Development of Protein-Based Inhibitors of SOD1 Misfolding, Aggregation and Cytotoxicity as Potential Therapeutics for ALS

Molecular agents that specifically bind and neutralize misfolded and toxic superoxide dismutase-1 (SOD1) mutant proteins are proposed as means to attenuate the disease progression of familial amyotrophic lateral sclerosis (fALS). We addressed the challenge of the high structural similarity between the wild-type and mutant SOD1 proteins by converting a promiscuous natural human IgG binding domain – the hyperthermophilic variant of protein G (HTB1) – into a highly specific aggregation inhibitor (designated HTB1_M) of two fALS-linked SOD1 mutants, SOD1^G93A and SOD1^G85R. For this purpose, we utilized a computational algorithm for mapping protein surfaces predisposed to HTB1 intermolecular interactions to construct a focused HTB1 library, complemented with a yeast surface display (YSD) experimental method for affinity and specificity screening. HTB1_M demonstrated high binding specificity toward SOD1 mutants, inhibiting their amyloid aggregation in vitro, preventing the accumulation of misfolded proteins in living cells, and reducing the cytotoxicity of SOD1^G93A expressed in the motor neuron-like cells. Competition assays and molecular docking simulations suggested that HTB1_M binds – via both its α-helical and β-sheet domains – to SOD1 at the native dimer interface that becomes exposed upon mutated SOD1 misfolding and monomerization. This study demonstrates the utility of computational mapping of the protein-protein interaction potential in designing the focused libraries used in directed evolution. It also provides new insight into the mechanism of the conversion of broad spectrum immunoglobulin binding proteins, such as HTB1, into target-specific proteins, thereby paving the way for the development of new selective drugs targeting the amyloidogenic proteins implicated in a variety of human diseases.