A novel mechanism by which small molecule inhibitors induce the DFG flip in Aurora A - PubMed (original) (raw)

. 2012 Apr 20;7(4):698-706.

doi: 10.1021/cb200508b. Epub 2012 Jan 27.

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A novel mechanism by which small molecule inhibitors induce the DFG flip in Aurora A

Mathew P Martin et al. ACS Chem Biol. 2012.

Abstract

Most protein kinases share a DFG (Asp-Phe-Gly) motif in the ATP site that can assume two distinct conformations, the active DFG-in and the inactive DFG-out states. Small molecule inhibitors able to induce the DFG-out state have received considerable attention in kinase drug discovery. Using a typical DFG-in inhibitor scaffold of Aurora A, a kinase involved in the regulation of cell division, we found that halogen and nitrile substituents directed at the N-terminally flanking residue Ala273 induced global conformational changes in the enzyme, leading to DFG-out inhibitors that are among the most potent Aurora A inhibitors reported to date. The data suggest an unprecedented mechanism of action, in which induced-dipole forces along the Ala273 side chain alter the charge distribution of the DFG backbone, allowing the DFG to unwind. As the ADFG sequence and three-dimensional structure is highly conserved, DFG-out inhibitors of other kinases may be designed by specifically targeting the flanking alanine residue with electric dipoles.

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Figures

Figure 1

Figure 1

Binding modes of bisanilinopyrimidine inhibitors with Aurora A. Crystal structures were determined for Aurora A liganded with different _ortho_-substituted bisanilinopyrimidine inhibitors (Supplementary Tables 1 and 2). The hinge region (residues 211–213) is indicated in orange, the DFG (residues 274–276) in cyan, the activation loop (residues 277–293) in magenta, other residues in grey, and the inhibitor in yellow. The dotted lines indicate the closest distances to the DFG. The 2Fo-Fc electron density, contoured at 1 σ, is shown as blue mesh around the inhibitor; the Fo-Fc electron density maps from refinements omitting the inhibitor are shown in the supporting material (Supplementary Fig. 5). The insets in the top right corners are surface representations of the overall structures. Compounds 1–5 are DFG-in inhibitors, compounds 6–9 are DFG-out inhibitors.

Figure 2

Figure 2

Structural changes in Aurora A induced by DFG-out inhibitors. a) Surface representation of Aurora A in the DFG-in state (left, liganded with 1) and DFG-out state (right, liganded with 7); the activation loop is highlighted in yellow and red, respectively, and the inhibitors are shown in green. b) Superposition reveals global conformational changes upon binding of 7 particularly of the activation loop and the C-helix, which harbors the catalytic residue Glu181. In the DFG-in state, the loop is oriented away from the ATP site and the inhibitor is exposed to solvent. In the DFG-out state, the loop flips by ~ 180° and the N-terminal flank is positioned above the active site, shielding the inhibitor from solvent. c) Conformation of the ADFGW segment in the DFG-in state liganded with 1 (yellow) and the DFG-out state liganded with 7 (red). The residue closest to the inhibitor is Ala273 (3.4 Å). The DFG-flip causes drastic conformational changes of the backbone, beginning with residue Asp274, forcing Trp277 and the entire activation loop to change direction. The binding interactions of Trp277 in the DFG-in and DFG-out states are shown in Supplementary Fig. 8. The C-helix of Fig. 2b gives way to accommodate the new conformation of Phe275. d) In the DFG-out conformation, the side chain of Asp274 interacts with residues Arg255 and Asp256, and the conformation of the activation loop is stabilized through hydrogen bonding interactions between the main chain atoms of His280 and Lys141. The loop is colored according to temperature factors from blue (low B-factor) to red (high B-factor). Potential hydrogen bonding interactions are indicated as black dotted lines. e) Comparison of the molecular mode of action of VX680 (blue, PDB 3E5A), compound 1 (yellow) and compound 7 (red) (stereo presentation).

Figure 3

Figure 3

Substitutions in other regions of the bisanilinopyrimidine scaffold do not affect the DFG-out mode of action (stereo presentations). a) Compounds 10 and 11 are analogues of the DFG-out inhibitor 7 (substitutions are highlighted in red). Both inhibitors induced the DFG flip and displayed the same general interaction pattern as 7. b) Introduction of a fluorine to the pyrimidine ring (10) fosters van-der-Waals interactions with hydrophobic residues around the gatekeeper residue Leu210, resulting in increased inhibitory activity. c) Substitution of tetrazole for carboxyl in _para_-position of the B-ring (11) preserves the electrostatic interaction with Arg137, and the inhibitory potency remains unchanged. Shown in blue mesh is the 2Fo-Fc electron density of the inhibitors, contoured at 1σ. Potential hydrogen bonding and hydrophobic interactions are indicated as black and green dotted lines, respectively.

Figure 4

Figure 4

Proposed dipole-induced mechanism of action for Aurora ADFG-out inhibitors. a) Model of the collision complex of the DFG-in state of Aurora A with the DFG-out inhibitor 7, based on superimposition of the co-crystal structures of 7 and 1. Displayed are the closest distances (Å) between the chlorine substituent and the enzyme. The ~ 0.8 Å reduced distance in the dead-end complex indicates attraction of Ala273, a feature observed for the DFG-out inhibitors 6–9 and, to a lesser degree, for the DFG-in inhibitors 4 and 5 (Supplementary Figs. 3 and 4). b) The electric dipoles along the C-R bonds (R= F, Cl, Br, C≡N) of the inhibitor may induce a dipole along the Cα-Cβ bond of Ala273. The dipole-dipole interaction is stabilized by altering the charge distribution along the DFG backbone, allowing or forcing the compact DFG-in state to unwind. c) Geometric arrangement of inhibitors 4–9 and Ala273 in the experimentally determined dead-end complexes. Substituents able to induce the DFG flip (6–9) align linearly with the Cα-Cβ bond of Ala273, whereas the C-F bonds of the DFG-in inhibitors 4 and 5 are positioned orthogonal.

Figure 5

Figure 5

Implications for the design of DFG-out inhibitors of other kinases. a) The ADFG-in states of Aurora A (wheat), ABL1 (blue), Rock1 (pink), and LCK (cyan) are highly similar, indicating that exogenous dipoles directed at the alanine residue may induce similar structural changes in these kinases. b) CDK2 (green), MAPK3 (orange), and MER (grey) adopt a different conformation in the C-terminal flank and therefore may respond to exogenous dipoles differently. The r.m.s.d. values for the ADFG-in state of various kinases with respect to Aurora A are shown in Table 2. c) The conformation of 7 bound to the active site of Aurora A is incompatible for efficient binding with CDK2. The model was generated by superimposition of the complexes of Aurora-7 and CDK2-7. d) The co-crystal structure of CDK2 in complex with 7 reveals that the bisanilinopyrimidine scaffold adopts an (s)-trans conformation (defined as the position of the groups colored red across the C-N bond), the A-ring pointing away from the DFG. e) The (s)-cis and (s)-trans conformation of 7 found in Aurora A and CDK2, respectively. The 2Fo-Fc electron density around the inhibitor in d) is contoured at 1 σ. Potential hydrogen bonding, van der Waals interactions, and steric clashes are indicated as black, green and red dotted lines, respectively. The structure of CDK2 with 1 is shown in the Supplementary Fig. 7.

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