Inhibitor SU-4984 Bound to the Tyrosine Kinase Domain of FGFR 1

by

Larry P. Taylor, Ph. D.

 

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Email: lpt

Molecular & Behavioral Neuroscience Institute

The University of Michigan

Ann Arbor, MI

 

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Inhibitor SU 4984 (3-[4-(1-formylpiperazin-4-yl)-benzylidenyl]-2-indolinone) Bound to the Tyrosine Kinase Domain of FGFR 1

 

Tyrosine Kinase activity has been observed in many different malignancies, so there is a therapeutic need for specific inhibitors of tyrosine kinase activity.

Most FGF receptors contain a tyrosine kinase motif  (Kinemage 5 ) downstream from the ligand binding domain and membrane spanning region. This crystal structure of the dimer unit cell  (Kinemage 1)  is from an engineered protein that represents residues 464-762 (sequence below) of the human FGF receptor 1 (FGFR 1).  Kinemage 2 adds ribbon rendering to a single tyrosine kinase domain. The inhibitor, SU-4984, resides in the ATP binding cleft which occurs between two distinctly different structural clusters. The characteristics of the unit cell for this structure are summarized at pdbsum.

 

The SYBYL rendered inhibitor (left) and the inhibitor docked into the ATP binding cleft of the Tyrosine Kinase Domain (right) are shown below:

 

 Stick View of SU-4984

 Bound Inhibitor Surface 

Atom

Color

Structure

Color

Carbon

white

alpha

orange

Hydrogen

cyan

beta

green

Oxygen

red

coil

white

Nitrogen

blue

Inhibitor Surface

magenta


The tyrosine kinase domain is composed of two distinct clusters or lobes separated by a "hinge region" (Tyr-563, Ala-564, Ser-565, Lys-566, and Gln-567). The N-terminal region has five anti-parallel beta sheets and one alpha helix. The C-terminal region has two beta strands and seven helices. The two-lobed cluster is a characteristic of tyrosine kinase activity. (This motif is easiest to visualize in Kinemage 3.) The "hinge region" accounts for a variety of conformational modes seen between the two distinct lobes of tyrosine kinases.

Some kinase enzymes contain a WE motif prior to the nucleotide binding loop. The WE motif in FGFR 1 is found at Trp-471 and Glu-472. The Glu-472 carboxyl oxygen hydrogen bonds to Thr-552. Kinases containing the WE motif  typically hydrogen bond to either a Thr or Ser residue (position 552  in FGFR 1) to stabilize the N-terminal lobe. The WE motif can be considered the boundary between the transmembrane spanning region and the start of the tyrosine kinase domain.

Unfortunately, in both the unbound and SU-4984 bound crystal structures, the endogenous nucleotide binding loop (Glu-486. Gly-587, Ala-488, Phe-489, and Gly-490) is not resolved.

The catalytic region contains the HRDLAARN sequence commonly found in tyrosine kinases (residues 621-628).

The tyrosines involved in the phosphate group transference are Tyr-653 and Tyr-654. It is believed that near-by residues Arg-661 and especially Pro-663 provide a barrier to peptide binding to the active kinase area. This barrier, while excluding peptides, allows the smaller nucleotides to fit into the cleft.

The ligand binding domain features a hydrophobic cleft (Val-492, Ala-512, Ile-545, Val-461, Ala-564, and Leu-630; Kinemage 4) that forms a slot for the incoming oxyindole ring portion of the inhibitor. Residues Leu-484 and Tyr-563 add additional hydrophobic contacts with the inhibitor phenyl ring proximal to the oxyindole. In addition, the inhibitor phenyl ring participates in an oxygen carbonyl to aromatic ring interaction with the carbonyl oxygen of Ala-564. The piperazine ring (most distal from the oxyindole ring) makes a Van der Waal's contact with the highly conserved Gly-567 of the receptor. 

The oxyindole portion of the inhibitor makes two hydrogen bonds to the peptide backbone "hinge" region (residues 563-568 between the two distinct lobes of the tyrosine kinase domain of the receptor). These involve the N1 of the ligand oxyindole interacting with the carbonyl oxygen of Glu-562 and the O2 of the oxyindole hydrogen bonding to the amide hydrogen of Ala-564. These same residues are involved in hydrogen bonding to the adenine portion of the endogenous ligand ATP.

