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Research Interests

Fluorescent Chemosensors for Biology: Visual Snapshots of Intramolecular Kinase Activity at the Onset of Mitosis Zhaohua Dai Department of Chemistry & Physical Sciences, NY. Research Interests. Fluorescent probes for kinase activity in live cells. Fluorescent and chiroptical

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Research Interests

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  1. Fluorescent Chemosensors for Biology: Visual Snapshots of Intramolecular Kinase Activity at the Onset of Mitosis Zhaohua DaiDepartment of Chemistry & Physical Sciences, NY

  2. Research Interests Fluorescent probes for kinase activity in live cells Fluorescent and chiroptical probes for metal ions Zn2+, Mn2+, Hg2+ Tyrosine Kinase, PKC Das, D.; Dai, Z.; Holmes, A. E.; Canary, J. W. Chirality, 2008, 20, 585-591. Dai, Z.; Canary, J. W. New J. Chem.2007, 31, 1708-1718. Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc.2005, 127, 1612-1613. Dai, Z.; Xu, X.; Canary, J.W. Chirality2005, 17, S227-233. Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004,126, 11760 Dai, Z.; Xu, X.; Canary, J. W. Chemical Communications2002, 1414-5. Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol.2007, 14, 1254-1260. Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc.2006, 128, 14016-14017.

  3. Zinc in Brain • More Zn2+ in brain than in any other organ • Zn2+ and Cu2+ are implicated in Alzheimer’s, Parkinson’s, and Amyotrophic Lateral Sclerosis (ALS) • Complicated roles • Tools needed to image Zn2+ distribution and kinetics High sensitivy Poor Zn(II)/Cu(II) selectivity TSQ, Zinquin

  4. Tailoring Tripodal Ligands for Zinc Sensing Zhaohua Dai and James W. Canary,  New J. Chem., 2007, 31, 1708-1718.

  5. Chiral Fluorescent Probes for Zn2+ Higher Zn2+/Cu2+Selectivity Stereochemical Control 2. Better contrast Fertile Optical Information: Differential Circularly Polarized Fluorescence Excitation (CPE)

  6. Stereochemical Approach to Improved Zn(II)/Cu(II) Selectivity Zn2+ 11.0 7.1 8.95 Cu2+ 16.157.17.0 10-5 1 90* logb Zn2+/Cu2+ Selectivity: 15% acetonitrile/aqueous buffer pH 7.19 * Z. Dai, et al. unpublished

  7. Fluorescence-detected Circular Dichroism (FDCD) DF = J-8100 Circular Dichroism System with FDCD Attachment Two channels of data Nehira; Berova; Nakanishi; et al. J. Am. Chem. Soc. 1999, 121, 8681

  8. Differential Circularly Polarized Fluorescence Excitation (CPE) CPE utilized only DF part of FDCD raw data for analysis. q: CD ellipticity; F: Fluorescence quantum yield. Changes in DF will be very large when changes in BOTH fluorescence AND circular dichroism are large.

  9. Zn2+ CPE Reduces Background from Free Ligand Ellipticity  /mdeg Relative Intensity If Zn2+ /nm /nm [Zn(L)]2+ Zn2+ CPE F Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004,126, 11760 Free ligand /nm

  10. CPE SELECTS AGAINST PROTEIN-BASED BACKGROUND FLUORESCENCE Zn2+ Relative Intensity If Ellipticity  /mdeg Zn2+ Lysozyme /nm /nm Lysozyme CPEF Lysozyme + [Zn(L)]2+ Zn2+ Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004,126, 11760 /nm

  11. Chiral Fluorescent Sensor for Hg2+ We intend to use these ligands to further develop CPE.

  12. ColorimetricMn(II)Sensor 5-Br-PAPS-Zn(II)-EGTA Displacement system

  13. Achieved solid Zn(II)/Cu(II) selectivity through a stereochemical approach Developed a new approach for analysis: CPE CPE may be used to improve contrast in detecting metal ions by fluorescent, chiral ligands with low background CPE may be used to diminish interference from fluorescent non-analytes CPE needs further development Summary for Metal Sensors

  14. Caged Sensors for Kinase Activity Snapshots of PKC Activity at the Onset of Mitosis Light-Regulated Sampling of Protein Tyrosine Kinase Activity Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol.2007, 14, 1254-1260. Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc.2006, 128, 14016-14017.

  15. Protein Kinase C • Cell proliferation, apoptosis, differentiation, migration • Cause cancer, etc. • Tools are needed for probing, therapeutics Nakashima, S. J. Biochem.2002, 132, 669-675.

  16. PKC in Early Mitosis (G2/M) Review: Black, J. D. Front. Biosci.2000, 5, 406-423 P. Collas et al J. Cell Sci. 1999, 112, 977-987.

  17. PKC bII in G2/M Transition 85K Target: lamin B Ser405 nocodazole Chelerythrine Km (mM): 4.9 (soluble) and 3.9 (envelope). IC50: 16 mM Chelerythrine (PKC b inhibitor ????) A. P. Fields et al. J. Biol. Chem.1994, 269, 19074-19080. A. P. Fields et al. J. Biol. Chem.1996, 271, 15045-15053.

