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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
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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. AURIGENE……Acccelerating Discovery
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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
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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
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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 N-terminal lobe C-terminal lobe AURIGENE……Acccelerating Discovery
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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 AURIGENE……Acccelerating Discovery
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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 Hinge Metal Substrate Phosphate H-bonds S T Y AURIGENE……Acccelerating Discovery
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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
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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 AURIGENE……Acccelerating Discovery
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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) AURIGENE……Acccelerating Discovery
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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 1nM 10nM 100nM 1μM 10μM Ref: Karaman et. al., NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 1 JANUARY 2008 AURIGENE……Acccelerating Discovery
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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
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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 AURIGENE……Acccelerating Discovery
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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 AURIGENE……Acccelerating Discovery
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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 : 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
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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 AURIGENE……Acccelerating Discovery
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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 AURIGENE……Acccelerating Discovery
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AURIGENE……Acccelerating Discovery
SBDD at Aurigene All the structural biology efforts are to aid in more focused medicinal chemistry AURIGENE……Acccelerating Discovery
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