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Our laboratory reported the chemical synthesis and stereochemical assignment of the recently discovered peptide antibiotic clovibactin. The current paper reports an improved, gram-scale synthesis of the amino acid building block Fmoc-(2R,3R)-3-hydroxyasparagine-OH that enables structure-activity relationship studies of clovibactin. An alanine scan reveals that residues Phe1, d-Leu2, Ser4, Leu7, and Leu8 are important for antibiotic activity. The side-chain amide group of the rare d-Hyn5 residue is not essential to activity and can be replaced with a methyl group with a moderate loss of activity. An acyclic clovibactin analogue reveals that the macrolactone ring is essential to antibiotic activity. The enantiomer of clovibactin is active, albeit somewhat less so than clovibactin. A conformationally constrained clovibactin analogue retains moderate antibiotic activity, while a backbone N-methylated analogue is almost completely inactive. X-ray crystallography of these two analogues reveals that the macrolactone ring adopts a crown-like conformation that binds anions.
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Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aß) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aß assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aß. These antibodies are the first mAbs generated against Aß oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aß42, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aß enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.
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This Note presents the X-ray crystallographic structure of the N-methylated teixobactin analogue N-Me-d-Gln4,Lys10-teixobactin (1). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel ß-sheet dimers in the crystal lattice, with residues 1-3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice.
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Antibodies that target the ß-amyloid peptide (Aß) and its associated assemblies are important tools in Alzheimer's disease research and have emerged as promising Alzheimer's disease therapies. This paper reports the creation and characterization of a triangular Aß trimer mimic composed of Aß17-36 ß-hairpins and the generation and study of polyclonal antibodies raised against the Aß trimer mimic. The Aß trimer mimic is covalently stabilized by three disulfide bonds at the corners of the triangular trimer to create a homogeneous oligomer. Structural, biophysical, and cell-based studies demonstrate that the Aß trimer mimic shares characteristics with oligomers of full-length Aß. X-ray crystallography elucidates the structure of the trimer and reveals that four copies of the trimer assemble to form a dodecamer. SDS-PAGE, size exclusion chromatography, and dynamic light scattering reveal that the trimer also forms higher-order assemblies in solution. Cell-based toxicity assays show that the trimer elicits LDH release, decreases ATP levels, and activates caspase-3/7 mediated apoptosis. Immunostaining studies on brain slices from people who lived with Alzheimer's disease and people who lived with Down syndrome reveal that the polyclonal antibodies raised against the Aß trimer mimic recognize pathological features including different types of Aß plaques and cerebral amyloid angiopathy.
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Amyloid-ß (Aß) forms heterogeneous oligomers, which are implicated in the pathogenesis of Alzheimer's disease (AD). Many Aß oligomers consist of ß-hairpin building blocksâAß peptides in ß-hairpin conformations. ß-Hairpins of Aß can adopt a variety of alignments, but the role that ß-hairpin alignment plays in the formation and heterogeneity of Aß oligomers is poorly understood. To explore the effect of ß-hairpin alignment on the oligomerization of Aß peptides, we designed and studied two model peptides with two different ß-hairpin alignments. Peptides Aßm17-36 and Aßm17-35 mimic two different ß-hairpins that Aß can form, the Aß17-36 and Aß17-35 ß-hairpins, respectively. These hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues that they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. In the crystal state, Aßm17-36 forms triangular trimers that further assemble to form hexamers, while Aßm17-35 forms tetrameric ß-barrels. In SDS-PAGE, Aßm17-36 assembles to form a ladder of oligomers, while Aßm17-35 either assembles to form a dimer or does not assemble at all. The differences in the behavior of Aßm17-36 and Aßm17-35 suggest ß-hairpin alignment as a source of the observed heterogeneity of Aß oligomers.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Modelos Moleculares , Conformação Proteica , Cristalografia por Raios X , Fragmentos de Peptídeos/químicaRESUMO
ß-Hairpins formed by the ß-amyloid peptide Aß are building blocks of Aß oligomers. Three different alignments of ß-hairpins have been observed in the structures of Aß oligomers or fibrils. Differences in ß-hairpin alignment likely contribute to the heterogeneity of Aß oligomers and thus impede their study at high-resolution. Here, we designed, synthesized, and studied a series of ß-hairpin peptides derived from Aß12-40 in one of these three alignments and investigated their solution-phase assembly and folding. These assays reveal the formation of tetramers and octamers that are stabilized by intermolecular hydrogen bonding interactions between Aß residues 12-14 and 38-40 as part of an extended ß-hairpin conformation. X-ray crystallographic studies of one peptide from this series reveal the formation of ß-barrel-like tetramers and octamers that are stabilized by edge-to-edge hydrogen bonding and hydrophobic packing. Dye-leakage and caspase 3/7 activation assays using tetramer and octamer forming peptides from this series reveal membrane-damaging and apoptotic properties. A molecular dynamics simulation of the ß-barrel-like tetramer embedded in a lipid bilayer shows membrane disruption and water permeation. The tetramers and octamers described herein provide additional models of how Aß may assemble into oligomers and supports the hypothesis that ß-hairpin alignment and topology may contribute directly to oligomer heterogeneity.
