Development of SPR Methods to Evaluate Peptide Inhibition of Cadherin Interactions

Abstract

The blood-brain barrier (BBB) is formed by vascular endothelial cells that selectively restricts the entry of molecules into the brain. Due to this selectivity, a lot of drug molecules are obstructed from entering the brain. Therefore, overcoming the BBB has been proven challenging in the field of medicine. Cadherin proteins (i.e., E- and VE-cadherins) are cell-cell adhesion molecules in the BBB that play a very important role in maintaining the adherens junction. Our group has shown that the BBB intercellular junctions can temporarily be disrupted by synthetic cadherin peptides (i.e. HAV6 and ADTC5) to improve passive penetration of molecules through the paracellular pathways of the BBB. Cyclic ADTC5 peptide binds to cadherins to block cadherin-cadherin interactions in an equilibrium and reversible fashion at the intercellular junctions to increase the porosity of paracellular pathways of the BBB. ADTC5 peptide has been used to deliver large molecules such as lysozyme, albumin, and monoclonal antibody (mAb) into the brains of animal models. This study develops Surface Plasmon Resonance (SPR) methods to confirm (a) interaction of EC1-to-EC1 domain of cadherins; (b) binding of ADTC5 peptide EC1 domain of cadherins; and (c) inhibition EC1-to-EC1 interaction by ADTC5. Our results showed that the EC1 domain of E-cadherin in the solution phase can bind to EC1 domain of E-cadherin immobilized on the surface of carboxylic acid SPR chip (KD of 1.45 μM in TBS and 44 μM in PBS). In addition, the EC1 domain of VE-cadherin in solution phase binds to the EC1 domain of E-cadherin immobilized on the surface of SPR chips (KD of 10.15 μM in TBS using gold chip; 47.7 μM in TBS and 1,340 μM in PBS using carboxylic acid chip). ADTC5 inhibits the EC1-to-EC1 interactions for E-cadherin with IC50 of 4.27 M and the EC1-to-EC1 interactions between E- and VE-cadherins with IC50 of 3.27 μM. In the future, this method could be used to evaluate inhibitory activities of new cadherin peptides designed using computational chemistry.