Mechanisms of transthyretin inhibition of ß-amyloid aggregation in vitro.

Tissue-specific overexpression of the human systemic amyloid precursor transthyretin (TTR) ameliorates Alzheimer's disease (AD) phenotypes in APP23 mice. TTR-ß-amyloid (Aß) complexes have been isolated from APP23 and some human AD brains. We now show that substoichiometric concentrations of TTR tetramers suppress Aß aggregation in vitro via an interaction between the thyroxine binding pocket of the TTR tetramer and Aß residues 18-21 (nuclear magnetic resonance and epitope mapping). The K(D) is micromolar, and the stoichiometry is <1 for the interaction (isothermal titration calorimetry). Similar experiments show that engineered monomeric TTR, the best inhibitor of Aß fibril formation in vitro, did not bind Aß monomers in liquid phase, suggesting that inhibition of fibrillogenesis is mediated by TTR tetramer binding to Aß monomer and both tetramer and monomer binding of Aß oligomers. The thousand-fold greater concentration of tetramer relative to monomer in vivo makes it the likely suppressor of Aß aggregation and disease in the APP23 mice.

Rational design of potent domain antibody inhibitors of amyloid fibril assembly.

Antibodies hold significant potential for inhibiting toxic protein aggregation associated with conformational disorders such as Alzheimer's and Huntington's diseases. However, near-stoichiometric antibody concentrations are typically required to completely inhibit protein aggregation. We posited that the molecular interactions mediating amyloid fibril formation could be harnessed to generate antibodies with potent antiaggregation. Here we report that grafting small amyloidogenic peptides (6-10 residues) into the complementarity-determining regions of a single-domain (V(H)) antibody yields potent domain antibody inhibitors of amyloid formation. Grafted AMyloid-Motif AntiBODIES (gammabodies) presenting hydrophobic peptides from Aß (Alzheimer's disease), α-Synuclein (Parkinson's disease), and islet amyloid polypeptide (type 2 diabetes) inhibit fibril assembly of each corresponding polypeptide at low substoichiometric concentrations (1:10 gammabody:monomer molar ratio). In contrast, sequence- and conformation-specific antibodies that were obtained via immunization are unable to prevent fibrillization at the same substoichiometric concentrations. Gammabodies prevent amyloid formation by converting monomers and/or fibrillar intermediates into small complexes that are unstructured and benign. We expect that our antibody design approach--which eliminates the need for immunization or screening to identify sequence-specific domain antibody inhibitors--can be readily extended to generate potent aggregation inhibitors of other amyloidogenic polypeptides linked to human disease.

Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions.

Antibodies commonly contain hydrophobic residues within their complementarity-determining regions (CDRs) that mediate binding to target antigens. Unfortunately, hydrophobic CDRs can also promote antibody aggregation, which is especially concerning for therapeutic antibodies due to the immunogenicity of antibody aggregates. Here we investigate how the sequences of CDRs within single-domain (V(H)) antibodies specific for the Alzheimer's amyloid ß peptide can be engineered to resist aggregation without reducing binding affinity. We find that domain antibodies containing clusters of hydrophobic residues within their third CDR (CDR3) are prone to aggregate within days at 25°C and minutes above 70°C. However, inserting two or more negatively charged residues at each edge of CDR3 potently suppresses antibody aggregation without altering binding affinity. We also find that inserting charged mutations at one edge of CDR3 (N- or C-terminal) prevents aggregation, but only if such mutations are located at the edge closest to most hydrophobic portion of CDR3. In contrast, charged mutations outside of CDR3 fail to suppress aggregation. Our findings demonstrate that the sequence of CDR loops can be engineered in a systematic manner to improve antibody solubility without altering binding affinity or specificity.

Conformational differences between two amyloid ß oligomers of similar size and dissimilar toxicity.

Several protein conformational disorders (Parkinson and prion diseases) are linked to aberrant folding of proteins into prefibrillar oligomers and amyloid fibrils. Although prefibrillar oligomers are more toxic than their fibrillar counterparts, it is difficult to decouple the origin of their dissimilar toxicity because oligomers and fibrils differ both in terms of structure and size. Here we report the characterization of two oligomers of the 42-residue amyloid ß (Aß42) peptide associated with Alzheimer disease that possess similar size and dissimilar toxicity. We find that Aß42 spontaneously forms prefibrillar oligomers at Aß concentrations below 30 µm in the absence of agitation, whereas higher Aß concentrations lead to rapid formation of fibrils. Interestingly, Aß prefibrillar oligomers do not convert into fibrils under quiescent assembly conditions but instead convert into a second type of oligomer with size and morphology similar to those of Aß prefibrillar oligomers. Strikingly, this alternative Aß oligomer is non-toxic to mammalian cells relative to Aß monomer. We find that two hydrophobic peptide segments within Aß (residues 16-22 and 30-42) are more solvent-exposed in the more toxic Aß oligomer. The less toxic oligomer is devoid of ß-sheet structure, insoluble, and non-immunoreactive with oligomer- and fibril-specific antibodies. Moreover, the less toxic oligomer is incapable of disrupting lipid bilayers, in contrast to its more toxic oligomeric counterpart. Our results suggest that the ability of non-fibrillar Aß oligomers to interact with and disrupt cellular membranes is linked to the degree of solvent exposure of their central and C-terminal hydrophobic peptide segments.

