Inhibitor Trapping in Kinases

Abstract

Recently, we identified a novel mechanism of enzyme inhibition in N-myristoyltransferases (NMTs), which we have named 'inhibitor trapping'. Inhibitor trapping occurs when the protein captures the small molecule within its structural confines, thereby preventing its free dissociation and resulting in a dramatic increase in inhibitor affinity and potency. Here, we demonstrate that inhibitor trapping also occurs in the kinases. Remarkably, the drug imatinib, which has revolutionized targeted cancer therapy, is entrapped in the structure of the Abl kinase. This effect is also observed in p38α kinase, where inhibitor trapping was found to depend on a 'magic' methyl group, which stabilizes the protein conformation and increases the affinity of the compound dramatically. Altogether, these results suggest that inhibitor trapping is not exclusive to N-myristoyltransferases, as it also occurs in the kinase family. Inhibitor trapping could enhance the binding affinity of an inhibitor by thousands of times and is as a key mechanism that plays a critical role in determining drug affinity and potency.

Keywords: Abl; Src; binding affinity; binding affinity prediction; drug design; drug potency; imatinib; kinases; protein conformation.

Figure 1

The structure of Abl in a complex with imatinib: (a) 2D structure of imatinib; (b) crystal structure of Abl in a complex with imatinib (PDB 1IEP). The Abl N-lobe is shown in green, the C-lobe in cyan, and the hinge region in yellow. The P-loop and A-loop are indicated in orange and blue. The carbon atoms of imatinib are shown in grey.

Figure 2

Inhibitor trapping in p38α: (a) The 2D structure of compounds 1 and 2. The removal of the indicated methyl group (grey circle) leads to a 208-fold reduction in potency. (b) The crystal structure of p38α in a complex with compound 1 (PDB 3D7Z). The image on the left shows the whole catalytic domain, and on the right, the binding site of compound 1 is shown. The P-loop, which adopts a closed conformation, is shown in orange. The A-loop is in blue, the hinge region is in cyan, and the hydrophobic pocket, where the ‘magic’ methyl group interacts, is in yellow. (c) Surface representation of the crystal structures depicted in “(b)”with the corresponding coloring scheme, showing that compound 1 is captured inside the p38α protein structure.

Figure 3

The role of the ‘magic’ methyl group in stabilizing the P-loop conformation: (a–h) RMSD of the heavy atoms in p38α complexes with compound 1 and compound 2 (without methyl) of the indicated structural elements. The numbers next to the legend indicate the average RMSD in Angstroms (Å).

Figure 4

Conformational changes in the P-loop observed during MD simulations of p38α complexes. Structural superimpositions of the structures at the beginning (0 ns) and the end (1000 ns) of the MD simulations are shown. The images on the left show the whole p38α kinase domain, and on the right the images depict zoomed views of the boxed areas. In time 0, the protein is shown as a green cartoon, with the P-loop in yellow and the inhibitor in dark grey. In time 1000 ns, the protein is in cyan, the P-loop is in orange, and the ligand is in light grey: (a) the complex between p38α and compound 1- the P-loop preserved its closed conformation during MD simulations. (b) the complex between p38α and compound 2 (without a methyl group). The P-loop transitions from the closed to open conformation, leading to disruption of the interaction between Y35 from the P-loop and D112 in the hinge region.

Figure 5

The P-loop adopts different conformations in Src and Abl complexes with imatinib. Images on top depict the whole kinase domain, and at the bottom, they depict the imatinib binding site. The dissociation constants Ki for imatinib with Abl and Src are shown on top: (a,c) crystal structure of Abl in a complex with imatinib (PDB 1IEP); (b,d) crystal structure of Src in a complex with imatinib (PDB 2OIQ). All polar interactions (black dots) are conserved in the two complexes. I360 in Abl corresponds to V383 in c-Src, D381 to D404, E286 to E310, T315 to T338, and M318 to M341. In Abl, imatinib forms stacking interactions with Y253 (red dots), which do not occur with the corresponding F278 in c-Src due to the different conformation of the P-loop. In Abl, Y253 also forms a hydrogen bond with N322 from the hinge region. The distances shown are in Angstroms (Å).

Figure 6

Imatinib trapping inside the structure of Abl. The surface representation of Abl’s crystal structure in a complex with imatinib (PDB 1IEP) is depicted in two different orientations. The carbon atoms of imatinib are shown in green, and the Abl protein is shown in grey. Blue and red correspond to electropositive and electronegative atoms.

Figure 7

Conformational dynamics of the Abl and Src complexes with imatinib: (a–h) RMSD of the heavy atoms of the indicated structural elements in Abl and Src complexes with imatinib during MD simulations. The numbers on the legend’s right indicate the average RMSD in Angstroms (Å).

Figure 8

A ‘magic’ methyl is present in the structure of imatinib. (a,b) The 2D structure of compound 1 and imatinib. Methyl groups are indicated by grey circles. (c,d) The structure of compound 1 and imatinib observed in their complexes with p38α (PDB 3D7Z) and Abl (PDB 1IEP). (e) The superimposition of the structures of compound 1 (grey) and imatinib (orange). (f) The superimposition of the binding pockets of compound 1 and imatinib. p38α is in green, and Abl is in cyan. The residues in the hydrophobic pocket binding the methyl group are conserved in p38α and Abl. Numbering is in p38α. V38, A51, K53, and T106 correspond to V255, A269, K271, and T315 in Abl.

Figure 9

Conformational changes in the P-loop in the complexes of Abl with imatinib alone or imatinib in which the methyl group is substituted with a hydrogen atom (imatinib–CH3/H) during MD simulations. (a) RMSD of the heavy atoms of the P-loop in Abl complexes. The numbers next to the legend indicate the average RMSD in Angstroms (Å). (b) In imatinib–Abl complexes, the P-loop is in the closed conformation at the beginning and remains closed during the MD simulations. (c) The P-loop opens during MD simulations of Abl complexes with imatinib–CH3/H. The hydrogen bond between Tyr253 in the P-loop and Asn322 in the hinge is indicated by black dots. In “(a)” the P-loop opens at around 500 ns and remains open until the end of the simulation.

Figure 10

Increased P-loop conformational dynamics in the imatinib–Y253F Abl complex. (a) RMSD of the heavy atoms of the P-loop in imatinib complexes with wt Abl (Abl) and Abl containing Y253F mutation. The numbers next to the legend indicate the average RMSD in Angstroms (Å). (b) In the Y253F mutant, initially, the P-loop is in the closed conformation and opens during the MD simulations.