Kinase Inhibitor Binding Sites

Kinase Inhibitor Binding Sites

Protein kinases are defined by their ability to catalyse the transfer of the terminal phosphate of ATP to substrates that usually contain a serine, threonine or tyrosine residue. They typically share a conserved arrangement of secondary structure elements that are arranged into 12 subdomains that fold into a bi-lobed catalytic core structure with ATP binding in a deep cleft located between the lobes.[30-32] ATP binds in the cleft with the adenine ring forming hydrogen bonds with the kinase 'hinge' — the segment that connects the amino- and carboxy-terminal kinase domains. The ribose and triphosphate groups of ATP bind in a hydrophilic channel extending to the substrate binding site that features conserved residues that are essential to catalysis. All kinases have a conserved activation loop, which is important in regulating kinase activity and is marked by conserved DFG and APE motifs (which refer to one-letter amino acid abbreviations) at the start and end of the loop, respectively. The activation loop can assume a large number of conformations with the extremes being a conformer that is catalytically competent and usually phosphorylated, and an 'inactive' conformer in which the activation loop blocks the substrate binding site. Most kinase inhibitors discovered to date are ATP competitive and present one to three hydrogen bonds to the amino acids located in the hinge region of the target kinase, thereby mimicking the hydrogen bonds that are normally formed by the adenine ring of ATP[32,33] (FIG. 1). The majority of kinase inhibitors do not exploit the ribose binding site (an exception being AZD0530, a novel Src and Abl dual family kinase inhibitor[34]) or the triphosphate binding site of ATP.

(Enlarge Image)

Figure 1.

Kinase inhibitor binding modes. Kinase inhibitor-protein interactions are depicted by ribbon structures (left) and chemical structures (right). The chemical structures depict hydrophobic regions I and II of ABL1 (shaded beige and yellow respectively) and hydrogen bonds between the kinase inhibitor (inhibitor atoms engaged in hydrogen bonds to hinge are highlighted in green or to allosteric site in red) and ABL1 are indicated by dashed lines. The DFG motif (pink), hinge and the activation loop of ABL1 are indicated in the ribbon representations. The kinase inhibitors are shown in light blue. a | ABL1 in complex with the type 1 ATP-competitive inhibitor PD166326 (Protein Data Bank (PDB) ID 1OPK).[104] Shown here is the DFG-in conformation of the activation loop (dark blue). b | The DFG-out conformation of the activation loop of ABL1 (dark blue) with the type 2 inhibitor imatinib (PDB ID 1IEP).[105] The allosteric pocket exposed in the DFG-out conformation is indicated by the blue shaded area (right).

[ CLOSE WINDOW ]

Figure 1.

Kinase inhibitor binding modes. Kinase inhibitor-protein interactions are depicted by ribbon structures (left) and chemical structures (right). The chemical structures depict hydrophobic regions I and II of ABL1 (shaded beige and yellow respectively) and hydrogen bonds between the kinase inhibitor (inhibitor atoms engaged in hydrogen bonds to hinge are highlighted in green or to allosteric site in red) and ABL1 are indicated by dashed lines. The DFG motif (pink), hinge and the activation loop of ABL1 are indicated in the ribbon representations. The kinase inhibitors are shown in light blue. a | ABL1 in complex with the type 1 ATP-competitive inhibitor PD166326 (Protein Data Bank (PDB) ID 1OPK).[104] Shown here is the DFG-in conformation of the activation loop (dark blue). b | The DFG-out conformation of the activation loop of ABL1 (dark blue) with the type 2 inhibitor imatinib (PDB ID 1IEP).[105] The allosteric pocket exposed in the DFG-out conformation is indicated by the blue shaded area (right).

Type 1 Inhibitors

This type of inhibitor constitutes the majority of ATP-competitive inhibitors and recognizes the so-called active conformation of the kinase (FIG. 1a), a conformation otherwise conducive to phosphotransfer.[33] The preponderance of type 1 inhibitors may be a consequence of many compounds having been discovered using enzymatic assays in which kinases were introduced in their active conformation and because many kinase inhibitors have been synthesized to mimic ATP (and each other). Type 1 inhibitors typically consist of a heterocyclic ring system that occupies the purine binding site, where it serves as a scaffold for side chains that occupy the adjacent hydrophobic regions I and II (FIG. 2).

(Enlarge Image)

Figure 2.

Diverse kinase inhibitors. The ATP binding site of AKT1 complexed with ATP (Protein Data Bank (PDB) ID 1O6L) is depicted with key regions indicated and hydrogen bonds indicated by red dotted lines. The middle ring shows commonly used heterocyclic core scaffolds (X = C, N). The outer ring shows examples of structurally diverse type 1 inhibitors and their reported kinase targets. Hydrogen bonds are indicated by hashed lines on these structures. EGFR, epidermal growth factor receptor; Eph, ephrin receptor tyrosine kinases; FAK, focal adhesion kinase; PDGFR, platelet-derived growth factor; PLK, Polo-like kinase; VEGFR, vascular endothelial growth factor receptor.

