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
catalyze phosphorylation
•
that
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.
AURIGENE……Acccelerating Discovery
www.cellsignal.com
2
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
Some Advanced Kinase Inhibitors
Druggable Genome
Structure
Identifier Target
F
F
Kinome
N
N
F
O
H
N
AMN-107 BCR/ABL
N
H
N
H 3C
N
N
N
N
H
N
Imatinib
CH 3
N
O
H 3C
CH 3
N
H
STI-571
N
BCR/ABL
N
Dasatinib
CH 3
Cl O
N
H
N
S
N
N
N
H
N
N
O
O
HC
H
N
N
BCR/ABL
+
OSI-774
Erbb
OH
CH 3
O
BMS354825
CH 3
O
CH 3
N
• 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
N-terminal lobe
• Bi-lobial structure
• N-termial lobe
– Mainly made of beta-sheets and
connecting loops
– One functionally important helix
C-terminal lobe
• 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 Nand C-terminal lobes
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Important Structural Elements
…GxGxxG…
Helix-C
Hinge
•
Plays an important role in catalysis
Hinge
–
•
Closes in on the ATP
Helix C
–
•
DFG……APE loop
Glycine rich loop
–
•
ATP
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
Orientation of the DFG motif critical for the phosphorylation
H-bonds
Hinge
Metal
Metal
γ-phosphate coordinates with the metal
S
•Activation loop (DFG……APE) provides docking site for the substrate
•Highly disordered and usually unresolved in the x-ray structures
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T
Substrate
Y
Phosphate
7
Important Residues
Close up of the catalytic machinery
N-terminal lobe
Helix-C
ATP
Lys
Salt bridge
Asp Water
Metal
C-terminal lobe
•
•
Glu
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
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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
ATP
Inhibitor
Ribose pocket
Phosphate pocket
ATP
Inhibitor
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Various Subsites in Kinases
Hinge
Gatekeeper
Deeper cavity
Inhibitor
Solvent
ATP
Schematic of the binding pockets
PDB: 2C6E/1MQ4
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
•
Deeper pocket
Hinge
•
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
Solvent
1nM
10nM
100nM
1μM
10μM
Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008
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DFG-IN vs DFG-OUT
Helix-C
Gly rich loop
DFG-Out
DFG-In
• 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
Helix-C
ATP
Gleevec
DFG OUT
DFG IN
• 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.
PDB:1T46
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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
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R
Some Known DFG OUT Inhibitors
2oo8
Tie – DFG out
J.Med.Chem. 50: 611-626
2p4i
Tie – DFG out
Bioorg.Med.Chem.Lett. 17: 2886-2889
2osc
Tie – DFG out
R
Bioorg.Med.Chem.Lett. 17: 2886-2889
N
H
2og8
Lck – DFG out
O
2ofv
Lck – DFG out
J.Med.Chem. 50: 611-626
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
Helix-C
ATP
DFG loop
• 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.
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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
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