is greater than the drug-efflux kineticsDrugs administered at a given time for a given periodRamping of redesigned doseWithdraw or washing offTissue/organ-specific targetingDomain-specific targeting of a protein2323