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Structural Insights into Kinase Inhibition Ramesh Sistla and Subramanya H.S. Aurigene Discovery Technologies Ltd. #39-40, KIADB Industrial Area, Electronic City Phase II Bangalore 560 100. Kinases - Introduction. Kinases are enzymes that catalyze phosphorylation

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Structural Insights into Kinase InhibitionRamesh Sistla and Subramanya H.S.Aurigene Discovery Technologies Ltd.#39-40, KIADB Industrial Area, Electronic City Phase IIBangalore 560 100


Kinases introduction l.jpg

Kinases - Introduction

  • Kinases are enzymes that catalyze phosphorylation

  • ATP + protein = ADP + phosphoprotein

  • Key signaling enzyme

  • Human genome encodes > 500 kinases - Kinome

  • They have been implicated in different diseases including cancer, metabolic disorders and central nervous system indications.

  • Depending on the amino acid a kinase phosphorylates, they are known as Serine/Threonine or Tyorsine kinases.

www.cellsignal.com

AURIGENE……Acccelerating Discovery


Signaling cascades l.jpg

Signaling Cascades

  • The figure shows the involvement of kinases in cell proliferation and survival.

  • In this cascade the phosphorylation of each kinase by its upstream kinase serves as a signal for downstream activity.

  • Inhibiting the pathway through inhibition of kinase involved in the pathway is an attractive proposition

Current Medicinal Chemistry, 2008 Vol. 15, No. 29 3037

AURIGENE……Acccelerating Discovery


Promise of kinase inhibitors l.jpg

Imatinib

Dasatinib

Promise of Kinase Inhibitors

Druggable Genome

Some Advanced Kinase Inhibitors

  • Kinases are an attractive target class

    • Druggability

    • Early successes (FDA approval of some of the kinase inhibitors)

  • Possibility of structure guided design

    • Large number of crystal structures in complex with inhibitors are available

Kinome

AURIGENE……Acccelerating Discovery


General structure of kinases l.jpg

General Structure of Kinases

  • Bi-lobial structure

  • N-termial lobe

    • Mainly made of beta-sheets and connecting loops

    • One functionally important helix

  • Both lobes joined by a loop called hinge.

  • ATP binding pocket is in the interface between the lobes

  • C-terminal lobe

    • Mainly made of α-helices

  • Activation loop spans both N- and C-terminal lobes

N-terminal lobe

C-terminal lobe

AURIGENE……Acccelerating Discovery


Important structural elements l.jpg

…GxGxxG…

Helix-C

Hinge

ATP

DFG……APE loop

Important Structural Elements

  • Glycine rich loop

    • Closes in on the ATP

  • Helix C

    • Plays an important role in catalysis

  • Hinge

    • Adenosine moiety of the ATP makes bidentate H-bond with this region

  • Activation loop

    • Starts with conserved sequence DFG and ends with APE.

AURIGENE……Acccelerating Discovery


Binding of atp and catalysis l.jpg

Orientation of the DFG motif critical for the phosphorylation

Hinge

Metal

Metal

γ-phosphate coordinates with the metal

Substrate

  • Activation loop (DFG……APE) provides docking site for the substrate

  • Highly disordered and usually unresolved in the x-ray structures

Phosphate

Binding of ATP and Catalysis

H-bonds

S

T

Y

AURIGENE……Acccelerating Discovery


Important residues l.jpg

Close up of the catalytic machinery

N-terminal lobe

Lys

Helix-C

ATP

Glu

Water

Asp

Metal

C-terminal lobe

Important Residues

  • In the active conformation of the kinases, a conserved Lys residue makes a salt bridge with a conserved Glu residue in the middle of the helix-C.

  • This interaction ensures the positioning of the amino acid Asp (of the DFG motif) to coordinate with the γ-phosphate, the divalent metal ion and catalytic water molecule to facilitate catalysis

Salt bridge

AURIGENE……Acccelerating Discovery


Kinase inhibitors l.jpg

ATP

ATP

Inhibitor

Inhibitor

Kinase Inhibitors

  • In most cases, inhibitors compete with ATP in order to inhibit the kinase

    • Such inhibitors are ATP mimetics in the sense that they make interactions similar to what ATP makes.

