Complexity of Signaling Networks

Old Protein, New Tricks

Biplab Bose

Exotoxin of Corynebacterium diphtheriae.

Diphtheria Toxin

Diphtheria Toxin in Therapeutics

Diphtheria Toxin in Cancer Therapeutics

HB-EGF: DT Receptor

Normal Function

• Cell proliferation

• Developmental process

• Wound-healing

In Oncogenesis:

• Increases proliferation

• Induction of migration and invasion

• Promotion of angiogenesis

Overexpressed in tumors: Pancreatic, Liver , Gastric and

Glioma

Heparin-binding epidermal growth factor (EGF)-like growth factor

Soluble & Membrane bound

HB-EGF Signaling

Receptor Binding Domain of DT (RDT)

Diphtheria ToxinR-domain of

Diphtheria Toxin

PDB ID: 1F0L

Cloning of RDT

SOURCE: Full Length DT cloned in pET-22b

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Expression & Purification of RDT

• E. coli BL21(DE3)

• Induction at 28 0C ,1 mM IPTG.

• Purification by His-Trap Column.

Western Blot using anti-His AbSDS-PAGE of purified RDT

RDT binds to HB-EGF

• HB-EGF Coated on 96-well ELISA plate.• Detection: anti-His Ab followed by anti-mouse Ab

Solid Phase ELISA:

RDT Binds to Cell Surface HB-EGF

• Cell line: U-87 MG.• Detection: anti-His Ab followed by FITC-Conjugated anti-mouse Ab

Immunofluorescence:

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Binding Affinity of RDT

• Single cycle Kinetics.

• CM5 Chip, HB-EGF immobilized

SPR, Biacore X-100

 

kon

(1/M.1/s) koff (1/s)

KD

(M)

 RDT 4.4 x 104 3.1 x 10-3 7.4 x 10-8

 DT 3.9 x 103 1.5 x 10-4 3.9 x 10-8

Can a Drug Bind to RDT ?

• blue = hydrophilic and • orange red = hydrophobic.

Curcumin: a Potential Therapeutic Agent

Curcumin: a Good Probe

Fluorescence of Curcumin depends upon environment

Water quenches curcumin fluorescence.

Curcumin binds to some proteins

Protein binding increases fluorescence of Curcumin

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Curcumin: a Good Probe

Docking of Curcumin on RDT

Docking server: SwissDockDocking Criteria: Blind, no flexibility for RDT.

RDT Structure of Curcumin (from ZINC)

Source: R-domain of B-chain of 1FOL17

• Blue = Hydrophilic

• Orange red = Hydrophobic.

Docking of Curcumin on RDT

Ribbon Diagram

Surface Diagram

A potential binding pose

Docking of Curcumin on RDT

Important interactions between RDT and curcumin:

Curcumin Binds to RDT

Fluorescence spectroscopy:• Curcumin (10 µM) in PBS, Molar ratio of

Curcumin:Protein (10:1)• Incubation at 4 0C, 2 hr• Excitation at 430 nm.

Curcumin Binds to RDT

Fluorescence spectroscopy:

• Curcumin (10 µM) in PBS, with RDT varied (0 to 2 µM)• Incubation at 4 0C, 2 hr• Excitation at 430 nm.

Curcumin Binds to RDT

Average Life time of

Fluorescence decay (ns)

Curcumin 0.86

Curcumin-RDT

1.04

Time-resolved fluorescence spectroscopy:

• Excitation at 405 nm•Curcumin (10 µM) in PBS, Molar

ratio of Curcumin:Protein(10:1)• Decay measured in ns/channel.• Exponential component analysis.

20X

Curcumin-RDT enhances accumulation of curcumin

Cell Line: U-87 MGCurcumin (2 µM); molar ratio curcumin:protein (10:1)

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24* No significant difference between these two (p = 0.143); **significantly different from others (p < 0.001). One-way ANOVA with pairwise comparison

Curcumin-RDT increases cellular uptake of curcumin

• Cell Line : U-87 MG• Curcumin:1 µM; • Curcumin-protein (molar ratio:10:1)• Incubation at 37 0C, 2 hr, Serum.• C18 column; • methanolic cell extract

HPLC:

*

*

**

Curcumin-RDT potentiate curcumin

Significant difference between Curcumin and Curcumin-RDT: 2-way ANOVA, p<0.001

• Cell line: U-87 MG• RDT: 0.1 µM• Incubation at 37 0C, 72 hr, Serum-Free

MTT assay:

Effect of Curcumin-RDT is not synergistic

• Cell line: U-87 MG

• RDT: 0.1µM; Curcumin:20 µM;

Curcumin-RDT: 20 µM:0.1 µM;

MTT Assay

** Significantly different from other treatment groups (p < 0.001)

Effect of Curcumin-RDT on cell cycle

Flowcytometry

• Cell line: U-87 MG

• RDT: 0.1 µM; Curcumin:20 µM;

Curcumin-RDT: 20 µM:0.1 µM;

• 48 hr.

Curcumin-RDT enhances apoptosis

PI

Annexin V

Flowcytometry

• Cell line: U-87 MG

• RDT: 0.1 µM; Curcumin:20 µM;

Curcumin-RDT: 20 µM:0.1 µM;

• Incubation at 37 0C, 48 hr.

Using RDT to enhance drug delivery