RESUMO
Alzheimer disease is a neurodegenerative disorder with limited treatment options. It is characterized by the presence of several biomarkers, including amyloid-ß aggregates, which lead to oxidative stress and neuronal decay. Targeted α-therapy (TAT) has been shown to be efficacious against metastatic cancer. TAT takes advantage of tumor-localized α-particle emission to break disease-associated covalent bonds while minimizing radiation dose to healthy tissues due to the short, micrometer-level, distances traveled. We hypothesized that TAT could be used to break covalent bonds within amyloid-ß aggregates and facilitate natural plaque clearance mechanisms. Methods: We synthesized a 213Bi-chelate-linked benzofuran pyridyl derivative (BiBPy) and generated [213Bi]BiBPy, with a specific activity of 120.6 GBq/µg, dissociation constant of 11 ± 1.5 nM, and logP of 0.14 ± 0.03. Results: As the first step toward the validation of [213Bi]BiBPy as a TAT agent for the reduction of Alzheimer disease-associated amyloid-ß, we showed that brain homogenates from APP/PS1 double-transgenic male mice (6-9 mo old) incubated with [213Bi]BiBPy exhibited a marked reduction in amyloid-ß plaque concentration as measured using both enzyme-linked immunosorbent and Western blotting assays, with a half-maximal effective concentration of 3.72 kBq/pg. Conclusion: This [213Bi]BiBPy-concentration-dependent activity shows that TAT can reduce amyloid plaque concentration in vitro and supports the development of targeting systems for in vivo validations.
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Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.
Assuntos
Bacteriocinas , Peptídeos , Conformação Proteica , Peptídeos/química , Bacteriocinas/química , Antibacterianos/químicaRESUMO
Lasso peptides are a structurally distinct class of biologically active natural products defined by their short sequences with impressively interlocked tertiary structures. Their characteristic peptide [1]rotaxane motif confers marked proteolytic and thermal resiliency, and reports on their diverse biological functions have been credited to their exceptional sequence variability. Because of these unique properties, taken together with improved technologies for their biosynthetic production, lasso peptides are emerging as a designable scaffold for peptide-based therapeutic discovery and development. Although the defined structure of lasso peptides is recognized for its remarkable properties, the role of the motif in imparting bioactivity is less understood. For example, sungsanpin and ulleungdin are natural lasso peptides that similarly exhibit encouraging cell migration inhibitory activities in A549 lung carcinoma epithelial cells, despite sharing only one-third of the sequence homology. We hypothesized that the shape of the lasso motif is beneficial for the preorganization of the conserved residues, which might be partially retained in variants lacking the threaded structure. Herein, we describe solid-phase peptide synthesis strategies to prepare acyclic, head-to-side chain (branched), and head-to-tail (macrocyclic) cyclic variants based on the sungsanpin (Sun) and ulleungdin (Uln) sequences. Proliferation assays and time-lapse cell motility imaging studies were used to evaluate the cell inhibitory properties of natural Sun compared with the synthetic Sun and Uln isomers. These studies demonstrate that the lasso motif is not a required feature to slow cancer cell migration and more generally show that these nonthreaded isomers can retain similar activity to the natural lasso peptide despite the differences in their overall structures.
Assuntos
Neoplasias Pulmonares , Peptídeos , Humanos , Peptídeos/farmacologia , Peptídeos/química , Peptídeo Hidrolases , Movimento CelularRESUMO
Ribosomally produced and post-translationally modified polypeptides (RiPPs) are a diverse group of natural products that are processed by a variety of enzymes to their biologically relevant forms. PapB is a member of the radical S-adenosyl-l-methionine (rSAM) superfamily that introduces thioether cross-links between Cys and Asp residues in the PapA RiPP. We report that PapB has high tolerance for variations in the peptide substrate. Our results demonstrate that branched side chains in the thiol- and carboxylate-containing residues are processed and that lengthening of these groups to homocysteine and homoglutamate does not impair the ability of PapB to form thioether cross-links. Remarkably, the enzyme can even cross-link a peptide substrate where the native Asp carboxylate moiety is replaced with a tetrazole. We show that variations to residues embedded between the thiol- and carboxylate-containing residues are tolerated by PapB, as peptides containing both bulky (e.g., Phe) and charged (e.g., Lys) side chains in both natural L- and unnatural D-forms are efficiently cross-linked. Diastereomeric peptides bearing (2S,3R)- and (2S,3S)-methylaspartate are processed by PapB to form cyclic thioethers with markedly different rates, suggesting the enzymatic hydrogen atom abstraction event for the native Asp-containing substrate is diastereospecific. Finally, we synthesized two diastereomeric peptide substrates bearing E- and Z-configured γ,δ-dehydrohomoglutamate and show that PapB promotes addition of the deoxyadenosyl radical (dAdoâ¢) instead of hydrogen atom abstraction. In the Z-configured γ,δ-dehydrohomoglutamate substrate, a fraction of the dAdo-adduct peptide is thioether cross-linked. In both cases, there is evidence for product inhibition of PapB, as the dAdo-adducts likely mimic the native transition state where dAdo⢠is poised to abstract a substrate hydrogen atom. Collectively, these findings provide critical insights into the arrangement of reacting species in the active site of the PapB, reveal unusual promiscuity, and highlight the potential of PapB as a tool in the development peptide therapeutics.