G-loop

Hinge

Phosphate pocket

Ribose pocket

AURIGENE……Acccelerating Discovery


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Various Subsites in Kinases

Schematic of the binding pockets

An example of a kinase inhibitor bound in the ATP pocket is shown.

Apart from hinge region interaction and solvent interaction, the inhibitor occupies a deeper hydrophobic cavity, also known as selectivity pocket

Size of an amino acid preceding the hinge region controls the accessibility to the deeper pocket – Gatekeeper, (Typically Met/Leu/Thr/Ile/Tyr)

AURIGENE……Acccelerating Discovery


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1nM

10nM

100nM

1μM

10μM

Type I Inhibitor- Dasatinib

  • Dasatinib was developed as a c-Src/BCR-Abl inhibitor but was found to hit many other kinases.

  • Cross reactivity mainly within the TK family; Approved by FDA

Deeper pocket

Hinge

Solvent

Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008

AURIGENE……Acccelerating Discovery


Dfg in vs dfg out l.jpg

Helix-C

Gly rich loop

DFG-Out

DFG-In

DFG-IN vs DFG-OUT

  • The activation loop (DFG….APE) has to be IN when the kinase is active – DFG “in” conformation

  • The DFG loop has been shown to be in an “out” position when kinases are inactive.

  • This can be exploited in the design of inhibitors.

AURIGENE……Acccelerating Discovery


Dfg in vs dfg out13 l.jpg

ATP

Gleevec

DFG OUT

DFG IN

DFG-IN vs DFG-OUT

  • Differences between DFG IN and DFG OUT structures are exemplified.

  • DFG loop in OUT position will clash with phosphate of ATP

  • When DFG moves to OUT helix-C also moves away creating the pocket shown by bold red arrow.

  • Gleevec binds to the DFG-OUT conformation of the C-Abl kinase.

Helix-C

PDB:1T46

AURIGENE……Acccelerating Discovery


Example of type ii inhbition l.jpg

Example of Type-II Inhbition

Hinge

Phe-out conformation

Schematic of the binding pockets

PDB:1KV1

BIRB-796 binds to p-38 in the Phe-out conformation

  • The doublet of H-bonds with E-111 (helix-C) and D-207 (DFG loop) backbone is very important

  • Hence a urea or amide is the common feature in these inhibitors

Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008

AURIGENE……Acccelerating Discovery


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Some Known DFG OUT Inhibitors

2ofv

Lck – DFG out

2og8

Lck – DFG out

2oo8

Tie – DFG out

Bioorg.Med.Chem.Lett. 17: 2886-2889

J.Med.Chem. 50: 611-626

Bioorg.Med.Chem.Lett. 17: 2886-2889

J.Med.Chem. 50: 611-626

2p4i

Tie – DFG out

2osc

Tie – DFG out

2p2i

KDR – DFG out

Apart from a hinge binding group, the common feature in these molecules is existence of the bi-aryl amide/urea group which makes interaction with Glu (helix-C) and Asp (DFG loop)

AURIGENE……Acccelerating Discovery


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Allosteric Kinase Inhibition – Type III

  • Certain kinases have an allosteric pocket in which an inhibitor can co-bind with ATP

  • The phosphorylation of the substrate is prevented by unavailability of the catalytic Asp

  • There are no hinge region interactions in these inhibitors.

Helix-C

ATP

DFG loop

AURIGENE……Acccelerating Discovery


A still different type of inhibitor l.jpg

A Still Different Type of Inhibitor?

  • Recently Merck published the co-crystal structure of CHK1 kinase with an inhibitor that is bounds far away from the active site.

  • DFG loop is has IN conformation, but the inhibitor probably occupies substrate binding site.

  • Such inhibitors are not being designed yet. They could be results of HTS campaigns.

PDB:3F9N

AURIGENE……Acccelerating Discovery


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SBDD at Aurigene

All the structural biology efforts are to aid in more focused medicinal chemistry

AURIGENE……Acccelerating Discovery


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