Polyphenolic disaccharides endow proteins with unusual resistance to aggregation.

Protein aggregation is a common problem during the purification and formulation of therapeutic proteins. Here we report that polyphenolic disaccharides are unusually effective at preventing protein aggregation. We find that two polyphenolic glycosides-naringin and rutin-endow diverse proteins with the ability to unfold without aggregating when heated, as well as the ability to refold without aggregating when cooled at low glycoside concentrations (<5 mM). This extreme solubilizing activity is a synergistic combination of the glycone and aglycone moieties, as combinations of polyphenols and sugars fail to suppress aggregation. Moreover, the activity of polyphenolic disaccharides is remarkably specific since their monosaccharide counterparts (as well as other common excipients such as arginine, trehalose, and cyclodextrin) fail to prevent aggregation at similar concentrations (<25 mM). We expect that polyphenolic disaccharides will be valuable additives for enhancing the solubility of proteins in applications plagued by protein aggregation.

Structure-based design of conformation- and sequence-specific antibodies against amyloid ß.

Conformation-specific antibodies that recognize aggregated proteins associated with several conformational disorders (e.g., Parkinson and prion diseases) are invaluable for diagnostic and therapeutic applications. However, no systematic strategy exists for generating conformation-specific antibodies that target linear sequence epitopes within misfolded proteins. Here we report a strategy for designing conformation- and sequence-specific antibodies against misfolded proteins that is inspired by the molecular interactions governing protein aggregation. We find that grafting small amyloidogenic peptides (6-10 residues) from the Aß42 peptide associated with Alzheimer's disease into the complementarity determining regions of a domain (V(H)) antibody generates antibody variants that recognize Aß soluble oligomers and amyloid fibrils with nanomolar affinity. We refer to these antibodies as gammabodies for grafted amyloid-motif antibodies. Gammabodies displaying the central amyloidogenic Aß motif (18VFFA21) are reactive with Aß fibrils, whereas those displaying the amyloidogenic C terminus (34LMVGGVVIA42) are reactive with Aß fibrils and oligomers (and weakly reactive with Aß monomers). Importantly, we find that the grafted motifs target the corresponding peptide segments within misfolded Aß conformers. Aß gammabodies fail to cross-react with other amyloidogenic proteins and scrambling their grafted sequences eliminates antibody reactivity. Finally, gammabodies that recognize Aß soluble oligomers and fibrils also neutralize the toxicity of each Aß conformer. We expect that our antibody design strategy is not limited to Aß and can be used to readily generate gammabodies against other toxic misfolded proteins.

Polyphenolic glycosides and aglycones utilize opposing pathways to selectively remodel and inactivate toxic oligomers of amyloid ß.

Substantial evidence suggests that soluble prefibrillar oligomers of the Aß42 peptide associated with Alzheimer's disease are the most cytotoxic aggregated Aß isoform. Limited previous work has revealed that aromatic compounds capable of remodeling Aß oligomers into nontoxic conformers typically do so by converting them into off-pathway aggregates instead of dissociating them into monomers. Towards identifying small-molecule antagonists capable of selectively dissociating toxic Aß oligomers into soluble peptide at substoichiometric concentrations, we have investigated the pathways used by polyphenol aglycones and their glycosides to remodel Aß soluble oligomers. We find that eleven polyphenol aglycones of variable size and structure utilize the same remodeling pathway whereby Aß oligomers are rapidly converted into large, off-pathway aggregates. Strikingly, we find that glycosides of these polyphenols all utilize a distinct remodeling pathway in which Aß oligomers are rapidly dissociated into soluble, disaggregated peptide. This disaggregation activity is a synergistic combination of the aglycone and glycone moieties because combinations of polyphenols and sugars fail to disaggregate Aß oligomers. We also find that polyphenolic glycosides and aglycones use the same opposing pathways to remodel Aß fibrils. Importantly, both classes of polyphenols fail to remodel nontoxic Aß oligomers (which are indistinguishable in size and morphology to Aß soluble oligomers) or promote aggregation of freshly disaggregated Aß peptide; thus revealing that they are specific for remodeling toxic Aß conformers. We expect that these and related small molecules will be powerful chemical probes for investigating the conformational and cellular underpinnings of Aß-mediated toxicity.

Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways.

In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aß42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aß. In contrast, a Class II molecule converts soluble Aß oligomers into fibrils, but is inactive against disaggregated and fibrillar Aß. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aß non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aß soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aß soluble oligomers and related aggregated conformers.

Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers.

Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Abeta1-42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Abeta monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their beta-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Abeta-mediated toxicity.