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The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/metabolismo , Conformação Proteica , Cristalografia por Raios X , Membrana Celular/metabolismo , Fragmentos de Peptídeos/químicaRESUMO
This paper describes the synthesis and stereochemical determination of Novo29 (clovibactin), a new peptide antibiotic that is related to teixobactin and is active against Gram-positive bacteria. Novo29 is an eight-residue depsipeptide that contains the noncanonical amino acid hydroxyasparagine of hitherto undetermined stereochemistry in a macrolactone ring. The amino acid building blocks Fmoc-(2R,3R)-hydroxyasparagine-OH and Fmoc-(2R,3S)-hydroxyasparagine-OH were synthesized from (R,R)- and (S,S)-diethyl tartrate. Novo29 and epi-Novo29 were then prepared by solid-phase peptide synthesis using these building blocks. Correlation with an authentic sample of Novo29 through 1H NMR spectroscopy, LC-MS, and in vitro antibiotic activity established that Novo29 contains (2R,3R)-hydroxyasparagine. X-ray crystallography reveals that epi-Novo29 adopts an amphiphilic conformation, with a hydrophobic surface and a hydrophilic surface. Four sets of epi-Novo29 molecules pack in the crystal lattice to form a hydrophobic core. The macrolactone ring adopts a conformation in which the main-chain amide NH groups converge to create a cavity, which binds ordered water and acetate anion. The amphiphilic conformation of epi-Novo29 is reminiscent of the amphiphilic conformation adopted by the related antibiotic teixobactin and its derivatives, which contains a hydrophobic surface that interacts with the lipids of the bacterial cell membrane and a hydrophilic surface that interacts with the aqueous environment. Molecular modeling suggests that Novo29 can adopt an amphiphilic conformation similar to teixobactin, suggesting that Novo29 may interact with bacteria in a similar fashion to teixobactin.
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Aminoácidos , Antibacterianos , Antibacterianos/farmacologia , Antibacterianos/química , Modelos Moleculares , Conformação Molecular , Aminoácidos/química , Espectroscopia de Ressonância MagnéticaRESUMO
Familial Alzheimer's disease (FAD) is associated with mutations in the ß-amyloid peptide (Aß) or the amyloid precursor protein (APP). FAD mutations of Aß were incorporated into a macrocyclic peptide that mimics a ß-hairpin to study FAD point mutations K16N, A21G, E22Δ, E22G, E22Q, E22K, and L34V and their effect on assembly, membrane destabilization, and cytotoxicity. The X-ray crystallographic structures of the four E22 mutant peptides reveal that the peptides assemble to form the same compact hexamer. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments reveal that the mutant FAD peptides assemble as trimers or hexamers, with peptides that have greater positive charge assembling as more stable hexamers. Mutations that increase the positive charge also increase the cytotoxicity of the peptides and their propensity to destabilize lipid membranes.
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Doença de Alzheimer , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Mutação , Fragmentos de Peptídeos/químicaRESUMO
Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Dissulfetos/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Dicroísmo Circular/métodos , Dissulfetos/química , Humanos , Ligação de Hidrogênio , Microscopia Eletrônica de Transmissão/métodos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Conformação Proteica em Folha betaRESUMO
The assembly of amyloidogenic peptides and proteins, such as the ß-amyloid peptide, α-synuclein, huntingtin, tau, and islet amyloid polypeptide, into amyloid fibrils and oligomers is directly linked to amyloid diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, frontotemporal dementias, and type II diabetes. Although amyloid oligomers have emerged as especially important in amyloid diseases, high-resolution structures of the oligomers formed by full-length amyloidogenic peptides and proteins have remained elusive. Investigations of oligomers assembled from fragments or stabilized ß-hairpin segments of amyloidogenic peptides and proteins have allowed investigators to illuminate some of the structural, biophysical, and biological properties of amyloid oligomers. Here, we summarize recent advances in the application of these peptide model systems to investigate and understand the structures, biological properties, and biophysical properties of amyloid oligomers.
