1. 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

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.
AURIGENE……Acccelerating Discovery

3. AURIGENE……Acccelerating Discovery
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
Cellular pathways explaining the involvement of kinases in disease
Current Medicinal Chemistry, 2008 Vol. 15, No
AURIGENE……Acccelerating Discovery

4. Promise of Kinase Inhibitors
Druggable Genome
Some Advanced Kinase Inhibitors
Imatinib
Dasatinib
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
AURIGENE……Acccelerating Discovery

5. 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

6. Important Structural Elements
…GxGxxG…
Helix-C
DFG……APE loop
Hinge
ATP
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.
Include Lys-Glu interaction
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7. 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
Hinge
Metal
Substrate
Phosphate
H-bonds S T Y
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8. AURIGENE……Acccelerating Discovery
Important Residues
N-terminal lobe
C-terminal lobe
ATP
Lys
Glu
Asp
Water
Metal
Helix-C
Close up of the catalytic machinery
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 γ-phosphate, the divalent metal ion and catalytic water molecule to facilitate catalysis
AURIGENE……Acccelerating Discovery

9. AURIGENE……Acccelerating Discovery
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
ATP
Inhibitor
ATP
Inhibitor
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10. Various Subsites in Kinases
Schematic of the binding pockets
Add schematic pharmacophore
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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11. 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
1nM
10nM
100nM
1μM
10μM
Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008
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12. AURIGENE……Acccelerating Discovery
DFG-IN vs DFG-OUT
Gly rich loop
Helix-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.
AURIGENE……Acccelerating Discovery

13. AURIGENE……Acccelerating Discovery
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.
ATP
Gleevec
DFG IN
DFG OUT
Helix-C
PDB:1T46
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14. 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

15. Some Known DFG OUT Inhibitors
2ofv
Lck – DFG out
2og8
Lck – DFG out
2oo8
Tie – DFG out
Bioorg.Med.Chem.Lett :
J.Med.Chem :
Bioorg.Med.Chem.Lett :
J.Med.Chem :
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

16. 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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17. 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

18. AURIGENE……Acccelerating Discovery
SBDD at Aurigene
All the structural biology efforts are to aid in more focused medicinal chemistry
AURIGENE……Acccelerating Discovery