Medchem and Chemical biology approach
MUC4 project: Development of new drug candidates targeting the MUC4-ErbB2 oncogenic complex in the treatment of pancreatic cancer.
Pancreatic cancer (PC) is one of the most deadly cancers in Western countries with an extremely poor prognosis (survival rate of 6 months) and is projected to become the second leading cause of cancer-related death by 2030. The MUC4 membrane-bound mucin and its membrane partner ErbB2 produce an oncogenic complex that mediate PC cell proliferation and migration. 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 MUC4b composed of three EGF-like domains, conserved throughout evolution, structurally equivalent to human EGF and biologically active (Fig. 1).
Figure 1: Schematic representation of the interaction between the EGF domains of MUC4 and ErbB2.
This discovery raises important mechanistic questions regarding the functionality of ErbB2 receptor and understanding the structure-function relationship of MUC4-ErbB2 complex may lead to the discovery of new therapeutic drugs.
Preliminary data and proof of principle
Using Microscale Thermophoresis (MST), a recent fluorescent biophysical technology, we have quantified for the first time the binding affinity between eGFP-labelled MUC4b, from CHO cellular lysates, and recombinant ErbB2 as a KD of 5 ± 3 nM (Fig. 2). Binding affinities for MUC4b fusion protein mutants, in which a single EGF domain is deleted at a time, were also quantified and provided critical insights for EGF domain targeting to better inhibit complex formation.
Fig. 2. KD measurement of eGFP-labelled MUC4ß fusion protein with recombinant ErbB2.
As no structural data of the MUC4-ErbB2 complex were available, we have undertaken molecular dynamics (MD) simulations to identify the specific binding hotspots between MUC4-EGF domains and ErbB2 extracellular domains which were validated by directed mutagenesis studies (Fig. 3A,B). In-house libraries (750 K small molecules and 16 K peptides) were screened by docking methods, combined to a pharmacophore approach, to identify virtual ligands of MUC4-EGF1 and MUC4-EGF2 respectively. The best 45 virtual hits were evaluated by MST and this process led to the identification of a peptide displaying a KD value of 500 µM for MUC4b and selective of EGF2 domain (Fig. 3C).
Fig. 3. (A) Identification of binding hotspots of MUC4-EGF1/ErbB2, (B) Mutation effect of EGF1-cluster 1 on Panc-1 cancer cell proliferation compared to EGF1 wild type, (C) KD measurements of peptide with MUC4ß fusion proteins.
Preliminary results showed that our approach based on homology models led to binding hotspots and ligands identification of MUC4-EGF domains. This strategy must be now applied from absolute structural data in order to identify new ligands with better i-PPI biological activities on MUC4-ErbB2 complex in vitro and in vivo.