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

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

c-Kit : key structural components

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”

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”

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

Active form of c-Kit: blue ; Inactive form : yellowThe molecular brake and the kinase hinge

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

Inactive of c-Kit formThe molecular brake and the kinase hinge

Active form of c-KitThe molecular brake and the kinase hinge

Inactive c-Kit + STI (1T46)

STI induces fit in molecular breakVan der Waals surface detailing the binding pocket for

1T46 + STI ; Red : Ile 653

STI induces fit in molecular break

Solvent accessible surface forInactive c-Kit + STI (1T46)

Solvent accessible surface forModified c-Kit (1T46+ brake on +STI)

Mechanism for release of the molecular brake through movement of Activation loop involves a similar

interaction between Ile653 and Phe 811

fesfer FES

matkcsk

frkfgr

fynyes

srcblklynhck

lcksrms

ptk6btk

bmxitk

tectxk

abl2abl1 epha8

ephb4ephb3ephb2

ephb1epha7epha6epha4epha5

epha3epha2epha1

ephazap-70

sykfak2

fak1tnk1

ack1 jak3jak2tyk2

jak1erbb3

erbb4erbb2

erbb1itk

alkros1

insrrigf1r

insr aatykaatk

ptk7ddr2ddr1ror2ror1

muskntrk3

ntrk2ntrk1

rykron

mettyro3

axlmertk

tektie

retfgfr4fgfr3

fgfr2fgfr1 vegfr3

vegfr2vegfr1pdgfrb

pdgfraflt3csf1r

ckit

CSK

SRC

BRK

TEC

ABL

EPH

SYKFAK

ACK

JAK

EGFR

ALKROSINSR

AATYKPTK7DDRRORMUSKTRKRYKMET

UFO

TIERET

FGFR

VEGFR

PDGFR

**K

NEK

*EK

***

Phylogenetic Tree of Human Tyrosine Kinases

fgfr2 481c-Kit 589abl1 242

fgfr2 541c-Kit 647abl1 293

fgfr2 582c-Kit 707abl1 335

fgfr2 601c-Kit 767abl1 338

fgfr2 661c-Kit 827abl1 397

fgfr2 720c-Kit 887abl1 456

541646292

581706334

600766337

660826396

719886455

770937493

1 2 3 -C helix

4 5 6 -D

-E 7 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

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

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

Inhibition of abl through the binding of ellipticines

Inhibition of c-Kit through the binding of ellipticines : competitive inhibition

HO-ellipticine: Blue

MeO-ellipticene: green

ADP: Cyan

Inhibition of c-Kit through the binding of ellipticines ; OMe-Phe401 hydrophobic

interaction stabilizing backbone h-bond