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1.
Nucleic Acids Res ; 51(17): 9356-9368, 2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37486777

RESUMO

RIG-I (retinoic acid inducible gene-I) can sense subtle differences between endogenous and viral RNA in the cytoplasm, triggering an anti-viral immune response through induction of type I interferons (IFN) and other inflammatory mediators. Multiple crystal and cryo-EM structures of RIG-I suggested a mechanism in which the C-terminal domain (CTD) is responsible for the recognition of viral RNA with a 5'-triphoshate modification, while the CARD domains serve as a trigger for downstream signaling, leading to the induction of type I IFN. However, to date contradicting conclusions have been reached around the role of ATP in the mechanism of the CARD domains ejection from RIG-I's autoinhibited state. Here we present an application of NMR spectroscopy to investigate changes induced by the binding of 5'-triphosphate and 5'-OH dsRNA, both in the presence and absence of nucleotides, to full length RIG-I with all its methionine residues selectively labeled (Met-[ϵ-13CH3]). With this approach we were able to identify residues on the CTD, helicase domain, and CARDs that served as probes to sense RNA-induced conformational changes in those respective regions. Our results were analyzed in the context of either agonistic or antagonistic RNAs, by and large supporting a mechanism proposed by the Pyle Lab in which CARD release is primarily dependent on the RNA binding event.


Assuntos
Transativadores , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Interferon Tipo I/genética , Estrutura Terciária de Proteína , RNA de Cadeia Dupla , RNA Viral/genética , RNA Viral/metabolismo , Transdução de Sinais , Transativadores/metabolismo
2.
J Org Chem ; 85(14): 9424-9433, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32614587

RESUMO

Herein we report a Cu-catalyzed, site-selective functionalization of peptides that employs an aspartic acid (Asp) as a native directing motif, which directs the site of O-arylation at a proximal tyrosine (Tyr) residue. Through a series of competition studies conducted in high-throughput reaction arrays, effective conditions were identified that gave high selectivity for the proximal Tyr in Asp-directed Tyr modification. Good levels of site-selectivity were achieved in the O-arylation at a proximal Tyr residue in a number of cases, including a peptide-small molecule hybrid.


Assuntos
Ácido Aspártico , Tirosina , Sequência de Aminoácidos , Peptídeos
3.
Org Biomol Chem ; 18(10): 1881-1885, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32100807

RESUMO

A convenient two-step method is reported for the ligation of alkoxyamine- or hydrazine-bearing cargo to proline N-termini. Using this approach, bifunctional proline N-terminal bioconjugates are constructed and proline N-terminal proteins are immobilized.


Assuntos
Aminas/química , Hidrazinas/química , Prolina/química , Proteínas/síntese química , Hidrazonas/síntese química , Cetonas/síntese química , Oxirredução , Oximas/síntese química , Pyrococcus furiosus/química , Vírus do Mosaico do Tabaco/química
4.
J Am Chem Soc ; 141(9): 3885-3892, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30726077

RESUMO

A convenient enzymatic strategy is reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus (abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.


Assuntos
Monofenol Mono-Oxigenase/metabolismo , Fenóis/metabolismo , Prolina/metabolismo , Quinonas/metabolismo , Agaricus/enzimologia , Modelos Moleculares , Estrutura Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/isolamento & purificação , Fenóis/química , Prolina/química , Quinonas/química
5.
Bioconjug Chem ; 30(4): 1127-1132, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30946565

RESUMO

A synthetic method to access novel azido-insulin analogs directly from recombinant human insulin (RHI) was developed via diazo-transfer chemistry using imidazole-1-sulfonyl azide. Systematic optimization of reaction conditions led to site-selective azidation of amino acids B1-phenylalanine and B29-lysine present in RHI. Subsequently, the azido-insulin analogs were used in azide-alkyne [3 + 2] cycloaddition reactions to synthesize a diverse array of triazole-based RHI bioconjugates that were found to be potent human insulin receptor binders. The utility of this method was further demonstrated by the concise and controlled synthesis of a heterotrisubstituted insulin conjugate.


Assuntos
Azidas/síntese química , Insulina/química , Sequência de Aminoácidos , Aminoácidos/química , Azidas/química , Reação de Cicloadição , Humanos , Proteínas Recombinantes/química , Triazóis/química
6.
Bioorg Med Chem Lett ; 26(18): 4513-4517, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27503684

RESUMO

Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.


