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