Abstract
Wild-type and mutant transthyretin (TTR) can misfold and deposit in the heart, peripheral nerves, and other sites causing amyloid disease. Pharmacological chaperones, Tafamidis® and diflunisal, inhibit TTR misfolding by stabilizing native tetrameric TTR; however, their minimal effective concentration is in the micromolar range. By immune-targeting sparsely populated TTR misfolding intermediates (i.e. monomers), we achieved fibril inhibition at substoichiometric concentrations. We developed an antibody (misTTR) that targets TTR residues 89–97, an epitope buried in the tetramer but exposed in the monomer. Nanomolar misTTR inhibits fibrillogenesis of misfolded TTR under micromolar concentrations. Pan-specific TTR antibodies do not possess such fibril inhibiting properties. We show that selective targeting of misfolding intermediates is an alternative to native state stabilization and requires substoichiometric concentrations. MisTTR or its derivative may have both diagnostic and therapeutic potential.
Highlights
A number of approved drugs for protein misfolding disease are available, they mainly offer symptomatic relief with few addressing the underlying root cause[5]
Protein misfolding diseases are caused by a multi-step pathway that results in the conversion of native proteins into abnormal conformations that lead to fibril formation and aggregation
We reasoned that an antibody targeted against this epitope would bind to misfolded TTR conformations with disrupted and exposed dimer interfaces, but not to the native tetramer. This antibody would bind to both monomeric (Fig. 1C) and non-native oligomeric forms of TTR. Since these misfolded species represent only a fraction of serum TTR, this may result in substoichiometric concentrations of the antibody being sufficient for activity
Summary
A number of approved drugs for protein misfolding disease are available, they mainly offer symptomatic relief with few addressing the underlying root cause[5]. Protein misfolding diseases are caused by a multi-step pathway that results in the conversion of native proteins into abnormal conformations that lead to fibril formation and aggregation. Current strategies in development include native state stabilization, fibril capping, and aggregate sequestration. These only indirectly affect the putative toxic oligomeric intermediates, and mostly target species in the misfolding pathway present at high concentrations, namely the native and fibrillar states. Native state stabilization strategies utilize small molecules called pharmacological chaperones that prevent aggregation by binding to the native protein, which imposes an energetic barrier to the formation of aggregation-prone misfolding intermediates. It is believed that the dissociation pathway of the native TTR tetramer is a multi-step process in which the TTR protein cycles through a series of misfolding intermediates before becoming an insoluble amyloid[16]. The mechanism of TTR misfolding is uncertain, studies with transgenic mice which over-express TTR suggest that it may be the result of complications either in TTR’s initial folding stages due to defective hepatic chaperones, or during its final stages of clearance as a result of defective proteasomes[17]
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