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


Research interests
Research Interests Intramolecular Kinase Activity at the Onset of Mitosis

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.


Zinc in brain
Zinc in Brain Intramolecular Kinase Activity at the Onset of Mitosis

  • 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


Tailoring tripodal ligands for zinc sensing
Tailoring Tripodal Ligands for Zinc Sensing Intramolecular Kinase Activity at the Onset of Mitosis

Zhaohua Dai and James W. Canary,  New J. Chem., 2007, 31, 1708-1718.


Chiral fluorescent probes for zn 2

Chiral Fluorescent Probes for Zn Intramolecular Kinase Activity at the Onset of Mitosis 2+

Higher Zn2+/Cu2+Selectivity

Stereochemical Control

2. Better contrast

Fertile Optical Information: Differential Circularly Polarized Fluorescence Excitation (CPE)


Stereochemical approach to improved zn ii cu ii selectivity
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


Fluorescence detected circular dichroism fdcd
Fluorescence-detected Circular Dichroism (FDCD) Selectivity

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


Differential circularly polarized fluorescence excitation cpe
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.


Cpe reduces background from free ligand

Zn (CPE)2+

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


Cpe selects against protein based background fluorescence
CPE SELECTS AGAINST PROTEIN-BASED BACKGROUND FLUORESCENCE (CPE)

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


Chiral fluorescent sensor for hg 2
Chiral Fluorescent Sensor for Hg (CPE)2+

We intend to use these ligands

to further develop CPE.


Colorimetric mn ii sensor
Colorimetric (CPE)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

Summary for Metal Sensors


Caged sensors for kinase activity
Caged Sensors for Kinase Activity stereochemical approach

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.


Protein kinase c
Protein Kinase C stereochemical approach

  • Cell proliferation, apoptosis, differentiation, migration

  • Cause cancer, etc.

  • Tools are needed for probing, therapeutics

Nakashima, S. J. Biochem.2002, 132, 669-675.


Pkc in early mitosis g 2 m
PKC in Early Mitosis (G stereochemical approach2/M)

Review: Black, J. D. Front. Biosci.2000, 5, 406-423

P. Collas et al J. Cell Sci. 1999, 112, 977-987.


Pkc b ii in g 2 m transition
PKC stereochemical approachbII 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.


Nbd based fluorescent sensor for pkc
NBD-based Fluorescent Sensor for PKC stereochemical approach

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


In vivo studies in hela cells
In vivo Studies in HeLa cells stereochemical approach


Caged pkc sensor
Caged PKC Sensor stereochemical approach

KVIP

Veldhuyzen, W. F. et alJ. Am. Chem. Soc.2003, 125, 13358-13359


Why caged sensors
Why Caged Sensors stereochemical approach

  • 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
Real-time temporal probing of PKC activity stereochemical approach

Veldhuyzen, W. F. et alJ. Am. Chem. Soc.2003, 125, 13358-13359


Studying mitosis
Studying Mitosis stereochemical approach

Microinjection

PtK2 Cells: flat

Kangroo rat didney

epithelial cells

KVIP


Pkc in ptk2
PKC in PtK2 stereochemical approach

S. Kumar


Vip pkc activity
VIP PKC Activity stereochemical approach

q*

Other kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP)

Nek2 (weakly)

S. Kumar


Coinjection of 200 stereochemical approachmM 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


5 min stereochemical approach

7 min

4 min

6 min


Coinjection of 200 stereochemical approachmM KVIP and 5 mM 70K dalton texas red-dextran

0 min injection

2 min uncaging

25 min


Injection with 200 m m kvip before nebd
Injection with 200 stereochemical approachmM KVIP before NEBD

1.PKC activity

accompanies

NEBD.

Which one?

2. PKC activity

levels off after

NEBD:

PKC off?

or

Sensor gone?


0 min injection stereochemical approach

2 min uncaging

11 min

Coinjection of 200 mM

KVIP and 5 mM 70K

dalton texas red-dextran

(uncaging after NEBD )


Injection with 200 m m kvip uncaging after nebd
Injection with 200 stereochemical approachmM KVIP (Uncaging after NEBD)

  • No PKC activity

  • right after NEBD?

  • 2. Both PKC and

  • phosphatase are active?


Incubation with 1 5 m m okadaic acid
Incubation with 1.5 stereochemical approachmM okadaic acid

Phosphatase inhibited

No PKC activity

right after NEBD.


High pkc b inhibitor concentration 12 m m induced or blocked cells at prophase
High PKC stereochemical approachb 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


Coinjection w 2 mm pkc a inhibitor and 200 m m kvip 5 m m 70k texas ted dextran
Coinjection w/ 2 mM PKC stereochemical approacha 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


Coinjection w/ 2 mM PKC stereochemical approacha inhibitor and 200 mM KVIP, 5 mM 70K rhodamine-dextran (No NEBD)

0 min injection

2 min uncaging

30 min


Coinjection of 2 mm pkc a inhibitor and 200 m m kvip
Coinjection of 2 mM PKC stereochemical approacha inhibitor and 200 mM KVIP

When PKCs are

shutdown, NEBD

is blocked w/o FL

enhancement.


Co injection of 1 m m pkc a inhibitor and 200 m m kvip
Co-injection of 1 stereochemical approachmM 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


Co injection of 1 m m pkc a inhibitor and 200 m m kvip1
Co-injection of 1 stereochemical approachmM PKCa inhibitor and 200 mM KVIP

PKC a shutdown

PKC b is responsible

for NEBD and FL

b1 or b2?


Redistribution of pkc b i and pkc b ii in cell cycle
Redistribution of PKC stereochemical approachbI 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


Conclusion for caged pkc sensor
Conclusion for Caged PKC Sensor stereochemical approach

  • 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.


Acknowledgement
Acknowledgement stereochemical approach

  • 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)


Mechanism of uncaging
Mechanism of Uncaging stereochemical approach


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