Modulation of N-cadherin-mediated homophilic trans interaction upon direct binding of ADH-1: Exploring the molecular basis of action of ADH-1 as N-cadherin inhibitor

Switching of Cadherin expression patterns, as linked to epithelial-to-mesenchymal transition (EMT), is one of the most notable characteristics of Cancer cell metastasis. Aberrant expression of N-Cadherin, a cell adhesion protein, on tumour cells, has made this protein a promising target for Cancer treatment. ADH-1 (commercial name Exherin) is a cyclic pentapeptide that targets and inhibits N-Cadherin. ADH-1 is the most utilized antagonist and the only inhibitor of N-Cadherin under clinical trial. By disrupting N-cadherin's function, ADH-1 aims to induce tumour cell death and disrupt tumour blood vessels. Unraveling the molecular basis of inhibiting action of ADH-1 on N-cadherin-mediated homophilic interaction along with a quantitative depiction of the drug-protein interaction is crucial for a detailed assessment of drug function. Herein, the impact of direct binding of ADH-1 on N-cadherin-mediated homophilic trans interaction has been studied at single-molecule level for the first time using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS), which is further compared with ensemble information. In SMFS experiments, a drastic reduction (3.5 times) in the unbinding events of N-Cadherin EC1-2_mut (on substrate surface) - N-Cadherin EC1-2_mut (onto SMFS tip) homophilic complex was observed in presence of ADH-1, compared to unbinding events in the absence of ADH-1. Comparative biophysical analysis of N-cadherin-N-cadherin and N-cadherin-ADH-1 interaction suggests a stronger and stabler interaction between the protein and the drug, indicating that ADH-1 could competitively bind to N-Cadherin, supporting its N-cadherin-N-cadherin interaction inhibiting action.