RESUMO
The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.
Assuntos
Amiloide , COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Amiloide/metabolismo , Proteínas Amiloidogênicas , Proteínas do Nucleocapsídeo , Peptídeos/química , Domínios Proteicos , SARS-CoV-2/metabolismoRESUMO
The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.
Assuntos
Atrofia de Múltiplos Sistemas , Doença de Parkinson , Sinucleinopatias , Camundongos , Animais , alfa-Sinucleína/metabolismo , Sinucleinopatias/patologia , Caenorhabditis elegans/metabolismo , Doença de Parkinson/patologia , Atrofia de Múltiplos Sistemas/patologia , Encéfalo/metabolismo , Amiloide/metabolismoRESUMO
Amyloid protein aggregation is commonly associated with progressive neurodegenerative diseases, however not all amyloid fibrils are pathogenic. The neuronal cytoplasmic polyadenylation element binding protein is a regulator of synaptic mRNA translation and has been shown to form functional amyloid aggregates that stabilize long-term memory. In adult Drosophila neurons, the cytoplasmic polyadenylation element binding homolog Orb2 is expressed as 2 isoforms, of which the Orb2B isoform is far more abundant, but the rarer Orb2A isoform is required to initiate Orb2 aggregation. The N terminus is a distinctive feature of the Orb2A isoform and is critical for its aggregation. Intriguingly, replacement of phenylalanine in the fifth position of Orb2A with tyrosine (F5Y) in Drosophila impairs stabilization of long-term memory. The structure of endogenous Orb2B fibers was recently determined by cryo-EM, but the structure adopted by fibrillar Orb2A is less certain. Here we use micro-electron diffraction to determine the structure of the first 9 N-terminal residues of Orb2A, at a resolution of 1.05 Å. We find that this segment (which we term M9I) forms an amyloid-like array of parallel in-register ß-sheets, which interact through side chain interdigitation of aromatic and hydrophobic residues. Our structure provides an explanation for the decreased aggregation observed for the F5Y mutant and offers a hypothesis for how the addition of a single atom (the tyrosyl oxygen) affects long-term memory. We also propose a structural model of Orb2A that integrates our structure of the M9I segment with the published Orb2B cryo-EM structure.
Assuntos
Peptídeos beta-Amiloides , Amiloide , Proteínas de Drosophila , Drosophila melanogaster , Agregados Proteicos , Fatores de Transcrição , Fatores de Poliadenilação e Clivagem de mRNA , Animais , Amiloide/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elétrons , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Neurônios/metabolismo , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Drosophila melanogaster/metabolismoRESUMO
Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-ß (Aß) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aß, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Agregação Patológica de Proteínas/tratamento farmacológico , alfa-Sinucleína/antagonistas & inibidores , Proteínas tau/antagonistas & inibidores , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Amiloidose , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/químicaRESUMO
The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19. ONE SENTENCE SUMMARY: Atomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.
RESUMO
vic -Dibromides containing the α-bromocarbonyl or α-bromoaromatic moieties were reductively debrominated to furnish alkenes in high yield. o- and m-Anisidines but not p-anisidine were found to be effective debrominating agents. The reductive debrominations were found to be trans-stereospecific.
RESUMO
A convenient and efficient method for the synthesis of N1-substituted orotic acid derivatives is reported. The synthetic route utilizes substituted maleimide as synthetic intermediate and takes only four simple steps from readily available starting materials. As a result, orotic acid derivatives with various alkyl and aromatic groups at N1 can be readily synthesized.
RESUMO
The "element effect" in nucleophilic aromatic substitution reactions (SNAr) is characterized by the leaving group order, L = F > NO2 > Cl ≈ Br > I, in activated aryl substrates. A different leaving group order is observed in the substitution reactions of ring-substituted N-methylpyridinium compounds with piperidine in methanol: 2-CN ≥ 4-CN > 2-F â¼ 2-Cl â¼ 2-Br â¼ 2-I. The reactions are second-order in [piperidine], the mechanism involving rate determining hydrogen-bond formation between piperidine and the substrate-piperidine addition intermediate followed by deprotonation of this intermediate. Computational results indicate that deprotonation of the H-bonded complex is probably barrier free, and is accompanied by simultaneous loss of the leaving group (E2) for L = Cl, Br, and I, but with subsequent, rapid loss of the leaving group (E1cB-like) for the poorer leaving groups, CN and F. The approximately 50-fold greater reactivity of the 2- and 4-cyano substrates is attributed to the influence of the electron withdrawing cyano group in the deprotonation step. The results provide another example of ß-elimination reactions poised near the E2-E1cB mechanistic borderline.
Assuntos
Elétrons , Hidrocarbonetos Aromáticos/química , Compostos de Piridínio/química , Ligação de Hidrogênio , Íons , Cinética , Conformação Molecular , Piperidinas/química , PrótonsRESUMO
Employing a genetically modified yeast strain as a screening tool, 4-dimethylaminobenzoic acid (5) was isolated from the marine sediment-derived Streptomyces sp. CP27-53 as a weak yeast sirtuin (Sir2p) inhibitor. Using this compound as a scaffold, a series of disubstituted benzene derivatives were evaluated to elucidate the structure activity relationships for Sir2p inhibition. The results suggested that 4-alkyl or 4-alkylaminobenzoic acid is the key structure motif for Sir2p inhibitory activity. The most potent Sir2p inhibitor, 4-tert-butylbenzoic acid (20), among the tested compounds in this study turned out to be a weak but selective SIRT1 inhibitor. The calculated binding free energies between the selected compounds and the catalytic domain of SIRT1 were well correlated to their measured SIRT1 inhibitory activities.
Assuntos
Benzoatos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/antagonistas & inibidores , Sirtuína 2/antagonistas & inibidores , Streptomyces/química , Benzoatos/química , Benzoatos/isolamento & purificação , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/isolamento & purificação , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
An improved method for the synthesis of N1-substituted orotic acid derivatives is reported. The method involves sequential incorporation of nitrogen atoms to the pyrimidine structure from simple starting materials and thus allows the synthesis of N1-substituted orotic acid derivatives with single 15N label at either N-1 or N-3.