Allo-targeting of the kinase domain: Insights from in silico studies and comparison with experiments

Abstract

The eukaryotic protein kinase domain has been a broadly explored target for drug discovery, despite limitations imposed by its high sequence conservation as a shared modular domain and the development of resistance to drugs. One way of addressing those limitations has been to target its potential allosteric sites, shortly called allo-targeting, in conjunction with, or separately from, its conserved catalytic/orthosteric site that has been widely exploited. Allosteric regulation has gained importance as an alternative to overcome the drawbacks associated with the indiscriminate effect of targeting the active site, and it turned out to be particularly useful for these highly promiscuous and broadly shared kinase domains. Yet, allo-targeting often faces challenges as the allosteric sites are not as clearly defined as its orthosteric sites, and the effect on the protein function may not be unambiguously assessed. A robust understanding of the consequence of site-specific allo-targeting on the conformational dynamics of the target protein is essential to design effective allo-targeting strategies. Recent years have seen important advances in in silico identification of druggable sites and distinguishing among them those sites expected to allosterically mediate conformational switches essential to signal transmission. The present opinion underscores the utility of such computational approaches applied to the kinase domain, with the help of comparison between computational predictions and experimental observations.

Figure 1.. Conserved structure of the kinase domain: Comparison of the active and inactive states illustrated for Src kinase domain.

(a) Ribbon diagram of Src kinase in the active conformation (PDB: 3DQW) [38]. Important residues are shown in sticks and the N- and C-lobes are shown in semi-transparent surface representation in cyan and yellow, respectively. ATPγS at the ATP binding site is shown in white spheres. (b) Src in the inactive, αC-helix-out / DFG Asp-in conformation (PDB: 2SRC) [39]. The red arrow indicates the change in the orientation of E310 side chain compared to that in the active form. (c) Src kinase in the inactive αC-helix-in / DFG-Asp-out conformation (PDB: 2OIQ). The change in D404 orientation with respect to the active form is indicated by the red arrow.

Figure 2.. Comparison of drug-binding sites on kinase domain observed in experiments and those predicted by druggability simulations and essential site analysis.

The ligand binding sites of the kinase domain are shown, as detected by experiments (a), and as predicted by computations (b-c). The top and bottom diagrams in each panel display the structure from two perspectives differing by a 90° rotation as indicated. In panel a, Src (PDB: 3DQW) [38] is shown in orange, ATP-bound PKA in yellow (PDB id: 1ATP) [38,46], ABL with ligand at myristate (MYR) site in green (PDB: 3K5V) [17,45], ATP-bound PDK1 with ligand at the PIF site in blue (PDB: 4AW1) [44], and RTKC8 with ligand at peptide-substrate site in salmon (PDB id: 8E4T) [47]. All five structures have ligands bound to the ATP site. Src with ligand at the G-loop site is shown in grey [42]. (b) Druggable hot spots based on druggability simulations using the active form of Src (PDB: 1Y57). Hot spots are shown in yellow spheres. (c) Essential sites predicted using ESSA. Identified essential residues are shown in sticks and transparent spheres. Two new sites are detected by druggability simulations and ESSA, highlighted by red ellipses in the top diagrams (panels b and c).

Fig. 3.. GNM analysis of Src kinase domain.

Residue movements along GNM mode 1 (a), mode 2 (b), and mode 3 (c) are shown in color-coded structures. Panels e-g display the distributions of residue movements when the structure reconfigures along the respective mode’s collective coordinate. Hinge residues in each mode are shown in transparent spheres in the diagrams and highlighted by yellow labels on the diagrams and on the curves. Active form of Src kinase (PDB: 1y57) [43] was used for GNM analysis. (a) Mode 1 enables the opening and closing of cleft between the N- and C-lobes, in line with the transition of the kinase domain between its active and inactive forms. (b) Mode 2 essentially mediates the movements of the G loop; several residues (E412, T440, R460, H492 surrounding (or lying in) the G-loop assume hinge roles. (c) Mode 3 enables the anticorrelated fluctuations between the G-loop and the activation loop revealing an allosteric coupling between those sites. (d) Ribbon diagram color-coded by the size of residue motions (square displacements) driven jointly by modes 1–3. The corresponding square displacements are plotted in panel h. The same panel also displays the motions driven by the individual modes 1, 2 and 3. The vertical yellow shades display the regions that undergo minimal displacements in modes 1–3, thus serving as hinges or anchors that control the global movements of the kinase domain and could serve for allotargeting. The box at the bottom of panel h shows the residue ranges corresponding to substrate-binding (S) and catalytic (C) sites, D₄₀₄FG motif (D), and to the activation loop (A) and G loop (G).