2. An investigation of the importance of the Kinase Hinge region on the structure and function of selected Tyrosine Kinases

3. c-Kit :active in blue, inactive in yellow. Activation involves a 6.7 degree tilt on the N-lobe towards the C-lobe N-lobe C-lobe

4. c-Kit : key structural components

5. Active form of c-Kit : juxtamembrane :green; P loop :magenta; C (control) –helix :cyan ; C (catalytic) loop :gray ; A (activation) loop: red  -C Glu is “in” DFG Phe is “in”

6. Inactive form of c-Kit : juxtamembrane :green; P loop :magenta; C (control) –helix :cyan ; C (catalytic) loop :gray ; A (activation) loop: red  -C Glu is “in” DFG Phe is “out”

7. Inactive form of c-Kit + Gleevec : juxtamembrane :green; P loop :magenta; C (control) –helix :cyan ; C (catalytic) loop :gray ; A (activation) loop: red

8. Active form of c-Kit: blue ; Inactive form : yellow The molecular brake and the kinase hinge

9. Induced fit of STI binding to c-Kit : 1T45 in yellow, 1T46 in gray

10. Inactive of c-Kit form The molecular brake and the kinase hinge

11. Active form of c-Kit The molecular brake and the kinase hinge

12. Inactive c-Kit + STI(1T46)

13. STI induces fit in molecular break Van der Waals surface detailing the binding pocket for 1T46 + STI ; Red : Ile 653

14. STI induces fit in molecular break Solvent accessible surface for Inactive c-Kit + STI (1T46) Solvent accessible surface for Modified c-Kit (1T46+ brake on +STI)

16. Mechanism for release of the molecular brake through movement of Activation loop involves a similar interaction between Ile653 and Phe 811

18. fgfr2 481 c-Kit 589 abl1 242 fgfr2 541 c-Kit 647 abl1 293 fgfr2 582 c-Kit 707 abl1 335 fgfr2 601 c-Kit 767 abl1 338 fgfr2 661 c-Kit 827 abl1 397 fgfr2 720 c-Kit 887 abl1 456 541 646 292 581 706 334 600 766 337 660 826 396 719 886 455 770 937 493 11 22 33  -C helix 44 55 66  -D  -E 77 88  -F  -G  -H  -I  -J  -K  -L The effect of Ligand binding on the “Molecular Brake” of the Kinase Hinge region of Tyrosine Kinases

19. No such interaction is present in abl since no h-bond stabilization is needed; molecular brake is off for hinge region of Inactive abl + STI (1OPJ) Leu 317 in abl compared to Ile 653 in c-Kit Gln 319 in abl compared to Asn 655 in c-Kit

20. Result#4: Inhibition of c-Kit through the binding of ellipticines

21. Inhibition of abl through the binding of ellipticines

22. Inhibition of c-Kit through the binding of ellipticines : competitive inhibition HO-ellipticine: Blue MeO-ellipticene: green ADP: Cyan

23. Inhibition of c-Kit through the binding of ellipticines ; OMe-Phe401 hydrophobic interaction stabilizing backbone h-bond