Medchem and Chemical biology approach
MUC4 project: Inhibition of the oncogenic MUC4-ErbB2 complex by small organic molecules targeting the MUC4-EGF domains.
The aberrant overexpression of the transmembrane MUC4 mucin in diverse pathologies of the epithelium, its partnership with ErbB2 and the negative consequences of the activity of MUC4-ErbB2 complex on epithelial homeostasis, has allowed identification of MUC4 as a highly attractive therapeutic target. MUC4-ErbB2 complex has started to be understood at the molecular level and biochemical studies conducted in UMR-S 1172 research group have shown that the physical interaction between human MUC4 and ErbB2 involves a region of the extracellular domain of MUC4 composed of three EGF-like domains, conserved throughout evolution, structurally equivalent to human EGF and biologically active (Figure 1). The aim of this project is to identify and synthesize small organic molecules targeting the MUC4-EGF domains to inhibit the oncogenic MUC4-ErbB2 complex.
Figure 1: Schematic representation of the interaction between the EGF domains of MUC4 and ErbB2.
For this purpose, Molecular dynamic (MD) simulations were then undertaken, from the high sequence homology (59%) with the crystallized hEGF-ErbB1 complex, to identify the specific binding hotspots, confirmed by directed mutagenesis, between MUC4-EGF1/2 domains and ErbB2 extracellular domains (Figure 2).
Figure 2: (A) Structural homology between crystallized hEGF-ErbB1 and MUC4-EGF1/2/ErbB2 complexes. (B) Final conformations from MD simulation of both MUC4-EGF1/ErbB2 and MUC4-EGF2/ErbB2 complex models.
Computational virtual screening was the chosen strategy to identify and design PPI inhibitors (i-PPI) that will directly compete with the MUC4-EGF domains of the MUC4-ErbB2 interface complex. Large compound libraries in the range of millions of compounds have been rationally screened by docking a pharmacophore approach, focusing a small subset of virtual hits.
On the anti-MUC4-EGF1 pharmacophore, this process led to the selection of a final list of 57 compounds, for experimental testing, which was ranked in four chemical series. In an in vitro cell proliferation assay on pancreatic cancer cell lines, several compounds induced 25 to 45% decrease of cell survival which for some of them were MUC4-dependents (Figure 3).
Figure 3: (A) Virtual screening campaign and (B) MUC4-expressing (CAPAN-2 Mock) and MUC4-non expressing (CAPAN-2 MUC4-KD) cell survival assays.
Pharmacological evaluation of these hits will be realized for measurement of binding affinities (MST, SPR, FRET) on MUC4-EGF1 domain and to quantify disruption of MUC4-ErbB2 complex. Compounds, from the virtual screening, displaying the potent binding affinities and biological activities have been selected as the starting point for a medicinal chemistry program for designing potent MUC4-ErbB2 inhibitors.