RESUMO
Inspired by nature's wide range of oxidation-induced modifications to install cross-links and cycles at tyrosine (Tyr) and other phenol-containing residue side chains, we report a Tyr-selective strategy for the preparation of Tyr-linked cyclic peptides. This approach leverages N4-substituted 1,2,4-triazoline-3,5-diones (TADs) as azo electrophiles that react chemoselectively with the phenolic side chain of Tyr residues to form stable C-N1-linked cyclic peptides. In the developed method, a precursor 1,2,4-triazolidine-3,5-dione moiety, also known as urazole, is readily constructed at any free amine revealed on a solid-supported peptide. Once prepared, the N4-substituted urazole peptide is selectively oxidized using mild, peptide-compatible conditions to generate an electrophilic N4-substituted TAD peptide intermediate that reacts selectively under aqueous conditions with internal and terminal Tyr residues to furnish Tyr-linked cyclic peptides. The approach demonstrates good tolerance of native residue side chains and enables access to cyclic peptides ranging from 3- to 11-residues in size (16- to 38-atom-containing cycles). The identity of the installed Tyr-linkage, a stable covalent C-N1 bond, was characterized using NMR spectroscopy. Finally, we applied the developed method to prepare biologically active Tyr-linked cyclic peptides bearing the integrin-binding RGDf epitope.
Assuntos
Peptídeos , Tirosina , Tirosina/química , Peptídeos/química , Peptídeos Cíclicos , Ligação ProteicaRESUMO
Progress toward the design and synthesis of ambiphilic aryl thiol-iminium-based small molecules for organocatalyzed thioacyl aminolysis is reported. Here we describe the synthesis of a novel tetrahydroisoquinoline-derived scaffold, bearing both thiol and iminium functionalities, capable of promoting the transthioesterification and subsequent amine capture reactions necessary to achieve organocatalyzed thioacyl aminolysis. Model studies demonstrate the ability of this designed organocatalyst to deliver critical intermediates capable of undergoing these individual reactions necessary for the proposed process. Future design improvements and directions toward cysteine-independent organocatalyzed native chemical ligation are discussed.
RESUMO
PURPOSE: Alzheimer's disease (AD) is a terminal neurodegenerative disease characterized by the buildup of amyloid fibrils, amorphous aggregates and tauopathies. Several treatment modalities, which rely on various biological processes to reduce disease burden, have been largely ineffective at treating Alzheimer's disease. Targeted alpha therapy (TAT) has demonstrated positive results in the treatment of cancer. Benzothiazole derivatives have been successfully shown to target these plaques and are used in several imaging applications. One such derivative, Flutemetamol (VizamylTM) is an FDA approved diagnostic tool for PET imaging of AD-associated plaques. We report the radiolabeling of benzothiazole derivatives with 211At, a 7.2 h alpha emitting radionuclide, using a copper catalyzed reaction with a boronic acid precursor molecule. Our final compound [211At]3'-At-PIB-OMe had a radiochemical yield of 55% and was found to be stable for at least 3 h in phosphate buffered saline.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Humanos , Placa Amiloide/diagnóstico por imagem , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/radioterapia , Benzotiazóis/uso terapêuticoRESUMO
Ribosomally synthesized post-translationally modified peptides (RiPPs) are ubiquitous and represent a structurally diverse class of natural products. The ribosomally encoded precursor polypeptides are often extensively modified post-translationally by enzymes that are encoded by coclustered genes. Radical S-adenosyl-l-methionine (SAM) enzymes catalyze numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of C-H, C-C, C-S, and C-O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein, SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for formation of oxazole-containing RiPP natural products.