The terminal formyl group of the receptor is poorly resolved which implies conformational mobility significant enough to prevent localization by the diffraction experiment. An identical compound lacking the formyl group was equally potent as an inhibitor of the kinase activity of this receptor.

The Kinemages:

The real-time visualization using KiNG of the structures on this site requires a java-enabled (JRE from Java) browser. 

 

Possible Icons to the left of molecular model image on the download page

 

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or message:

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KiNG Inactive KiNG Inactive KiNG Full Functional

 

A single click on the KiNG logo will launch the appropriate kinemage. 

Kinemage 1: The Unit Cell Dimer: FGFR 1 bound to the Inhibitor SU-4984

 



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47 K

Click on KiNG to see SU-4984 Bound To Tyrosine Kinase Domain of FGFR 1


Kinemage 2:  Inhibitor SU-4984 bound to the monomer unit (Chain A) of FGFR 1

 



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456 K

Click on KiNG to see   SU-4984 Bound to FGFR 1


  Kinemage 3:  Secondary structure ribbon  rendering of the monomer unit of FGFR 1 bound to SU-4984

View 1  the cartoon rendering

View 2  shows an arbitrary "top" view of the ligand binding site

View 3  shows an arbitrary "side" view of the ligand binding site.

 



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305 K

Click on KiNG to see    Cartoon of SU-4984 Bound to FGFR 1

 

 Kinemage 4:Residues of FGFR 1 that interact with SU-4984

The ligand binding domain features a hydrophobic cleft (Val-492, Ala-512, Ile-545, Val-461, Ala-564, and Leu-630). Residues Leu-484 and Tyr-563 add additional hydrophobic contacts with the inhibitor phenyl ring proximal to the oxyindole. In addition, the inhibitor phenyl ring participates in an oxygen carbonyl to aromatic ring interaction with the carbonyl oxygen of Ala-564. The piperazine ring (most distal from the oxyindole ring) makes a Van der Waal's contact with the highly conserved Gly-567 of the receptor. 

View 1  the complex

View 2: closer view of the hydrophobic cleft

View 3  another angle looking at the hydrophobic cleft.



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392 K

Click on KiNG to see    Residues of FGFR 1 Interacting with SU-4983

 

 Kinemage 5 : The Unbound Tyrosine Kinase Domain of FGFR 1

View 1  the unbound tyrosine kinase domain of FGFR 1
View 2  shows the "top" of the ATP binding site.
View 3  shows a "side" view of the ATP binding region
View 4  shows the WE motif
View 5  displays the "catalytic loop:" His-621, Arg-622, Asp-623, Leu-624, Ala-625, Ala-626, Arg-627 and Asn-628.
View 6  highlights the tyrosine residues that participate in the phosphate transfer reaction: Tyr-653 and Tyr-654.



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308 K

Click on KiNG to see    Tyrosine Kinase Domain of Human FGFR 1

 

Sequence: (X-ray resolved residues spanning  464-762 are from the human FGFR 1)

Unresolved N-Terminal: MVAGVSEY

 

X-Ray Resolved: ELPEDPRWELPRDRLVLGKPLG

 

X-Ray Unresolved: EGAFG

X-Ray Resolved: QVVLAEAIGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKMIGKHKNIINLLGAC

TQDGPLYVIVEYASKGNLREYLQARRPPGLEYSYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLA

ARNVLVTEDNVMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGS

PYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRIVALTS

Unresolved C-Terminal: NQE

Source:

Human sequence expressed in spodoptera frugiperda insect cell line: sf9; Structural Coordinates from the Brookhaven Database file1AGW.

The engineered sequence contained three residues that were different from human:
Cys-488 and 584 were changed to Ser to prevent disulfide oligiomerization.
Leu-457 was changed to Val to create a Ncol cloning site.

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Copyright 2005-2020 by Larry P. Taylor
Molecular & Behavioral Neuroscience Institute
University of Michigan

All Rights Reserved

Supported by the Pritzker Neuropsychiatric Disorders Research Consortium, and by NIH Grant 5 P01 MH42251, Conte Center Grant #L99MH60398, RO1 DA13386 and the Office of Naval Research (ONR) N00014-02-1-0879 to Huda Akil & Stanley J. Watson. at the Molecular & Behavioral Neuroscience Institute.