Structural Insights into Kinase Inhibition Ramesh Sistla and Subramanya H.S. Aurigene Discovery...

Structural Insights into Kinase InhibitionRamesh Sistla and Subramanya H.S.

Aurigene Discovery Technologies Ltd.#39-40, KIADB Industrial Area, Electronic City Phase II

Bangalore 560 100

AURIGENE……Acccelerating Discovery 2

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


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


Promise of Kinase InhibitorsDruggable Genome

• 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

Structure Identifier Target

AMN-107 BCR/ABL

STI-571 BCR/ABL

BMS-354825

BCR/ABL+

OSI-774 Erbb

O

NH

F

FF

N CH3NCH3

NH

N

N

N

O

NH

CH3

NH

N

N

N

N

NCH3

O N

NH

N

CH3

N

N

NOH

SNH

CH3

Cl

CH3OO

NN

NHCH

OOCH3

Imatinib

Dasatinib

Some Advanced Kinase Inhibitors

Kinome


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

C-terminal lobe

N-terminal lobe


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.

…GxGxxG…

Helix-C

DFG……APE loopHinge ATP


Binding of ATP and Catalysis

γ-phosphate coordinates with the metal

•Activation loop (DFG……APE) provides docking site for the substrate•Highly disordered and usually unresolved in the x-ray structures

Orientation of the DFG motif critical for the phosphorylation

HingeMetal

Metal

SubstratePhosphate

H-bonds

S T

Y


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

N-terminal lobe

C-terminal lobe

ATP

Lys

GluAsp Water

Metal

Helix-C

Close up of the catalytic machinery

Salt bridge


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.

Hinge

G-loop

ATPInhibitor ATP

Inhibitor

Ribose pocketPhosphate pocket


Various Subsites in Kinases

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)

Hinge

ATP

Inhibitor

Deeper cavity

Solvent

Gatekeeper

PDB: 2C6E/1MQ4

Hinge

ATP

Inhibitor

Deeper cavity

Solvent

Gatekeeper

PDB: 2C6E/1MQ4

Schematic of the binding pockets


Type I Inhibitor- Dasatinib

1nM10nM100nM1μM10μM

• 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

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

Hinge

Solvent

Deeper pocket


DFG-IN vs DFG-OUT

Gly rich loopHelix-C

DFG-In

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.


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.

ATPGleevec

DFG IN

DFG OUT

Helix-C

PDB:1T46


Example of Type-II Inhbition

Phe-out conformation

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

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

• 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

PDB:1KV1

Hinge

Schematic of the binding pockets


Some Known DFG OUT Inhibitors

2ofvLck – DFG out

2og8Lck – DFG out

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

2oo8Tie – DFG out

2p4iTie – DFG out

J.Med.Chem. 50: 611-626

2oscTie – DFG out

Bioorg.Med.Chem.Lett. 17: 2886-2889 J.Med.Chem. 50: 611-626

2p2iKDR – 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)

N H

O

R

R


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.

DFG loop

Helix-C

ATP


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


SBDD at Aurigene

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

Structural Insights into Kinase Inhibition Ramesh Sistla and Subramanya H.S. Aurigene Discovery...

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