High throughput testing of drug library substances and monoclonal antibodies for capacity to reduce formation of cystatin C dimers to identify candidates for treatment of hereditary cystatin C amyloid angiopathy

Abstract

Objective: To establish a high-throughput system for testing the ability of drugs or monoclonal antibodies to reduce the in vitro formation of cystatin C dimers to identify substances potentially useful for treatment of patients with hereditary cystatin C amyloid angiopathy (HCCAA). Methods: Various combinations of incubation temperature, time period, guanidinium chloride concentration and concentration of cystatin C monomers were tested in low-volume formats to induce dimer formation of recombinant cystatin C. The extent of dimerization was analysed by gel filtration chromatography and agarose gel electrophoresis. Results: A high-throughput system based upon agarose gel electrophoresis was developed and used to test 1040 drugs in a clinical drug library for their capacity to reduce cystatin C dimer formation in vitro. Seventeen substances reducing dimer formation by more than 30% were identified. A similar system for testing the capacity of monoclonal antibodies against cystatin C to reduce the in vitro formation of cystatin C dimers was also developed and used to test a panel of 12 monoclonal antibodies. Seven antibodies reducing dimer formation by more than 30% were identified and the two most potent, Cyst28 and HCC3, reduced dimerization by 75 and 60%, respectively. Conclusion: We constructed a simple high-throughput system for testing the capacity of drugs and monoclonal antibodies to reduce the in vitro formation of cystatin C dimers and several candidates for treatment of HCCAA could be identified.

Key Words::

• Amyloidosis
• brain hemorrhage
• drug library screening

Acknowledgements

The investigation was supported by grants from the Swedish Science Research Council (Project No. 5196), the Alfred Österlund Foundation, Greta and Johan Kock Foundation, the Faculty of Medicine at Lund University and from Skåne Regional Council.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.