Abstract A142: Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction

Abstract

Abstract eIF4E is the rate limiting factor for cap-dependent protein translation, forming an essential part of the translation initiation complex, eIF4F and is a key signalling convergence point for the major oncogenic pathways MAPK and PI3K/AKT/MTOR. eIF4E has been shown to be overexpressed in cancer, which has been associated with poor prognosis and resistance to chemotherapeutics and targeted agents such as mTOR and MAPK inhibitors. eIF4E is therefore a promising target for cancer treatment but has so far been difficult to target with small molecules due to a lack of druggable pockets. Fragment based crystallographic screening is a powerful technique for probing the surface of proteins to identify potentially druggable binding sites. Here, we describe our fragment-based approach and hit optimisation campaign to generate potent compounds binding to eIF4E For the fragment screen, we generated an engineered form of eIF4E, where the N-terminal (residues 1-35) were removed and replaced a short, tethered peptide based on the canonical binding sequence of the eIF4E binding protein 4E-BP1, and screened 1400 fragments using a combination of Xray crystallography and NMR. Several low affinity (mM) fragment hits were identified, binding to both the mRNA cap- binding site and at an additional binding site of unknown functional relevance. Using a combination of biophysical and biochemical assays, subsequent rounds of iterative structure-based drug design of yielded potent lead molecules against both binding sites. For the cap-binding site, lead compounds of low nM potency were generated but due to the polar nature of the binding site, the molecules had very poor permeability properties and did not inhibit cell proliferation. For the non-canonical binding site, lead compounds with potency of 100nM alongside less potent enantiomers were generated which provided useful tools for probing the function of the binding site in vitro. We developed a MSD (Meso-scale discovery) assay to determine that the lead compound and not the inactive enantiomer could disrupt the eIF4E:eIF4G interaction in HeLa cell lysates with an IC50 of 1uM. Using a HeLa extract in vitro translation assay, we could also demonstrate that both the cap site and non-canonical site lead compounds could inhibit cap-dependent but not IRES driven protein translation. This work demonstrates the power of fragment screening to identify novel functional pockets on difficult to drug proteins such as eIF4E. Citation Format: Caroline J Richardson, Mladen Vincovic, Charlotte East, Nicola Wallis, George Ward, Andrew Woodhead. Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A142.