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Structural Insights into Kinase Inhibition Ramesh Sistla Aurigene ...
Transcript of Structural Insights into Kinase Inhibition Ramesh Sistla Aurigene ...
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
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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
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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 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
FFF
N CH3NCH3
NH
N
N
N
O
NH
CH3
NH
N
N
NN
NCH3
O N
NH
N
CH3
N
NN
OH
SNH
CH3
Cl
CH3OO
NN
NHCH
OOCH3
Imatinib
Dasatinib
Some Advanced Kinase Inhibitors
Kinome
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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 α-helices
• Activation loop spans both N- and C-terminal lobes
C-terminal lobe
N-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.
…GxGxxG…
Helix-C
DFG……APE loopHinge ATP
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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
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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
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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.
Hinge
G-loop
ATPInhibitor ATP
Inhibitor
Ribose pocketPhosphate pocket
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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
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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
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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.
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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.
ATPGleevec
DFG IN
DFG OUT
Helix-C
PDB:1T46
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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
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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
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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.
DFG loop
Helix-C
ATP
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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.
PDB:3F9N
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SBDD at Aurigene
All the structural biology efforts are to aid in more focused medicinal chemistry