Assuntos
Inativação Gênica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , beta Catenina/genética , Células HEK293 , Humanos
7.
Bioconjug Chem ; 25(2): 296-307, 2014 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-24409989

RESUMO

Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.


Assuntos
Concentração de Íons de Hidrogênio , Polímeros/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Animais , Polímeros/química , Polímeros/farmacocinética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , Ratos
8.
J Nat Prod ; 77(6): 1280-6, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24933689

RESUMO

The Candida albicans fitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the DRE2 heterozygote and hyposensitivity of the DIP5 heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin.


Assuntos
Antifúngicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Pirróis/isolamento & purificação , Streptomyces/química , Antifúngicos/química , Antifúngicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Candida albicans/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Ferro-Enxofre/genética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirróis/química , Pirróis/farmacologia
9.
Viruses ; 16(7)2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39066320

RESUMO

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).


Assuntos
Antivirais , SARS-CoV-2 , Animais , Camundongos , SARS-CoV-2/efeitos dos fármacos , Humanos , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Tratamento Farmacológico da COVID-19 , Inibidores de Proteases/farmacologia , COVID-19/virologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia
10.
J Med Chem ; 67(5): 3935-3958, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38365209

RESUMO

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.


Assuntos
COVID-19 , Glutamina , Humanos , Glutamina/química , SARS-CoV-2 , Cisteína Endopeptidases/química , Invenções , Inibidores de Proteases/farmacologia , Amidas , Antivirais/farmacologia , Antivirais/química
11.
Antimicrob Agents Chemother ; 56(9): 4662-70, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22710113

RESUMO

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Bifenilo/farmacologia , Depsipeptídeos/farmacologia , Glicopeptídeos/farmacologia , Glicosídeos/farmacologia , Lipopeptídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oligopeptídeos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , beta-Lactamas/farmacologia , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Compostos de Bifenilo/síntese química , Depsipeptídeos/isolamento & purificação , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/isolamento & purificação , Glicosídeos/isolamento & purificação , Humanos , Lipopeptídeos/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Família Multigênica , Oligopeptídeos/síntese química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Infecções Estafilocócicas/microbiologia , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
12.
Chem Biol ; 15(4): 363-74, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18420143

RESUMO

Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.


Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Alelos , Sequência de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/isolamento & purificação , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/metabolismo , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Candida albicans/metabolismo , Candidíase/tratamento farmacológico , Candidíase/metabolismo , Misturas Complexas/farmacologia , Desoxiadenosinas/metabolismo , Desoxiadenosinas/farmacologia , Farmacorresistência Fúngica , Fermentação , Heterozigoto , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Poliadenilação/efeitos dos fármacos , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Resultado do Tratamento
13.
Bioorg Med Chem Lett ; 19(4): 1224-7, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19147347

RESUMO

Parnafungins, natural products containing an isoxazolidinone ring, have been isolated from Fusarium larvarum and have been shown to be potent inhibitors of the fungal polyadenosine polymerase. The extraction and analysis of fermentation broths of taxonomically related organisms identified as closely related Fusarium spp. produce not only parnafungin A and B, but also significant quantities of two related components. These members of the paranfungin family of natural products have been isolated and the structure of each has been elucidated. While structurally analogous to parnafungin A, parnafungin C is further elaborated by methylation of a phenolic hydroxyl group, and parnafungin D has both the methyl phenol ether as well as an epoxide in the xanthone ring system. Parnafungin C and D have potent, broad spectrum antifungal activity and also have been shown to target fungal mRNA cleavage and polyadenylation.


Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Fusarium/química , Oxazolidinonas/isolamento & purificação , Oxazolidinonas/farmacologia , Antifúngicos/química , Produtos Biológicos/química , Candida albicans/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oxazolidinonas/química
14.
J Nat Prod ; 72(1): 59-62, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19102658

RESUMO

Antisense-based screening strategies can be used to sensitize a microorganism and selectively detect inhibitors against a particular cellular target of interest. A strain of Staphylococcus aureus that generates an antisense RNA against SecA,a central member of the protein secretion machinery, has been used to screen for novel antibacterials. Possible inhibitors of the SecA ATP-ase were selected with a high-throughput, two-plate agar-based whole cell differential sensitivity screen. After screening a library of over 115 000 natural products extracts with the SecA antisense strain, an extract of Geomyces pannorum was identified as providing increased activity against the sensitized strain as compared with the wild-type control. Bioassay-guided isolation of the active component from this fungal extract provided a new cis-decalin secondary metabolite, which we have named pannomycin.