Assuntos
Peptídeos Antimicrobianos/metabolismo , Geobacter/metabolismo , S-Adenosilmetionina/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/fisiologia , Peptídeos Antimicrobianos/fisiologia , Produtos Biológicos/metabolismo , Catálise , Geobacter/patogenicidade , Espectrometria de Massas/métodos , Oxazóis , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Ribossomos , S-Adenosilmetionina/metabolismoRESUMO
The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered "undruggable" due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all-l- and all-d-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-d peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target.
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Proteínas Proto-Oncogênicas p21(ras)/síntese química , Sequência de Aminoácidos , Variação Genética , Humanos , Dobramento de Proteína , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation-desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Peptídeos/química , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência MolecularRESUMO
Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.
Assuntos
Ferroquelatase/genética , Genes Recessivos , Ceratodermia Palmar e Plantar/complicações , Ceratodermia Palmar e Plantar/genética , Protoporfiria Eritropoética/complicações , Protoporfiria Eritropoética/genética , Adolescente , Adulto , Criança , Feminino , Ferroquelatase/fisiologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Estações do AnoRESUMO
Haem controls its own synthesis in non-erythroid cells primarily by regulation of ALAS1 mRNA stability. Alternative splicing of human ALAS1 generates two mRNAs with different 5'-UTRs: a major one, where exon 1B is omitted, and a minor form containing exon 1B. We show that, unlike the major ALAS1 mRNA, the minor form was resistant to haem-mediated decay. Furthermore, we demonstrate that the ALAS1 5'-UTR alone did not confer haem-mediated decay upon a heterologous mRNA and the inclusion of exon 1B inhibited translation. These data suggest that translation of ALAS1 mRNA itself might be required for destabilisation in response to haem.
Assuntos
Regiões 5' não Traduzidas/genética , 5-Aminolevulinato Sintetase/genética , Processamento Alternativo/genética , Éxons/genética , Heme/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , 5-Aminolevulinato Sintetase/biossíntese , Linhagem Celular Tumoral , Dactinomicina/farmacologia , Humanos , RNA Mensageiro/biossínteseRESUMO
PGHS-2 (prostaglandin H synthase-2) is induced in mammalian cells by pro-inflammatory cytokines in tandem with iNOS [high-output ('inducible') nitric oxide synthase], and is co-localized with iNOS and nitrotyrosine in human atheroma macrophages. Herein, murine J774.2 macrophages incubated with lipopolysaccharide and interferon gamma showed induction of PGHS-2 and generated NO using iNOS that could be completely depleted by 12(S)-HPETE [12(S)-hydroperoxyeicosatetraenoic acid; 2.4 muM] or hydrogen peroxide (500 microM) (0.42+/-0.084 and 0.38+/-0.02 nmol x min(-1) x 10(6) cells(-1) for HPETE and H2O2 respectively). COS-7 cells transiently transfected with human PGHS-2 also showed HPETE- or H2O2-dependent NO decay (0.44+/-0.016 and 0.20+/-0.04 nmol x min(-1) x 10(6) cells(-1) for 2.4 microM HPETE and 500 microM H2O2 respectively). Finally, purified PGHS-2 consumed NO in the presence of HPETE or H2O2 (168 and 140 microM x min(-1) x microM enzyme(-1) for HPETE and H2O2 respectively), in a haem-dependent manner, with 20 nM enzyme consuming up to 4 microM NO. K(m) (app) values for NO and 15(S)-HPETE were 1.7+/-0.2 and 0.45+/-0.16 microM respectively. These data indicate that PGHS-2 catalytically consumes NO during peroxidase turnover and that pro-inflammatory cytokines simultaneously upregulate NO synthesis and degradation pathways in murine macrophages. Catalytic NO consumption by PGHS-2 represents a novel interaction between NO and PGHS-2 that may impact on the biological effects of NO in vascular signalling and inflammation.