Studies on Recombinant Insulin Analogs Interactions with Partner Cell Membrane Receptors Using the SPR Technique ^†

Abstract

The rapid progress in the field of chemical sensors has made it possible to carry out quick, accurate, and precise monitoring of interactions between biological compounds, enabling the development of models describing the processes that occur at the cellular level. Measurement techniques utilizing biosensors coupled with optical detection play a special role in this type of research. Label-free techniques, such as surface plasmon resonance, are particularly useful for studying intermolecular interactions in real time and for determining the thermodynamic parameters that characterize them. The study presented in this work concerns the use of the surface plasmon resonance (SPR) technique to evaluate the biological activity and potential mutagenicity of recombinant insulin analogs exhibiting a prolonged period of action (compared to human insulin)—these are innovative drug candidates for the treatment of diabetes. The project’s aim was achieved by the analysis of kinetic parameters describing the interactions of cell surface receptors with a wide range of recombinant proteins with amino acid sequences that differ from the natural hormone in only a few positions. The presented results include the construction of the receptor layer with immobilized membrane receptors, obtained by their covalent conjugation with -COOH terminated SPR chips. The selection of appropriate parameters of the conjugation process with the use of carbodiimide chemistry could obtain good-quality layers with a sensitivity that enabled the direct monitoring of the receptor’s interactions with insulin and its analogues. The research covers the optimization of the receptor layer to reach high-ligand surface density. The results of molecular modelling with the use of the kinetic curves obtained for the interaction of insulin and long-acting analogues were also shown. Determined kinetic parameters (especially KD) became the starting point for the comparative characteristics of studied proteins’ interactions with an insulin receptor.