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Amiloide , Diabetes Mellitus Tipo 2 , Amiloide/química , Peptídeos beta-Amiloides/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/químicaRESUMO
Chemically constrained peptides that self-assemble can be used to better understand the molecular basis of amyloid diseases. The formation of small assemblies of the amyloidogenic peptides and proteins, termed oligomers, is central to amyloid diseases. The use of chemical model systems can help provide insights into the structures and interactions of amyloid oligomers, which are otherwise difficult to study. This chapter describes the use of macrocyclic ß-hairpin peptides as model systems to study amyloid oligomers. The first part of the chapter describes the chemical synthesis of the macrocyclic ß-hairpin peptides and covalent assemblies thereof. The second part of the chapter describes the characterization of the oligomers formed by the macrocyclic ß-hairpin peptides, focusing on SDS-PAGE, size-exclusion chromatography (SEC), and X-ray crystallography. The procedures provided focus on the ß-amyloid peptide, but these strategies are applicable to a broad range of amyloid-derived peptides and proteins.
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Peptídeos beta-Amiloides , Amiloide , Cristalografia por Raios X , Modelos MolecularesRESUMO
This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. The cyclic peptide contains a [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) linker that is designed to enforce a conformation that mimics a peptide substrate of Mpro. In vitro evaluation of the cyclic peptide inhibitor reveals that the inhibitor exhibits modest activity against Mpro and does not appear to be cleaved by the enzyme. Conformational searching predicts that the cyclic peptide inhibitor is fairly rigid, adopting a favorable conformation for binding to the active site of Mpro. Computational docking to the SARS-CoV-2 Mpro suggests that the cyclic peptide inhibitor can bind the active site of Mpro in the predicted manner. Molecular dynamics simulations provide further insights into how the cyclic peptide inhibitor may bind the active site of Mpro. Although the activity of the cyclic peptide inhibitor is modest, its design and study lays the groundwork for the development of additional cyclic peptide inhibitors against Mpro with improved activities.
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Proteases 3C de Coronavírus/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Inibidores de Proteases/farmacologia , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Desenho de Fármacos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/toxicidade , Conformação ProteicaRESUMO
Fluorescent derivatives of the ß-amyloid peptides (Aß) are valuable tools for studying the interactions of Aß with cells. Facile access to labeled expressed Aß offers the promise of Aß with greater sequence and stereochemical integrity, without impurities from amino acid deletion and epimerization. Here, we report methods for the expression of Aß42 with an N-terminal cysteine residue, Aß(C1-42), and its conjugation to generate Aß42 bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aß(MC1-42) gene yields the Aß(C1-42) peptide, because the N-terminal methionine is endogenously excised by Escherichia coli. Conjugation of Aß(C1-42) with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aß42. The expression affords â¼14 mg of N-terminal cysteine Aß from 1 L of bacterial culture. Subsequent conjugation affords â¼3 mg of labeled Aß from 1 L of bacterial culture with minimal cost for labeling reagents. High-performance liquid chromatography analysis indicates the N-terminal cysteine Aß to be >97% pure and labeled Aß peptides to be 94-97% pure. Biophysical studies show that the labeled Aß peptides behave like unlabeled Aß and suggest that labeling of the N-terminus does not substantially alter the properties of the Aß. We further demonstrate applications of the fluorophore-labeled Aß peptides by using fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. We anticipate that these methods will provide researchers convenient access to useful N-terminally labeled Aß, as well as Aß with an N-terminal cysteine that enables further functionalization.
