Discovery of Lysine-Targeted eIF4E Inhibitors through Covalent Docking

Abstract

Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5'-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.

Figure 1.

Covalent docking to eIF4E Lys162. (a) Arylsulfonyl fluorides 1–7 prioritized by covalent docking. (b) eIF4E (1 μM) was treated with compound 2 (100 μM) with or without m7GTP (100 μM). At the indicated time points, eIF4E labeling by 2 was quantified by LC–MS. (c) Docked pose of compound 2 covalently bound to Lys162 (green) of eIF4E (docked to PDB ID 4DT6; pink side chains, gray surface).

Figure 2.

Structure-guided optimization of covalent eIF4E inhibitors. (a) Secondary docking hits 8 and 9. (b) WT and K162R eIF4E were treated with 8 and 9, followed by LC–MS analysis. (c) Cocrystal structure of 9/eIF4E (PDB ID 6U09). Electron density (2F_o – F_c) is shown at a contour level of 1σ. (d) Overlay of compound 9/eIF4E (cyan) with the ligand from PDB ID 4DUM (yellow), which guided the design of compounds 10–12 (e). (f) eIF4E was treated with 9–12 for 1 h at pH 8.1 (left) or 15 min at pH 7 (right), followed by LC–MS analysis. (g) Kinetic parameters for modification of eIF4E by compounds 10–12 (pH 8.1).

Figure 3.

Covalent inactivation of eIF4E in cells. (a) Jurkat cells were treated with 9–12. After 3 h, cell lysates were prepared, and an aliquot of each sample was analyzed by Western blotting for eIF4E and tubulin (“lysates”). Remaining lysate samples were enriched with m7GTP-agarose beads, and the bound (“elution”) and unbound (“supernatant”) fractions were analyzed by Western blotting. (b) HEK293T cells stably overexpressing WT or K162R FLAG-eIF4E or nontransduced cells (NT) were treated with 12 for 30 min. Cell lysates were prepared and analyzed by Western blotting (* denotes endogenous eIF4E). (c) Stable cell lines from (b) were treated with 12 prior to transfection with a bicistronic plasmid comprising a cap-dependent cistron (Renilla luciferase) followed by a cap-independent cistron (firefly luciferase). Cells were incubated with 12 or DMSO for 6 h, after which the Renilla and firefly luciferase activities were measured (Figure S9). Data are means ± SEM (n = 3). **, P < 0.01; ****, P < 0.0001.