Assuntos
Antibacterianos/isolamento & purificação , Ascomicetos/química , Naftalenos/isolamento & purificação , RNA Antissenso/genética , Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Estrutura Molecular , Naftalenos/química , Naftalenos/farmacologia , RNA Antissenso/metabolismo , Canais de Translocação SEC , Proteínas SecA , Staphylococcus aureus/efeitos dos fármacos , Estereoisomerismo
15.
Mycologia ; 101(4): 449-72, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19623926

RESUMO

Evaluation of fungal fermentation extracts with whole cell Candida albicans activity resulted in the identification of a novel class of isoxazolidinone-containing metabolites named parnafungins. Chemical-genetic profiling with the C. albicans fitness test identified the biochemical target as inhibition of polyadenosine polymerase, a component of the mRNA cleavage and polyadenylation complex. Parnafungins were discovered from fermentation extracts of fungi resembling F. larvarum isolated from plants, plant litter and lichens. Furthermore authentic strains of F. larvarum var. larvarum and F. larvarum var. rubrum could be induced to produce parnafungins and their degradation products in low titers. Relationships among strains of the F. larvarum complex (FLC), including parnafungin-producing strains, were examined by cladistic analyses of rDNA, mitochondrial rDNA, and two protein-coding genes, comparisons of antifungal activity and antifungal metabolite profiles, and morphological phenotypes. Integrated analyses of these data led to the conclusion that the diversity within the FLC exceeded the one-to-one correspondence between F. larvarum and its teleomorph Cosmospora aurantiicola. Based on multiple gene sequence analyses, strains of the FLC formed a monophyletic clade inclusive of the parnafungin-producing strains. The FLC, including newly discovered parnafungin-producing strains, could be resolved into at least six different lineages, possibly representing cryptic' species, of which one was not fully resolved from F. larvarum var. rubrum. Fusarium larvarum var. rubrum represents a species distinct from var. larvarum. Finally we report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms.


Assuntos
Fusarium/classificação , Fusarium/metabolismo , Oxazolidinonas/metabolismo , Poliadenilação , RNA Mensageiro/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , DNA Ribossômico/genética , Fusarium/genética , Genes Fúngicos , Variação Genética , Espectrometria de Massas , Filogenia , Análise de Sequência de DNA , Esporos Fúngicos/citologia
16.
J Am Chem Soc ; 130(22): 7060-6, 2008 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-18461935

RESUMO

The Candida albicans Fitness Test, a whole-cell screening platform, was used to profile crude fermentation extracts for novel antifungal natural products with interesting mechanisms of action. An extract with intrinsic antifungal activity from the fungus Fusarium larvarum displayed a Fitness Test profile that strongly implicated mRNA processing as the molecular target responsible for inhibition of fungal growth. Isolation of the active components from this sample identified a novel class of isoxazolidinone-containing natural products, which we have named parnafungins. These natural products were isolated as an interconverting mixture of four structural- and stereoisomers. The isomerization of the parnafungins was due to a retro-Michael ring-opening and subsequent reformation of a xanthone ring system. This interconversion was blocked by methylation of an enol moiety. Structure elucidation of purified parnafungin derivatives was accomplished by X-ray crystallography and NMR analysis. The biochemical target of these natural products has been identified as the fungal polyadenosine polymerase. Parnafungins demonstrated broad spectrum antifungal activity with no observed activity against gram-positive or gram-negative bacteria. The intact isoxazolidinone ring was required for antifungal activity. In addition, the natural products were efficacious in a mouse model of disseminated candidiasis.


Assuntos
Antifúngicos/isolamento & purificação , Fusarium/química , Oxazolidinonas/isolamento & purificação , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cristalografia por Raios X , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxazolidinonas/química , Oxazolidinonas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta
17.
Chem Sci ; 9(17): 4168-4175, 2018 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-29780547

RESUMO

Two radical-based approaches have been developed to effect the trifluoromethylation of aryl C-H bonds in native peptides either using stoichiometric oxidant or visible light photoredox catalysis. The reported methods are able to derivatize tyrosine and tryptophan sidechains under biocompatible conditions, and a number of examples are reported involving fully unprotected peptides with up to 51 amino acids. The development of this chemistry adds to the growing array of chemical methods for selectively modifying amino acid residues in the context of complex peptides. The direct incorporation of trifluoromethyl groups into biopolymers enables the study of a range of biological and biochemical systems, and preliminary results indicate this method can be extended to the incorporation of other fluoroalkyl groups for bioconjugation applications.

