2. From Signal Transduction to Targeted Therapy (Fall 2010) Pin Ling ( 凌 斌 ), Ph.D. Department of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw

3. Outline Signaling Transduction - Definition - History - Mechanisms - Example Targeted Therapy - Mechanisms - Examples - Current Trend Your involvement is the key to success in this lecture.

4. What is Signaling Transduction? Conversion of a signal from one physical or chemical form into another. The process initiated by recognition a Signal by a Sensor (receptor, kinase or enzyme) in the cell, then converting to one or more cellular responses through a series of signal transmission.

5. Receptors Signal Transducers Effectors A simple scheme of signal transduction Fig 15-1 Adopted from Molecular Biology of The Cell Q: Who first gets the idea about “ Signal Transduction”? Molecules involved in this process, called Signaling Molecules

6. History of signaling transduction Adopted from Nobelprize.org

7. Rodbell’s findings Adopted from Nobelprize.org

8. Gilman’s findings Adopted from Nobelprize.org

9. Adopted from Molecular Cell Biology Current view of GPCR signaling

10. A simple scheme of signal transduction Fig 15-1 Adopted from Molecular Biology of The Cell Receptors Signal Transducers Effectors

11. Four types of surface receptors Adopted from Molecular Cell Biology GPCR Ion Channel Receptor w/o Enzyme Receptor w/ Enzyme

12. Adopted from Molecular Cell Biology Four common second messengers

13. cAMP is the first 2 nd Messenger Adopted from Nobelprize.org (1 st messenger) (2 nd messenger)

14. A simple scheme of signal transduction Fig 15-1 Adopted from Molecular Biology of The Cell Receptors Signal Transducers Effectors

15. Two types of signal transducers Enzymatic proteins: Kinase, GTPase,….etc Non-Enzymatic proteins: Adaptors, Scaffolds,...etc Post-Translation Modifications (PTMs): Phosphorylation….. Protein-Protein Interactions, Signalsome Formation Two major biochemical events in signal transduction

16. Examples of enzymatic proteins Adopted from Molecular Cell Biology

17. Adaptors in signal transduction Adopted from Molecular Cell Biology

18. Ras activation following EGFR signaling Adopted from Molecular Cell Biology

19. Ras activates the MAPK/ERK pathway Adopted from Cell Signaling

20. A simple scheme of signal transduction Fig 15-1 Adopted from Molecular Biology of The Cell Receptors Signal Transducers Effectors

21. Types of Post-Translation Modifications Phosphorylation Methylation Acetylation Ubiquitination Sumoylation Chemical groups Small peptides Palmitoylation Myristoylation Lipid groups Glycosylation Sugar groups

22. Features of Post-Translation Modifications 1.Most are Reversible 2.Regulate Protein Activity, Protein Localization, Protein Interaction,……etc. 3. Focus on “Protein Phosphorylation” today

23. Protein Phosphorylation Adopted from Nobelprize.org

24. Activation of a enzyme by phosphorylation

25. Mechanism of Phosphorylation by cAPK (PKA) Adopted from Molecular Cell Biology

26. G. Manning et al., Science. 2002, 298:1912-34. Human Kinome 1.518 protein kinases 2.Tyr & Ser/Thr kinases 3.Involve many processes 4.Dysregulation => diseases 5.Targets for therapy

27. (Charles Swyers, Nature 2004) Examples of kinase-associated diseases

28. Post-translational modifications of human nucleosomal histones

29. Modular interaction domains in signaling transduction (Pawson et al, Science 2003) Check more details in BIND database (Biomolecular Interaction Network Database) www.bind.ca

30. Cell. 2004 Jan 23;116(2):191-203. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Pawson Tony Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada. pawson@mshri.on.ca Science 18 April 2003: Vol. 300. no. 5618, pp. 445 – 452 Assembly of Cell Regulatory Systems Through Protein Interaction Domains Tony Pawson 1,2* and Piers Nash 1 1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. 2 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. * To whom correspondence should be addressed. E-mail: pawson@mshri.on.capawson@mshri.on.ca Signaling Specificity