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Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Cisteína/química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/genética , Biotinilação , Expressão Gênica , Humanos , Fragmentos de Peptídeos/genéticaRESUMO
This paper describes the structure-based design of a preliminary drug candidate against COVID-19 using free software and publicly available X-ray crystallographic structures. The goal of this tutorial is to disseminate skills in structure-based drug design and to allow others to unleash their own creativity to design new drugs to fight the current pandemic. The tutorial begins with the X-ray crystallographic structure of the main protease (Mpro) of the SARS coronavirus (SARS-CoV) bound to a peptide substrate and then uses the UCSF Chimera software to modify the substrate to create a cyclic peptide inhibitor within the Mpro active site. Finally, the tutorial uses the molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV Mpro and the highly homologous SARS-CoV-2 Mpro. The supporting information provides an illustrated step-by-step protocol, as well as a video showing the inhibitor design process, to help readers design their own drug candidates for COVID-19 and the coronaviruses that will cause future pandemics. An accompanying preprint in bioRxiv [https://doi.org/10.1101/2020.08.03.234872] describes the synthesis of the cyclic peptide and the experimental validation as an inhibitor of SARS-CoV-2 Mpro.
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Antivirais/química , Proteases 3C de Coronavírus , Desenho de Fármacos , Descoberta de Drogas , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Sítios de Ligação , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Humanos , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , Software , Tratamento Farmacológico da COVID-19RESUMO
Oligomers of the ß-amyloid peptide, Aß, play a central role in the pathogenesis and progression of Alzheimer's disease. Trimers and higher-order oligomers composed of trimers are thought to be the most neurotoxic Aß oligomers. To gain insights into the structure and assembly of Aß oligomers, our laboratory has previously designed and synthesized macrocyclic peptides derived from Aß17-23 and Aß30-36 that fold to form ß-hairpins and assemble to form trimers. In this study, we found that mutating Phe20 to cyclohexylalanine (Cha) in macrocyclic Aß-derived peptides promotes crystallization of an Aß-derived peptide containing the Aß24-29 loop (peptide 3F20Cha) and permits elucidation of its structure and assembly by X-ray crystallography. X-ray crystallography shows that peptide 3F20Cha forms a hexamer. X-ray crystallography and SDS-PAGE further show that trimer 4F20Cha, a covalently stabilized trimer derived from peptide 3F20Cha, forms a dodecamer. Size exclusion chromatography shows that trimer 4F20Cha forms higher-order assemblies in solution. Trimer 4F20Cha exhibits cytotoxicity against the neuroblastoma cell line SH-SY5Y. These studies demonstrate the use of the F20Cha mutation to further stabilize oligomers of Aß-derived peptides that contain more of the native sequence and thus better mimic the oligomers formed by full-length Aß.
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Peptídeos beta-Amiloides/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Mutação , Conformação Proteica em Folha beta , Multimerização ProteicaRESUMO
This paper describes the structure-based design of a preliminary drug candidate against COVID-19 using free software and publicly available X-ray crystallographic structures. The goal of this tutorial is to disseminate skills in structure-based drug design and to allow others to unleash their own creativity to design new drugs to fight the current pandemic. The tutorial begins with the X-ray crystallographic structure of the main protease (Mpro) of the SARS coronavirus (SARS-CoV) bound to a peptide substrate and then uses the UCSF Chimera software to modify the substrate to create a cyclic peptide inhibitor within the Mpro active site. Finally, the tutorial uses the molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV Mpro and the highly homologous SARS-CoV-2 Mpro. The supporting information (supplementary material) provides an illustrated step-by-step guide for the inhibitor design, to help readers design their own drug candidates for COVID-19 and the coronaviruses that will cause future pandemics. An accompanying preprint in bioRxiv [https://doi.org/10.1101/2020.08.03.234872] describes the synthesis of the cyclic peptide and the experimental validation as an inhibitor of SARS-CoV-2 Mpro.
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High-resolution structures of oligomers formed by the ß-amyloid peptide, Aß, are important for understanding the molecular basis of Alzheimer's disease. Dimers of Aß are linked to the pathogenesis and progression of Alzheimer's disease, and tetramers of Aß are neurotoxic. This paper reports the X-ray crystallographic structures of dimers and tetramers, as well as an octamer, formed by a peptide derived from the central and C-terminal regions of Aß. In the crystal lattice, the peptide assembles to form two different dimers-an antiparallel ß-sheet dimer and a parallel ß-sheet dimer-that each further self-assemble to form two different tetramers-a sandwich-like tetramer and a twisted ß-sheet tetramer. The structures of these dimers and tetramers derived from Aß serve as potential models for dimers and tetramers of full-length Aß that form in vitro and in Alzheimer's disease-afflicted brains.