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

Fluorescent and chiroptical probes for metal ions Research Interests Fluorescent probes for kinase activity in live cells Zn 2+, Mn 2+, Hg 2+ Das, D.; Dai, Z.; Holmes, A. E.; Canary, J. W. Chirality, 2008, 20, Dai, Z.; Canary, J. W. New J. Chem. 2007, 31, Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, Dai, Z.; Xu, X.; Canary, J.W. Chirality 2005, 17, S Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, Dai, Z.; Xu, X.; Canary, J. W. Chemical Communications 2002, Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol. 2007, 14, Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, Tyrosine Kinase, PKC

Zinc in Brain More Zn 2+ in brain than in any other organ Zn 2+ and Cu 2+ are implicated in Alzheimer’s, Parkinson’s, and Amyotrophic Lateral Sclerosis (ALS) Complicated roles Tools needed to image Zn 2+ distribution and kinetics TSQ, Zinquin High sensitivy Poor Zn(II)/Cu(II) selectivity

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

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

Zn Cu * Stereochemical Approach to Improved Zn(II)/Cu(II) Selectivity 15% acetonitrile/aqueous buffer pH 7.19 * Z. Dai, et al. unpublished Zn 2+ /Cu 2+ Selectivity: log 

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

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

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

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

Chiral Fluorescent Sensor for Hg 2+ We intend to use these ligands to further develop CPE.

Colorimetric Mn(II) Sensor 5-Br-PAPS-Zn(II)-EGTA Displacement system

Summary for Metal Sensors 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

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

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

PKC in Early Mitosis (G 2 /M) Review: Black, J. D. Front. Biosci. 2000, 5, P. Collas et al J. Cell Sci. 1999, 112,

PKC  II in G 2 /M Transition A. P. Fields et al. J. Biol. Chem. 1994, 269, A. P. Fields et al. J. Biol. Chem. 1996, 271, Target: lamin B Ser K K m (  M): 4.9 (soluble) and 3.9 (envelope). IC50: 16  M nocodazole Chelerythrine Chelerythrine (PKC  inhibitor ????)

NBD-based Fluorescent Sensor for PKC NBD-peptide Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem. 2002, 277, Assay PKC  PKC  PKC  Radioact.9.0± ± ±1.0 Fluoresc.29 ±327 ±430 ±5 K m (  M) VIP

In vivo Studies in HeLa cells

Caged PKC Sensor Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125, KVIP

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

Real-time temporal probing of PKC activity Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125,

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

PKC in PtK2 S. Kumar

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

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

4 min 5 min 6 min 7 min

0 min injection2 min uncaging25 min Coinjection of 200  M KVIP and 5  M 70K dalton texas red-dextran

Injection with 200  M KVIP before NEBD Total cells NEBDLarge enhancement (>40%) Small enhancement (<40%) No enhancement 18Yes No 33 1.PKC activity accompanies NEBD. Which one? 2. PKC activity levels off after NEBD: PKC off? or Sensor gone?

0 min injection2 min uncaging11 min Coinjection of 200  M KVIP and 5 mM 70K dalton texas red-dextran (uncaging after NEBD )

Injection with 200  M KVIP (Uncaging after NEBD) Total cellsLarge enhancemen t (>40%) Small enhancemen t (<40%) No enhancemen t (within 5%) Very small Enhancement (within 15%) No PKC activity right after NEBD? 2. Both PKC and phosphatase are active?

Incubation with 1.5  M okadaic acid No PKC activity right after NEBD. Total cellsNEBDLarge enhancement (>40%)Small enhancement (<40%) No enhancement Little enhancement (around 15%) 10Yes Phosphatase inhibited

High PKC  inhibitor concentration (12  M) induced or blocked cells at prophase 65% of the cells (20 out of 31) are stuck at prophase  II  Nek2 IC  M 11 nM no obs. inhibition Tanaka, M. et al. Bioorg. Med. Chem. Lett. 2004, 14, S. Kumar PKC ,  might be implicated in NEBD. Which one?

Coinjection w/ 2 mM PKC  inhibitor and 200  M KVIP, 5  M 70K Texas ted-dextran PKC  IC 50 (  M) Ki (  M) PKC  385-fold PKC  580-fold PKC  2730-fol PKC  600-fol PKC  1310-fold PKC  1210-fold PKC  940-fold PKC  640-fold Lee, Nandy, Lawrence. JACS, 2004

0 min injection2 min uncaging 30 min Coinjection w/ 2 mM PKC  inhibitor and 200  M KVIP, 5  M 70K rhodamine-dextran (No NEBD)

Coinjection of 2 mM PKC  inhibitor and 200  M KVIP Total cellsNEBDLarge enhancement (>30%) Small enhancement (<30%) No enhancement 10Yes No When PKCs are shutdown, NEBD is blocked w/o FL enhancement.

Co-injection of 1  M PKC  inhibitor and 200  M KVIP 0 min injection 2 min 3 min 4 min 5 min 6 min 7 min 9 min 13 min 14 min Texas-red fluorescence

Co-injection of 1  M PKC  inhibitor and 200  M KVIP Total cellsNEBDLarge enhancement (>30%) Small enhancement (<30%) No ehancement (within 1%) 15Yes No 3003 PKC  is responsible for NEBD and FL  1 or  2? PKC  shutdown

Redistribution of PKC  I and PKC  II In Cell Cycle N. G. Dulyaninova  1: associated w/ nucleus in interphase and prophase.  2: everywhere in interphase Partial relocation to nuclear boundary in prophase. Significant for NEBD?

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. PKC  is implicated in NEBD of PtK2 cells. It is active just prior to NEBD, not immediately after.

Acknowledgement Mike Isaacman Cho Tan Amanda Mickley Patrick Carney Nikhil Khosla Pace Colleagues Prof JaimeLee I. Rizzo 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 NSF (JWC) NIH (DSL, ARB, JWC) Pace University (Startup Fund, Scholarly Research Fund, Kenan Award)

Mechanism of Uncaging