18.
Front Microbiol ; 8: 343, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28321210

RESUMO

Ramoplanin is a glycolipodepsipeptide antibiotic obtained from fermentation of Actinoplanes sp. ATCC 33076 that exhibits activity against clinically important multi-drug-resistant, Gram-positive pathogens including vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-intermediate resistant Clostridium difficile. It disrupts bacterial cell wall through a unique mechanism of action by sequestering the peptidoglycan intermediate Lipid II and therefore does not show cross-resistance with other antibiotics. However, while demonstrating excellent antimicrobial activity in systemic use in animal models of infection, ramoplanin presents low local tolerability when injected intravenously. As a consequence of this limitation, new derivatives are desirable to overcome this issue. During a natural product screening program developed to discover compounds that disrupt bacterial cell wall synthesis by inhibiting peptidoglycan transglycosylation through binding to the intermediate Lipid II, 49 actinomycete strains were identified by HR-LCMS as producers of ramoplanin-related compounds. The producing strains were isolated from environmental samples collected worldwide comprising both tropical and temperate areas. To assess the diversity of this microbial population, the 49 isolates were initially identified to the genus level on the basis of their micromorphology, and 16S sequencing confirmed the initial identification of the strains. These analyses resulted in the identification of members of genus Streptomyces, as well as representatives of the families Micromonosporaceae, Nocardiaceae, Thermomonosporaceae, and Pseudonocardiaceae, suggesting that the production of ramoplanins is relatively widespread among Actinomycetes. In addition, all of these isolates were tested against a panel of Gram-positive and Gram-negative bacteria, filamentous fungi, and yeast in order to further characterize their antimicrobial properties. This work describes the diversity of actinomycete strains that produced ramoplanin-related compounds, and the analysis of the antimicrobial activity exhibited by these isolates. Our results strongly suggest the presence of new ramoplanin-analogs among these actinomycete producers.

19.
J Am Soc Mass Spectrom ; 24(8): 1315-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23740032

RESUMO

With the development of new synthesis procedures, an ever increasing number of chemical modifications can now be incorporated into synthetic oligonucleotides, representing new challenges for analytical chemists to efficiently identify and characterize such molecules. While conventional mass spectrometry (MS) has proven to be a powerful tool to study nucleic acids, new and improved methods and software are now needed to address this emerging challenge. In this report, we describe a simple yet powerful program that affords great flexibility in the calculation of theoretical masses for conventional as well as modified oligonucleotide molecules. This easy to use program can accept input oligonucleotide sequences and then calculate the theoretical mass values for full length products, process impurities, potential metabolites, and gas phase fragments. We intentionally designed this software so that modified nucleotide residues can be incorporated into oligonucleotide sequences, and corresponding mass values can be rapidly calculated. To test the utility of this program, two oligonucleotides that contain a large number of chemical modifications were synthesized. We have analyzed these samples using a Q-TOF mass spectrometer and compared the calculated masses to the observed ones. We found that all of the data matched very well with less than 30 ppm mass errors, well within the expectation for our instrument operated in its current mode. These data confirmed the validity of calculations performed with this new software.


Assuntos
Oligonucleotídeos/química , Cromatografia Líquida de Alta Pressão , Enzimas/química , Espectrometria de Massas , Peso Molecular , Oligonucleotídeos/síntese química , Fosfatos/análise , Espectrofotometria Ultravioleta
20.
Chem Biol ; 18(2): 148-64, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21338914

RESUMO

Starting with the discovery of penicillin, the pharmaceutical industry has relied extensively on natural products (NPs) as an unparalleled source of bioactive small molecules suitable for antibiotic development. However, the discovery of structurally novel and chemically tractable NPs with suitable pharmacological properties as antibiotic leads has waned in recent decades. Today, the repetitive "rediscovery" of previously known NP classes with limited antibiotic lead potential dominates most industrial efforts. This limited productivity, exacerbated by the significant financial and resource requirements of such activities, has led to a broad de-emphasis of NP research by most pharmaceutical companies, including most recently Merck. Here we review our strategies--both technological and philosophical--in addressing current antifungal discovery bottlenecks in target identification and validation and how such efforts may improve NP-based antimicrobial discoveries when aligned with NP screening and dereplication.


Assuntos
Antifúngicos/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Animais , Antifúngicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos
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