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1、Wei DUAN School of Medicine Deakin University Melbourne, Australia,Molecular Medicine for Heart Failure Isozyme-specific Modulation of PKC,Heart Failure in Australia,The prevalence of heart failure in Australia is 6.3% . The heart failure prevalence is 10% in people aged over 65 years (this populati
2、on 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 ed,Molecular Cell Biology underlying heart failure: PKC Activation and Heart Fai
3、lure,Circulation Research. 2007;101:195,J. Clin. Invest., 115:527,2005,Activation of PKC and Heart Failure PKC is activated by Gq/G11 -PLC -IP3-SR-released Ca+ and DAG,Sarcoplasmic Reticulum),wo,Nature Medicine, 10:239,2004,Protein Phosphatase-1,Phospholamban P ca+ uptake, increased Ca+ release & co
4、ntractile force),Sarcoplasmic reticular calcium uptake protein,PKCa,Ca+ cycling,Reduced 1-contraction strength 2ralaxation 3Cardiac reserve,PKCa is activated by Ca+, stress agonists, pressure load, myocardial infarction, angiotensin,Transition in failing hearts from adequate compensation To advanced
5、 failure,Protein Phosphatase Inhibitor-1,Calcium handling proteins: PLB, SERCA-2a,Domain Structure of PKC,Most available kinase inhibitors are ATP-binding site analogues,Lack of specificity: Nat. Biotechnol. 23:329-336, 2005 Biochem. J. 351:95-105, 2000.,Staurosporine (originally developed as a PKC
6、inhibitor),Is there such a thing as a “Specific” kinase inhibitor?,3-D structure of PKA,DIFFICULTIES in structural determination of C-tail,Role of the very C-termini of PKCs,N-Lobe,C-Lobe,Study of the function of C-terminus of PKC-alpha,The last 10 amino acid residues are essential for the catalytic
7、 competence of PKC-alpha,Intrinsic activity, protamine sulfate as substrate.,DAG- activation Histone H1 as substrate,S.S. Yeong, et al., J. Biol. Chem. 281:30768, 2006,The 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, 2006,The c
8、ritical role of the very C-terminus of PKC-a in Augmenting Melatonin-stimulated Neurite Outgrowth in Neuronal Cells,S.S. Yeong, et al., J. Biol. Chem. 281:30768, 2006,Targeting “intrinsically disordered segment” instead of “druggable pocket”.,Proof-of-principle data,S.S. Yeong, et al., J. Biol. Chem
9、., 2006,Aptamers (from Latin aptus, means “fitting”),Synthetic RNA, DNA or peptide that binds to protein targets with high specificity and affinity,-Fold into well-defined 3-D structure -Bind by complementary shape interactions -Not toxic or immunogenic -Effectively inhibit the function of target pr
10、otein -No prior knowledge of the 3-D structure of the target is required,thrombin,Current Opinion in Chemical Biology, 9:336-342, 2005,Nucleic Acids Base Pairing,tRNA,SELEX: Systemic Evolution of Ligands by Exponential enrichment.,Develop Aptamers Targeting the very C-terminus of PKC ,C-terminus of
11、PKC is exposed (accessible) from IP experiments,From Aptamer to Small Molecule Drugs: Aptamer-guided HTS,Fluorescence Polarization-based screening. Homogenous assay system.,Fluorescence Intensity-based screening,Prof. Michael Famulok Laboratory of Chemical Biology University of Bonn,Source of Chemic
12、al Libraries,Commercial sources (free of royalty) -NCI (http:/dtp.nci.nih.gov/docs/misc/available_samples/dtp_indsamples.html) Structural Diversity Set, version 1 (1,991 compounds) Structural Diversity Set, version 2 (1,986 compounds) Mechanistic Diversity Set (879 compounds) Open Collection 1 (90,0
13、00 compounds) Open Collection 2 (10,000 compounds) -ChemDiv (http:/) -ChemBridge Corporation (http:/ Partnership with pharmas Partnership with academia MOLECULAR LIBRARIES SCREENING CENTERS NETWORK (Established in 2004) 9 Centers,Sources of Current or Off-patient medications -Prestwick Chemical Co.
14、(1120 off-patient drugs) -Microsources: The Spectrum Collection (2000) -Sequoia (a large collection) -NIH Brain Bioactive Compound collection,Aptamer-guided Heart-Specific Delivery,Molecular Medicine for Heart Diseases,Engineering nanoparticles Drug loading Targeting Controlled Release (heat, magnet
15、ic field, pH), engineering the nanoparticle system such that the intracellular drug-release kinetics is greater than the drug-efflux kinetics,Drugs administered at a given time for a given period Ramping of redesigned dose Withdraw or washing off Tissue/organ-specific targeting Domain-specific targeting of a protein,