[ CLOSE WINDOW ]

Figure 2.

Diverse kinase inhibitors. The ATP binding site of AKT1 complexed with ATP (Protein Data Bank (PDB) ID 1O6L) is depicted with key regions indicated and hydrogen bonds indicated by red dotted lines. The middle ring shows commonly used heterocyclic core scaffolds (X = C, N). The outer ring shows examples of structurally diverse type 1 inhibitors and their reported kinase targets. Hydrogen bonds are indicated by hashed lines on these structures. EGFR, epidermal growth factor receptor; Eph, ephrin receptor tyrosine kinases; FAK, focal adhesion kinase; PDGFR, platelet-derived growth factor; PLK, Polo-like kinase; VEGFR, vascular endothelial growth factor receptor.

Type 2 Inhibitors

By contrast, type 2 kinase inhibitors recognize the inactive conformation of the kinase (FIG. 1b). The conformation that is recognized by type 2 inhibitors is sometimes referred to as DFG-out owing to the rearrangement of this motif. Movement of the activation loop to the DFG-out conformation exposes an additional hydrophobic binding site directly adjacent to the ATP binding site. The original discovery that inhibitors such as imatinib and sorafenib bind in the type 2 conformation was serendipitous, but subsequent analysis of multiple type 2 kinase inhibitor co-crystal structures has revealed that all share a similar pharmacophore and exploit a conserved set of hydrogen bonds (FIG. 1b). Examples of type 2 inhibitors that are approved by the US Food and Drug Administration include the ABL1, KIT and platelet-derived growth factor receptor (PDGFR) inhibitors imatinib and nilotinib,[35] and the KIT, PDGFR and Raf inhibitor sorafenib.[36] The inhibitor-stabilized conformational rearrangement of the activation loop observed in type 2 kinase inhibitor co-crystal structures demonstrates that the kinase active site is highly mobile and can remodel to accommodate a variety of inhibitors.[33] The ability to induce dramatic conformational change is not unique to type II kinase inhibitors, for example the type I inhibitor PIK-39 exhibits selectivity towards PI3Kγ over other PI3K isoforms by inducing a conformational rearrangement of the side chain of M804 to form a novel pocket at the entrance to the kinase active site in a manner similar to the DFG-out kinase conformation.[37] Although the methionine residue with which PIK-39 interacts is conserved among other PI3Ks, selectivity towards PI3γ is achieved as only this isoform permits an inhibitor-induced conformational rearrangement.

Allosteric Inhibitors

The third class of compounds binds outside the ATP-binding site — at an allosteric site — and modulates kinase activity in an allosteric manner. Inhibitors belonging to this category tend to exhibit the highest degree of kinase selectivity because they exploit binding sites and regulatory mechanisms that are unique to a particular kinase. The most well-characterized allosteric kinase inhibitor is CI-1040, which inhibits MEK1 and MEK2 by occupying a pocket adjacent to the ATP binding site[38] (FIG. 3a, b). Other examples include GNF2, which binds to the myristate binding site of BCR-ABL1 (Ref. 39); the pleckstrin homology domain-dependent Akt inhibitor Akt-I-1 (Refs 40, 41); and the IKK (inhibitor of nuclear factor-κB kinase) inhibitor BMS-345541 (Ref. 42) (FIG. 3b). Allosteric activators of kinase activity have also been discovered, including RO0281675 (Ref. 43) and numerous analogues[44] that activate glucokinase, as well as AICAR[45] and A-769662 (Ref. 46), which activate AMP-activated protein kinase. More allosteric inhibitors are likely to be uncovered in the future as a greater emphasis is placed on cell-based assays in which kinases are interrogated in their native context. This approach presents the advantage of allowing compounds to be identified that may require an accessory protein for function. For example, the requirement of the mTOR inhibitor rapamycin for the intracellular protein FKBP1A would not have been discovered in a biochemical mTOR assay.

(Enlarge Image)

Figure 3.

Allosteric kinase activity modulators. a | The figure shows the chemical structures of MEK1 inhibitors. AZD-6244 and PD334581 are second-generation MEK1 inhibitors, currently under clinical development. b | CI-1040 (indicated by a blue circle) binds MEK1 (green ribbon, Protein Data Bank (PDB) ID 1S9J)[38] immediately adjacent to the ATP binding site (indicated by red circle). c | GNF2 binds the myristate binding site

...