Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective death of motor neurons in the brainstem, motor cortex, and spinal cord, leading to muscle atrophy and eventually to death. It is currently held that various oligomerization‐inducing mutations in superoxide dismutase 1 (SOD1), an amyloid‐forming protein, may be implicated in the familial form of this fast‐progressing highly lethal neurodegenerative disease. A possible therapeutic approach could therefore lie in developing inhibitors to SOD1 mutants. By screening a focused mutagenesis library, mutated randomly in specific “stability patch” positions of the B1 domain of protein G (HTB1), we previously identified low affinity inhibitors of aggregation of SOD1G93A and SOD1G85R mutants. Herein, with the aim to generate a more potent inhibitor with higher affinity to SOD1 mutants, we employed an unbiased, random mutagenesis approach covering the entire sequence space of HTB1 to optimize as yet undefined positions for improved interactions with SOD1. Using affinity maturation screens in yeast, we identified a variant, which we designated HTB1M3, that bound strongly to SOD1 misfolded mutants but not to wild‐type SOD1. In‐vitro aggregation assays indicated that in the presence of HTB1M3 misfolded SOD1 assembled into oligomeric species that were not toxic to NSC‐34 neuronal cells. In addition, when NSC‐34 cells were exposed to misfolded SOD1 mutants, either soluble or preaggregated, in the presence of HTB1M3, this inhibitor prevented the prion‐like propagation of SOD1 from one neuronal cell to another by blocking the penetration of SOD1 into the neuronal cells.
Intra- and extraneuronal
deposition of amyloid β (Aβ)
peptides have been linked to Alzheimer’s disease (AD). While
both intra- and extraneuronal Aβ deposits affect neuronal cell
viability, the molecular mechanism by which these Aβ structures,
especially when intraneuronal, do so is still not entirely understood.
This makes the development of inhibitors challenging. To prevent the
formation of toxic Aβ structural assemblies so as to prevent
neuronal cell death associated with AD, we used a combination of computational
and combinatorial-directed evolution approaches to develop a variant
of the HTB1 protein (HTB1M2). HTB1M2 inhibits in vitro self-assembly of Aβ42 peptide and shifts
the Aβ42 aggregation pathway to the formation of oligomers that
are nontoxic to neuroblastoma SH-SY5Y cells overexpressing or treated
with Aβ42 peptide. This makes HTB1M2 a potential
therapeutic lead in the development of AD-targeted drugs and a tool
for elucidating conformational changes in the Aβ42 peptide.
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