Using Directed Evolution for Developing Novel SOD1 Binding Proteins for the Treatment of Neurodegenerative Diseases

Neurodegenerative diseases are incurable diseases that are characterized by gradual death of neurons. The exact biochemical mechanism of neurodegenerative diseases has not been elucidated, but toxicity has been linked to amyloid aggregates of proteins in the central nervous system (CNS) that form insoluble inclusions. Although toxicity is not a product of aggregation in all cases, elucidation and inhibition of the aggregation of proteins into amyloids is a major goal in the study of neurodegenerative diseases and the search for possible therapies. Superoxide Dismutase 1 (SOD1) is an amyloid-forming protein linked to Amyotrophic Lateral Sclerosis (ALS) with mutations such as SOD1^G93A and SOD1^G85R that lead to SOD1 misfolding and aggregation. Previously in our lab, a variant of hyperthermophilic variant of Protein G (HTB1) was developed using computational and combinatorial methods and was found to bind SOD1^G93A and SOD1^G85R and inhibit their aggregation and toxicity in-vitro. Herein, to generate higher affinity binders, we generated a second-generation random mutagenesis library based on the structure of the novel protein and used yeast surface display (YSD) to screen the library for high affinity binders of SOD1^G93A and SOD1^G85R, respectively. Affinity maturation yielded several clones selected based on high expression yield and target affinity and one unique clone containing the mutation K12E was expressed in BL21 bacteria and purified using affinity chromatography. This protein variant binds SOD1 unfolded mutants with high affinity as determined by SPR, and is more potent in inhibiting aggregation of SOD1^G85R compared to the previously developed variant. Transmission electron microscopy (TEM) was used to verify the absence of fibrillar aggregates in SOD1^G85R samples treated with the inhibitor. To further characterize the ability of the novel protein to inhibit aggregation and toxicity of mutant SOD1, in vivo viability experiments is performed using motor neuron like cell line NSC-34 overexpressing SOD1 mutants and SOD1^G93A transgenic mice.