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

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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
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Kinases - Introduction

• Kinases are enzymes that catalyze phosphorylation
• ATP protein ADP phosphoprotein
• Key signaling enzyme
• Human genome encodes gt 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
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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
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Promise of Kinase Inhibitors
Druggable Genome
Some Advanced Kinase Inhibitors
Kinome

• 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

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

N-terminal lobe
C-terminal lobe
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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.

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Binding of ATP and Catalysis
H-bonds
S
T
Y
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Important Residues
Salt bridge

• 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 ?-phosphate, the divalent metal ion and
  catalytic water molecule to facilitate catalysis

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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
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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)
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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
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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.

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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
PDB1T46
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Example of Type-II Inhbition
Hinge
Phe-out conformation
Schematic of the binding pockets
PDB1KV1
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
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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)
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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
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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.

PDB3F9N
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SBDD at Aurigene
All the structural biology efforts are to aid in
more focused medicinal chemistry