University of Maryland Baltimore Experimental Therapeutics Symposium 2009

1. 1 Carey Lab InterestsE.T. 10/02/09 Gregory B. Carey, Ph.D. Asst. Prof. Microbiology and Immunology Center For Vascular And Inflammatory Diseases Rm 313, Biopark1, 800W Baltimore St. gcarey@som.umaryland.edu, x68191

2. Carey Lab interests RedOxBiology: ROS: NO, O2-, H2O2 ROS no longer metabolic waste byproducts Homeostatic Act as 2ndmessengers Switching functions?? Mechanism of Action of Manumycin-A (Man-A) natural tumoricide Antimicrobial agent ca 1960 Ras Inhibitor ca 1990 Natural compounds generally well tolerated Broader spectrum of benefits Use to understand cell death resulting from RedOxchanges

3. Collaborative Interests Tumor biology and Immunobiology: Role PI-3K/Akt pathway in tumor development, survival, progression etc. Dr. Achsah Keegan (UMB) Metabolomics Switching or skewing of pathways in cancer? Unique metabolites, markers etc? IL-4 conditioning causes exclusive dependence on glycolytic respiration Intervene w/ 3-Br Pyruvate and 2-DOG Drs. Keegan UMB) and Wendy Davidson (NIH) Gld mouse work Why does incidence and number of autoreactive immune cells increase w/ aging? (   geriatric lymphomatosis) = deletion failure? Metabolic advantage? Both? What are persistent antigens? RedOx Biology and Proteomics Superoxide lethal for tumors and normal cells (not NO and H2O2) Compartmental ROS overload promotes death in tumors Preceded by biochemical changes on key proteins Are changes cause or effect or unrelated?

4. Lab InterestsMechanism of Action of Manumycin-A Manumycin-A (Man-A) Stimulates death via ROS/PPase/Caspase cascade PP1 activated, CDK2 deactivated Have also seen potent anti-tumor activity in Nigerian cola nut fractions Dr. Chris Okunje (Formely Walter Reed. Presently U. Penn) Manumycin A Potent anti-tumor and pleiotropic effects in animals Reduced trypanosome infection Reduced cholesterolemia and arterial plaque formation in mice Tumor rejection Broad spectrum, plieotropic effects of Natural cmpds. HOW IS IT WORKING? Usable or adaptable for potential use in humans?

5. Loss pm Cyt - C Ox NO *, â Dephos Casp DY integrity H O H O · m , <2N â - O PPase 2 2 2 2 2 Response 0.5h 4h 1h Time Figure1. Manumycin - A - Stimulated Cascade of Events. Research Summary I Akt cleavage CDK2 Activity Loss? H2O2 good? O2- bad? 5

6. H2O2 X Research Summary II PP1a stimulates O2- generating system? PP1a suppresses H2O2 generator? PP1a Man-A ? O2- CDK2 modulates PP1a (Thr320) What is Role of CDK2 in survival pathways? 6

7. Research Summary III ? O2- X ? Man-A CDK2 ? H2O2 CDK2 stimulates H2O2 generator? CDK2 inhibits O2- generator? 7

8. Research Summary IV CDK2 and PP1 antagonism determines cell fate? Cal-A Thr320PP1a O2- X Thr160-CDK2 Roscovitine H2O2 8

9. Research Summary IV SOD regulated by CDK2? X Thr320PP1a Cal-A O2- Rosc. Growth Factors Thr160-CDK2 SOD H2O2 Tonic H2O2 Includes: -Activation of JAK/STATs -Inhibition of caspases -Inhibition of Tyr and Ser/Thr PPase 9

10. P-Akt, P-MEK Working Model Of Man-A Action Man-A Cal-A NAC CDK2 PP1a-POFF PP1a ON ROS Akt, MEK Caspases Akt and MEK cleavage Apoptosis, Quiescence etc. 10

11. Central Hypotheses Controlled RedOx changes dephosphorylation of select, critical targets? RedOx change without dephosphorylation  Uqsticking and proteasomaltargeting? (CDK2 major protagonist) Redox damage + dephosphorylation  caspase targeting? PP1 protagonist? 11

12. Future Directions and Questions R01 CA-138994-A1 Role of ROS in Signal Protein Targeting During Apoptosis How are MEK and Akt targeted and not ERK (and others)? ROS Sources? Probing Proteins for Thioreactive Sites DMPO and IAA labeling Spin trap and thiol label Western blot, IP and Mass Spec. 12

13. Future Directions: Questions Is Man-A a trigger or participant in process? Spectral analyses will identify Man-A metabolites Proteomics to examine Man-A adduction to proteins IF HAPPENS!! Anti-Man-A Antibody? Or, Conjugate Man-A to agarose or sepharose Purify targets then subject to Mass Spec. Analysis Man-A important if CDK2 inhibitor or PPase activator 13

15. Ph.D. candidate, U-MD, Dept. Microbiology and Immunology (’10)

16. Mr. Allan Carey

17. Humanities Scholar UMBC

18. Siddiq al Wahhab

19. MHHS, Ellicott City

20. Ms. Kateila Miles

21. Medical School ‘10

22. Dr. Hanako Daino

23. Postdoctoral Fellow, Carey lab 06-08

24. Dr. Mark Williams

25. Micro. And Immunol

26. Dr. Jaeyul Kwon

27. NIH

28. Mr. Brian Hampton,

29. Protein Core CVID

30. Dr. Austin Yang

31. Proteomic Core, Greenebaum

32. Dr. Len Neckers

33. NIH

34. Dr. Alexandra Newton

35. UCSD

36. Dr. Brian Polster

37. UMBFunding: NCI -CA94027 (’01-’07) -CA12882 (’07-’11) Dean’s Office -Sm. Intramural grant ‘05-’07) 14

38. Mechanism of Damaged Protein Processing? - - - - - - - Tonic ROS? + Kinase - - - - - - - - - - - - - - -Ser473-P -Ser473 - - - - - - - Ppase And Trx? - - - - - - -Cys460 - - - - - - -Cys460-O - - - - ROS - - - - - - - - - - - -Ser473-P - - - Ppases and Caspases - - - - - - -Cys460-O - -O - - - - - - - - - - - - -