31. Multiple signaling cascades form signaling networks Cell. 2000 Oct 13;103(2):193-200.

32. Curr Opin Cell Biol. 2002 Apr;14(2):173-9. M. Synder et al, Approaches to studying signaling networks

33. Trends in Therapies 1.Gene Therapy – genetic diseases, cancer,…etc 2.Cell Therapy – degenerative diseases (Alzheimer, Myocardial disorders….etc) 3.Targeted Therapy – cancer, immune disorders,…etc => Each has its pros and cons. Signaling Transduction => Molecular Targets => Targeted Therapy

34. Fig 15-1 Adopted from Molecular Biology of The Cell Receptors Signal Transducers Effectors Dysregulation of signaling molecules leads to disorders A simple scheme of signal transduction

35. Q1: How to do targeted therapy? Two major biochemical events in signal transduction: (1) Post-Translation Modifications: protein phosphorylation (2) Protein-Protein Interactions: ligand- receptor, protein-dimeriztion Molecules designed to block these two biochemical events

36. Abl, BCR-Abl, & Chronic Myelogenous Leukemia (CML) Nat Rev Cancer. 2005 Mar;5(3):172-83.

37. Leukemogenic signaling of BCR-Abl

38. Development of Chronic Myelogenous Leukemia (CML) Nat Rev Cancer. 2005 Mar;5(3):172-83.

39. Copyright ©2001 AlphaMed Press Mauro, M. J. et al. Oncologist 2001;6:233-238 Figure 1. Schematic representation of the mechanism of action of STI571 Gleevec (STI 571, Imatinib): A Small Molecule with a Big Impact

40. Q2: How to deal with drug resistance ? Some CML patients develop resistance or relapse to targeting small molecule (Imatinib). (1) Develop modified drug (2 nd generation kinase inhibitor) (2) Combination therapy

41. 2 nd generation TKI -Imatinib-like compound J. Cortes et al Blood, 2009 IC 50, lower is better.

42. Molecules for targeted therapies (1)Small molecules: target the ATP binding site or other regions in protein kinase domain, e.g. Gleevec (to BCR-Abl) (2) Monoclonal Abs: target receptors, cytokines, other surface proteins, e.g. Herceptin (to Her), Erbitux (to EGFR) (3) Others: Decoy receptors (soluble CTLA4-Ig), Vaccines, RNAi,..etc

43. Targeting drugs in clinical trials

44. Ab-mediated signaling inhibition Adopted from Nature Biotechnology 23, 1147 - 1157 (2005)

45. Approved mAb Cancer therapeutics

46. Summary 1. Signaling transduction is essential for cells to communicate with environmental stimuli. 2. It usually includes three major components: Receptor, Transducer, & Effector. 3. Two key biochemical events during signaling transduction: PTMs & Protein Interactions 4. Dysregulation of signaling molecules perturbs cellular processes then leading to disease develpoment. 5. Targeted therapy are mostly based on targeting two biochemical events.

47. Kinase: www.kinase.com (seq, evolution & kinomes) Protein kinase resource: www.kinasenet.org (kinase structure) Alliance for cell signaling: www.signaling-gateway.org Phosphosite database: www.phosphosite.org (in vivo phosphorylation sites)www.phosphosite.org Websites for signaling transduction

48. Homework Gleevec has also been found to effectively treat other cancer cells such as gastrointestinal stromal tumors (GIST). However, scientists found no mutation of Abl kinase in these tumor cells. Please explain the underlying mechanism of how Gleevec is still working in this kind of cancer cells even without c-Abl mutation.

50. B. Druker, Cancer Cell, 2002 2 nd generation TKI STI 571-like compound

51. Kantarjian et al. Nature Reviews Drug Discovery 5, 717–718 (September 2006) | doi:10.1038/nrd2135