Monoamine Oxidase-A (MAO-A) Inhibitors Screened from the Autodisplayed Fv-Antibody Library.

ABSTRACT

Serotonin-like mimotopes were screened from the Fv-antibody library to be used as inhibitors against monoamine oxidase A (MAO-A). The Fv-antibody [corresponding to the VH region of immunoglobulin G (IgG)] consists of three complementarity-determining regions and four frame regions. The Fv-antibody library was prepared by site-directed mutagenesis of CDR3, which consists of 11 amino acid residues. Three target clones were screened from the Fv-antibody library, and the binding affinity of the screened clones to the monoclonal anti-serotonin antibody was analyzed using fluorescence-activated cell sorting. The screened Fv-antibodies were expressed as soluble proteins fused with green fluorescence protein. Additionally, the screened CDR3 regions (11 residues) of the selected Fv-antibodies were synthesized as peptides with linking amino acid residues. The binding constants (KD) of the three serotonin-like mimotopes (Fv-antibodies and peptides) were estimated using a surface plasmon resonance biosensor. The inhibitory activity (IC50) of the serotonin-like mimotopes (Fv-antibodies and peptides) was estimated separately for MAO-A and MAO-B enzymes and compared with that of conventional inhibitors. Finally, the screened serotonin-like mimotopes were used to treat a cell line (SH-SY5Y, ATCC code CRL-2266) expressing serotonin receptors. This was done to confirm the following two aspects (1) the binding of mimotopes to the serotonin receptors on the cell surface and (2) the inhibitory activity of mimotopes against MAO-A enzymes in the cell lysates.