1. Complexity of Signaling Networks Old Protein, New Tricks Biplab Bose

2. Exotoxin of Corynebacterium diphtheriae. Diphtheria Toxin

3. Diphtheria Toxin in Therapeutics

4. Diphtheria Toxin in Cancer Therapeutics

5. 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

6. HB-EGF Signaling

7. Receptor Binding Domain of DT (RDT) Diphtheria Toxin R-domain of Diphtheria Toxin PDB ID: 1F0L

8. Cloning of RDT SOURCE: Full Length DT cloned in pET-22b 8

9. Expression & Purification of RDT E. coli BL21(DE3) Induction at 28 0 C,1 mM IPTG. Purification by His-Trap Column. Western Blot using anti-His Ab SDS-PAGE of purified RDT

10. 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:

11. RDT Binds to Cell Surface HB-EGF Cell line: U-87 MG. Detection: anti-His Ab followed by FITC-Conjugated anti-mouse Ab Immunofluorescence: 11

12. Binding Affinity of RDT Single cycle Kinetics. CM5 Chip, HB-EGF immobilized SPR, Biacore X-100 k on (1/M.1/s) k off (1/s) K D (M) RDT 4.4 x 10 4 3.1 x 10 -3 7.4 x 10 -8 DT 3.9 x 10 3 1.5 x 10 -4 3.9 x 10 -8

13. Can a Drug Bind to RDT ? blue = hydrophilic and orange red = hydrophobic.

14. Curcumin: a Potential Therapeutic Agent

15. Curcumin: a Good Probe Fluorescence of Curcumin depends upon environment  Water quenches curcumin fluorescence.

16. Curcumin binds to some proteins Protein binding increases fluorescence of Curcumin 16 Curcumin: a Good Probe

17. Docking of Curcumin on RDT Docking server: SwissDock Docking Criteria: Blind, no flexibility for RDT. RDT Structure of Curcumin (from ZINC) Source: R-domain of B-chain of 1FOL 17

18. Blue = Hydrophilic Orange red = Hydrophobic. Docking of Curcumin on RDT Ribbon Diagram Surface Diagram A potential binding pose

19. Docking of Curcumin on RDT Important interactions between RDT and curcumin:

20. Curcumin Binds to RDT Fluorescence spectroscopy: Curcumin (10 µM) in PBS, Molar ratio of Curcumin:Protein (10:1) Incubation at 4 0 C, 2 hr Excitation at 430 nm.

21. Curcumin Binds to RDT Fluorescence spectroscopy: Curcumin (10 µM) in PBS, with RDT varied (0 to 2 µM) Incubation at 4 0 C, 2 hr Excitation at 430 nm.

22. Curcumin Binds to RDT Average Life time of Fluorescence decay (ns) Curcumin0.86 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.

23. 20X Curcumin-RDT enhances accumulation of curcumin Cell Line: U-87 MG Curcumin (2 µM); molar ratio curcumin:protein (10:1) 23

24. 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 0 C, 2 hr, Serum. C18 column; methanolic cell extract HPLC : * * **

25. 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 0 C, 72 hr, Serum-Free MTT assay:

26. 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)

27. 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.

28. 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 0 C, 48 hr.

29. Using RDT to enhance drug delivery