  18. NBD-based Fluorescent Sensor for PKC VIP NBD-peptide Km(mM) Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem.2002, 277, 11527-11532

  19. In vivo Studies in HeLa cells

  20. Caged PKC Sensor KVIP Veldhuyzen, W. F. et alJ. Am. Chem. Soc.2003, 125, 13358-13359

  21. Why Caged Sensors • In cuvette: investigator controls the start and stop of enzyme catalyzed rxns • In live cell: the cell controls the timing and during • Caged sensors can be delivered in inert forms and activated on demand • Give precise temporal control over sensor activity

  22. Real-time temporal probing of PKC activity Veldhuyzen, W. F. et alJ. Am. Chem. Soc.2003, 125, 13358-13359

  23. Studying Mitosis Microinjection PtK2 Cells: flat Kangroo rat didney epithelial cells KVIP

  24. PKC in PtK2 S. Kumar

  25. VIP PKC Activity q* Other kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP) Nek2 (weakly) S. Kumar

  26. Coinjection of 200 mM KVIP and 5 mM 70K dalton texas red-dextran Green Fl NBD Red Fl 70K dextran- Texas red 2 min uncaging 0 min injection 3 min before

  27. 5 min 7 min 4 min 6 min

  28. Coinjection of 200 mM KVIP and 5 mM 70K dalton texas red-dextran 0 min injection 2 min uncaging 25 min

  29. Injection with 200 mM KVIP before NEBD 1.PKC activity accompanies NEBD. Which one? 2. PKC activity levels off after NEBD: PKC off? or Sensor gone?

  30. 0 min injection 2 min uncaging 11 min Coinjection of 200 mM KVIP and 5 mM 70K dalton texas red-dextran (uncaging after NEBD )

  31. Injection with 200 mM KVIP (Uncaging after NEBD) • No PKC activity • right after NEBD? • 2. Both PKC and • phosphatase are active?

  32. Incubation with 1.5 mM okadaic acid Phosphatase inhibited No PKC activity right after NEBD.

  33. High PKCb inhibitor concentration (12 mM) induced or blocked cells at prophase a bII i m q z Nek2 1.3 mM 11 nM no obs. inhibition IC50 S. Kumar PKC a, b might be implicated in NEBD. Which one? 65% of the cells (20 out of 31) are stuck at prophase Tanaka, M. et al. Bioorg. Med. Chem. Lett. 2004, 14, 5171-5174

  34. Coinjection w/ 2 mM PKCa inhibitor and 200 mM KVIP, 5 mM 70K Texas ted-dextran PKCa IC50 (mM) Ki (mM) PKCb385-fold PKCg580-fold PKCd 2730-fol PKCe 600-fol PKCh 1310-fold PKCq 1210-fold PKCi 940-fold PKCz 640-fold 0.0019 0.00080 Lee, Nandy, Lawrence. JACS, 2004

  35. Coinjection w/ 2 mM PKCa inhibitor and 200 mM KVIP, 5 mM 70K rhodamine-dextran (No NEBD) 0 min injection 2 min uncaging 30 min

  36. Coinjection of 2 mM PKCa inhibitor and 200 mM KVIP When PKCs are shutdown, NEBD is blocked w/o FL enhancement.

  37. Co-injection of 1 mM PKCa inhibitor and 200 mM KVIP 2 min 5 min 9 min 3 min 6 min 13 min 0 min injection 4 min 7 min 14 min Texas-red fluorescence

  38. Co-injection of 1 mM PKCa inhibitor and 200 mM KVIP PKC a shutdown PKC b is responsible for NEBD and FL b1 or b2?

  39. Redistribution of PKCbI and PKCbIIIn Cell Cycle b1: associated w/ nucleus in interphase and prophase. b2: everywhere in interphase Partial relocation to nuclear boundary in prophase. Significant for NEBD? N. G. Dulyaninova

  40. Conclusion for Caged PKC Sensor • Caged sensors can be used to probe PKC activity at G2/M in live cells with temporal precision, providing a way to interrogate enzymatic activity at any point during the cell-division cycle. • PKCb is implicated in NEBD of PtK2 cells. It is active just prior to NEBD, not immediately after.

  41. Acknowledgement • Prof. James W. Canary (NYU) • Prof. David S. Lawrence (Einstein, UNC) • Dr. Williem Veldhuyzen, Dr. Sandip Nandy • Prof. Sanjai Kumar • Prof. Anne R. Bresnick (Einstein) • Dr. Natalya G. Dulyaninova • Dr. Zhonghua (Alice) Li • Mike Isaacman • Cho Tan • Amanda Mickley • Patrick Carney • Nikhil Khosla • Pace Colleagues • Prof JaimeLee I. Rizzo NSF (JWC) NIH (DSL, ARB, JWC) Pace University (Startup Fund, Scholarly Research Fund, Kenan Award)

  42. Mechanism of Uncaging

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