Identification of small molecule inhibitors of protein aggregation

Abstract

Therapeutic antibodies utilize the adaptive immune response to treat complex disease states. Antibodies’ success in therapeutic applications has led to their domination of total biologics revenue. With more therapeutic antibodies currently in development, as well as many new drug targets being discovered, there is little doubt that this sector will continue to grow. However, bottlenecks exist in therapeutic antibody development. One constraint in antibody development is antibody aggregation. Formulating antibodies can be problematic, as in vitro preparations require high protein concentrations that can promote aggregation. Each antibody peptide sequence is unique within complimentary determining regions (CDRs); this region provides antibody specificity to an antigen target. However the CDR region can also determine the aggregation properties of the molecule. The resulting difference in aggregation properties between antibodies prevents development of a general approach to inhibit aggregation occurring in vitro. Antibody aggregation can also cause disease. Light Chain Deposition Disease (LCDD) is a condition caused by aggregation of light chain antibodies in vivo. It often results in amyloid deposits affecting vital organ function. The primary aim of this thesis was to discover a chemical compound that could inhibit in vitro antibody aggregation. A tertiary aim was to establish that a candidate compound could also serve as a potential drug candidate in LCDD treatment. To achieve this, CDR mediated aggregation was modeled with variable light chain domain antibodies (VLs) displayed on phage and in soluble form. A method utilizing phage-display was developed to detect FDA approved compounds that could inhibit CDR mediated VL aggregation. Specifically, The Prestwick Chemical Library of 1200 compounds was screened with phage expressing aggregation prone VL DPK9 using a ‘heat-cool’ ELISA method. This screen identified Levodopa and Apomorphine as compounds that could prevent aggregation of DPK9. Further testing demonstrated that Levodopa and Apomorphine could also successfully prevent aggregation of soluble VL proteins. Structural analysis demonstrated that the catechol moiety present on both Levodopa and Apomorphine prevented aggregation of VL protein. This led to the discovery of four other polyphenol compounds also capable of preventing VL aggregation. Further experimentation also allowed for the mechanism of action of the catechol moiety to be partially understood. All candidate compounds identified were either FDA approved or naturally occurring polyphenols. Consequently such compounds may have direct implications as excipients in antibody in vitro formulation and as drug candidates for treatment of LCDD.