Study of proteins as drug targets by NMR spectroscopy [Elektronische Ressource] / von Sridhar Sreeramulu

Sridhar Sreeramulu

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• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 16 Mo

Extrait

Study of Proteins as Drug Targets by
NMR spectroscopy

Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt beim
Fachbereich Biochemie, Chemie und Pharmazie der
Johann Wolfgang Goethe - Universität
zu Frankfurt am Main
von

Sridhar Sreeramulu

aus
Ambur (Indien)

Frankfurt 2009
D30



 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vom Fachbereich Biochemie, Chemie und Pharmazie der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.


DEKAN: Prof. Dr. Dieter Steinhilber

1. GUTACHTER: Prof. Dr. Harald Schwalbe
2. GTER: Prof. Dr. Volker Dötsch


DATUM DER DISPUTATION: 2009
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
To my parents and wife
 
 
 
 
 
 

 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 



































This thesis was prepared under the supervision of Prof. Dr. Harald Schwalbe
between Sept 2003 and May 2009 at the Institute for Organic Chemistry and
Chemical Biology of the Johann-Wolfgang Goethe-University Frankfurt. Contents
List of Figures v
List of Tables vii
1 Overview and Summary 1
2 Structural Proteomics: An Overview 7
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Global structural efforts and target selection strategies . . . . . . . . 8
2.2.1 Japan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2.2 United States of America (USA) . . . . . . . . . . . . . . . . . 9
2.2.3 North America . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.4 Structural proteomics in Europe (SPINE) . . . . . . . . . . . . 10
2.3 Role of NMR in structural proteomics . . . . . . . . . . . . . . . . . . 12
2.4 Protein Data Bank (PDB) statistics for structures solved by NMR . . . 14
3 Importance of Study of Various Protein Families of Biomedical Relevance 17
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.2 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2.1 Molecular basis of cancer phenotypes . . . . . . . . . . . . . . 19
3.2.2 Oncogenes as therapeutic targets . . . . . . . . . . . . . . . . 21
3.3 Protein Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.3.1 Classification of the kinome superfamily . . . . . . . . . . . . 22
3.3.2 Structural analysis of cAMP-dependent protein kinase (PKA)
and major classes of protein kinases . . . . . . . . . . . . . . 23
3.3.3 Substrate specificity: How does a kinase recognize its substrate? 26
iii
3.3.4 Mechanism of regulation of protein kinase activity . . . . . . 26
3.3.5 Oncogenic kinases in cancer . . . . . . . . . . . . . . . . . . . 28
3.3.6 NMR studies on protein kinases . . . . . . . . . . . . . . . . . 28
3.4 Heat shock protein of 90kDa (Hsp90)-a kinome chaperone . . . . . . 30
3.4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4.2 Chaperone alteration in cancer . . . . . . . . . . . . . . . . . 32
3.4.3 Hsp90 structure and function . . . . . . . . . . . . . . . . . . 34
3.4.4 Inhibition of Hsp90 . . . . . . . . . . . . . . . . . . . 35
3.5 Cell division cycle protein 37 (Cdc37)-a kinome co-chaperone . . . . 36
3.5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.5.2 Cdc37 promotes proliferation . . . . . . . . . . . . . . . . . . 40
3.5.3 Cdc37 structure and function . . . . . . . . . . . . . . . . . . 40
3.5.4 Targeting Cdc37 in cancer . . . . . . . . . . . . . . . . . . . . 43
4 Small Molecule Inhibitors for Disrupting Protein-Protein Interaction 47
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.1.1 Major challenges and approaches used for targeting protein-
protein interactions . . . . . . . . . . . . . . . . . . . . . . . . 50
4.1.2 Small molecule inhibitor of Hsp90-Cdc37 complex . . . . . . 51
5 NMR Methods to Study Protein-Protein interactions 53
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5.2 Nuclear Magnetic Resonance (NMR) methods to study proteins and
protein complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.2.2 Methods to study large proteins . . . . . . . . . . . . . . . . . 57
5.2.3 NMR methods . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.2.4 Optimization of protein domains for NMR studies . . . . . . . 58
5.2.5 NMR based methods for the study of protein-protein interaction 61
5.2.5.1 Nuclear Overhauser Effect (NOE) . . . . . . . . . . 61
5.2.5.2 Chemical Shift Perturbation (CSP) . . . . . . . . . . 62
5.2.5.3 Cross-Saturation Transfer (CST) . . . . . . . . . . . 64
5.2.5.4 Mapping with dynamics . . . . . . . . . . . . . . . . 67
5.2.5.5 with amide-proton exchange (H-D
Exchange) . . . . . . . . . . . . . . . . . . . . . . . 67
5.2.5.6 Mapping with paramagnetics . . . . . . . . . . . . . 67
5.2.5.7 with site-specific spin labeling . . . . . . . 68