Small-molecule sequestration of amyloid-β as a drug discovery strategy for Alzheimer's disease.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-β (Aβ) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aβ-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

10 pags., 5 figs.The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.This work was supported by the Cambridge Centre for Misfolding Diseases (R.L., B.M., F.S.R., C.K.X., M.P., S.C., S.W.C., J.H., T.K., J.R.K., T.P.J.K., M.V., and C.M.D.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), the Wellcome Trust (203249/Z/16/Z to T.P.J.K and M.V.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana – FAS Salute, project SUPREMAL (R.C., A.B., C.C., and F.C.), the Gates Cambridge Trust and St. John’s College Cambridge (R.L.), Darwin College Cambridge (F.S.R.), the Herchel Smith Fund (C.K.X.), a Faculty Development Research Fund grant from the United States Military Academy, West Point (R.L.) and a DTRA Service Academy Research Initiative grant (HDTRA1033862 to R.L.)

Rational design of a conformation-specific antibody for the quantification of Aβ oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid β (Aβ) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aβ oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine

12 pags, 3 figs. -- The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00466.The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.This work was supported by the Boehringer Ingelheim Fonds (P.F.), the Studienstiftung des Deutschen Volkes (P.F.), Gates Cambridge Scholarships (R.L. and G.T.H) and a St. John’s College Benefactors’ Scholarship (R.L.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), a Senior Research Fellowship award from the Alzheimer’s Society, UK, grant number (317, AS-SF-16-003) (F.A.A.), the Wellcome Trust (C.M.D., M.V., and T.P.J.K.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana—FAS Salute—Supremal project (R.C., C.C., and F.C.), a Marie Skłodowska-Curie Actions—Individual Fellowship (C.G.), Sidney Sussex College Cambridge (G.M.), the Spanish Government—MINECO (N.C.), and by the Cambridge Centre for Misfolding Diseases (M.P., P.F., R.L., F.A.A., C.G., G.T.H., S.W.C., J.R.K., T.P.J.K., M.V., and C.M.D)

Impaired Brain Dopamine and Serotonin Release and Uptake in Wistar Rats Following Treatment with Carbotplatin

Chemotherapy-induced cognitive impairment, known also as “chemobrain”, is a medical complication of cancer treatment that is characterized by a general decline in cognition affecting visual and verbal memory, attention, complex problem solving skills, and motor function. It is estimated that one-third of patients who undergo chemotherapy treatment will experience cognitive impairment. Alterations in the release and uptake of dopamine and serotonin, central nervous system neurotransmitters that play important roles in cognition, could potentially contribute to impaired intellectual performance in those impacted by chemobrain. To investigate how chemotherapy treatment affects these systems, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to measure dopamine and serotonin release and uptake in coronal brain slices containing the striatum and dorsal raphe nucleus, respectively. Measurements were taken from rats treated weekly with selected doses of carboplatin and from control rats treated with saline. Modeling the stimulated dopamine release plots revealed an impairment of dopamine release per stimulus pulse (80% of saline control at 5 mg/kg and 58% at 20 mg/kg) after 4 weeks of carboplatin treatment. Moreover, Vmax, the maximum uptake rate of dopamine, was also decreased (55% of saline control at 5 mg/kg and 57% at 20 mg/kg). Nevertheless, overall dopamine content, measured in striatal brain lysates by high performance liquid chromatography, and reserve pool dopamine, measured by FSCV after pharmacological manipulation, did not significantly change, suggesting that chemotherapy treatment selectively impairs the dopamine release and uptake processes. Similarly, serotonin release upon electrical stimulation was impaired (45% of saline control at 20 mg/kg). Measurements of spatial learning discrimination were taken throughout the treatment period and carboplatin was found to alter cognition. These studies support the need for additional neurochemical and behavioral analyses to identify the underlying mechanisms of chemotherapy-induced cognitive disorders

Rationally Designed Antibodies as Research Tools to Study the Structure–Toxicity Relationship of Amyloid-β Oligomers

Alzheimer’s disease is associated with the aggregation of the amyloid-β peptide (Aβ), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which Aβ aggregates induce neuronal dysfunction has highlighted the importance of the Aβ oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure−toxicity relationship of Aβ oligomers. For this purpose, we use Zn2+-stabilized oligomers of the 40-residue form of Aβ (Aβ40) as models of brain Aβ oligomers and two single-domain antibodies (DesAb18-24 and DesAb34-40), designed to bind to epitopes at residues 18−24 and 34−40 of Aβ40, respectively. We found that the DesAbs induce a change in structure of the Zn2+-stabilized Aβ40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure−toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of Aβ oligomers

Rationally Designed Antibodies as Research Tools to Study the Structure–Toxicity Relationship of Amyloid-β Oligomers

Alzheimer’s disease is associated with the aggregation of the amyloid-β peptide (Aβ), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which Aβ aggregates induce neuronal dysfunction has highlighted the importance of the Aβ oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure−toxicity relationship of Aβ oligomers. For this purpose, we use Zn2+-stabilized oligomers of the 40-residue form of Aβ (Aβ40) as models of brain Aβ oligomers and two single-domain antibodies (DesAb18-24 and DesAb34-40), designed to bind to epitopes at residues 18−24 and 34−40 of Aβ40, respectively. We found that the DesAbs induce a change in structure of the Zn2+-stabilized Aβ40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure−toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of Aβ oligomers

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine.

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders

A rationally designed bicyclic peptide remodels Aβ42 aggregation in vitro and reduces its toxicity in a worm model of Alzheimer’s disease

Funder: Centre for Misfolding DiseasesAbstract: Bicyclic peptides have great therapeutic potential since they can bridge the gap between small molecules and antibodies by combining a low molecular weight of about 2 kDa with an antibody-like binding specificity. Here we apply a recently developed in silico rational design strategy to produce a bicyclic peptide to target the C-terminal region (residues 31–42) of the 42-residue form of the amyloid β peptide (Aβ42), a protein fragment whose aggregation into amyloid plaques is linked with Alzheimer’s disease. We show that this bicyclic peptide is able to remodel the aggregation process of Aβ42 in vitro and to reduce its associated toxicity in vivo in a C. elegans worm model expressing Aβ42. These results provide an initial example of a computational approach to design bicyclic peptides to target specific epitopes on disordered proteins