Download Curcumin Binds to RDT

Complexity of Signaling Networks
Old Protein, New Tricks
Biplab Bose
Diphtheria Toxin
Exotoxin of Corynebacterium diphtheriae.
Diphtheria Toxin in Therapeutics
Diphtheria Toxin in Cancer Therapeutics
HB-EGF: DT Receptor
Heparin-binding epidermal growth factor (EGF)-like growth factor
Soluble & Membrane bound
Normal Function
In Oncogenesis:
• Cell proliferation
• Increases proliferation
• Developmental process
• Induction of migration and invasion
• Wound-healing
• Promotion of angiogenesis
Overexpressed in tumors:
Pancreatic, Liver , Gastric and
Glioma
HB-EGF Signaling
Receptor Binding Domain of DT (RDT)
Diphtheria Toxin
R-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.
SDS-PAGE of purified RDT
Western Blot using anti-His Ab
RDT binds to HB-EGF
Solid Phase ELISA:
• HB-EGF Coated on 96-well ELISA plate.
• Detection: anti-His Ab followed by anti-mouse Ab
RDT Binds to Cell Surface HB-EGF
Immunofluorescence:
• Cell line: U-87 MG.
• Detection: anti-His Ab followed by FITC-Conjugated anti-mouse Ab
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Binding Affinity of RDT
SPR, Biacore X-100
• Single cycle Kinetics.
• CM5 Chip, HB-EGF immobilized
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: a Good Probe
Curcumin binds to some proteins
Protein binding increases fluorescence
of Curcumin
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Docking of Curcumin on RDT
Docking server: SwissDock
Docking Criteria: Blind, no flexibility for RDT.
RDT
Structure of Curcumin (from ZINC)
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Source: R-domain of B-chain of 1FOL
Docking of Curcumin on RDT
A potential binding pose
Surface Diagram
Ribbon Diagram
•
Blue = Hydrophilic
•
Orange red = Hydrophobic.
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
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.
Average Life
time of
Fluorescence
decay (ns)
Curcumin
0.86
CurcuminRDT
1.04
Curcumin-RDT enhances accumulation of curcumin
Cell Line: U-87 MG
Curcumin (2 µM); molar ratio curcumin:protein (10:1)
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20X
Curcumin-RDT increases cellular uptake of curcumin
HPLC:
• 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
**
*
*
24 between these two (p = 0.143); **significantly
* No significant difference
different from others (p < 0.001). One-way ANOVA with pairwise comparison
Curcumin-RDT potentiate curcumin
MTT assay:
• Cell line: U-87 MG
• RDT: 0.1 µM
• Incubation at 37 0C,
72 hr, Serum-Free
Significant difference between Curcumin and Curcumin-RDT: 2-way ANOVA, p<0.001
Effect of Curcumin-RDT is not synergistic
MTT Assay
• Cell line: U-87 MG
•
RDT: 0.1µM; Curcumin:20 µM;
Curcumin-RDT: 20 µM:0.1 µM;
** 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
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.
PI
Annexin V
Using RDT to enhance drug delivery