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1.
Cell ; 165(4): 813-26, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27114034

RESUMO

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.


Assuntos
HIV-1/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Cristalografia por Raios X , Glicosilação , HIV-1/classificação , HIV-1/imunologia , Evasão da Resposta Imune , Modelos Moleculares , Simulação de Dinâmica Molecular , Polissacarídeos/análise , Polissacarídeos/metabolismo
2.
Immunity ; 48(3): 500-513.e6, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29548671

RESUMO

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Polissacarídeos/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/química , Antígenos Virais/imunologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Polissacarídeos/química , Ligação Proteica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Relação Estrutura-Atividade
3.
Acc Chem Res ; 56(4): 414-424, 2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36731116

RESUMO

development of antibiotics, antineoplastics, and therapeutics for other diseases. Natural products are unique among all other small molecules in that they are produced by dedicated enzymatic assembly lines that are the protein products of biosynthetic gene clusters. As the products of chiral macromolecules, natural products have distinct three-dimensional shapes and stereochemistry is often encoded in their structures through the presence of stereocenters, or in the case of molecules that lack a stereocenter, the presence of an axis or plane of chirality. In the latter forms of chirality, if the barrier to rotation about the chiral axis or chiral plane is sufficiently high, stable conformers may exist allowing for isolation of discrete conformers, also known as atropisomers. Importantly, the diverse functions and biological activities of natural products are contingent upon their structures, stereochemistry and molecular shape. With continued innovation in methods for natural products discovery, synthetic chemistry, and analytical and computational tools, new insights into atropisomerism in natural products and related scaffolds are being made. As molecular complexity increases, more than one form of stereoisomerism may exist in a single compound (for example, point chirality, chiral axes, and chiral planes), sometimes creating atypical or noncanonical atropisomers, a term used to distinguish physically noninterconvertable atropisomers from typical atropisomers.Here we provide an account of the discovery and unusual structural and stereochemical features of the chrysophaentins, algal derived inhibitors of the bacterial cytoskeletal protein FtsZ and its associated protein partners. Eleven members of the chrysophaentin family have been discovered to date; seven of these are macrocyclic bis-bibenzyl ethers wherein the site of the ether linkage yields either a symmetrical or asymmetrical macrocyclic ring system. The asymmetrical ring system is highly strained and corresponds to the compounds having the most potent antimicrobial activity among the family. We review the structure elucidation and NMR properties that indicate restricted rotation between axes of two biaryl ethers, and the plane represented by the substituted 2-Z-butene bridge common to all of the macrocycles. Computational studies that corroborate high barriers to rotation about one representative plane, on the order of 20+ kcal/mol are presented. These barriers to rotation fix the conformation of the macrocycle into a bowl-like structure and suggest that an atropisomer should exist. Experimental evidence for atropisomerism is presented, consistent with computational predictions. These properties are discussed in the context of the total synthesis of 9-dechlorochrysophaenin A and its ring C isomers. Last, we discuss the implications for the presence of enantiomers in the biological activity and macrocyclization of the natural product.


Assuntos
Produtos Biológicos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Estereoisomerismo , Éteres
4.
Angew Chem Int Ed Engl ; 62(38): e202307053, 2023 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-37335229

RESUMO

Determination of the absolute configuration of chiral molecules is a prerequisite for obtaining a fundamental understanding in any chirality-related field. The interaction with polarised light has proven to be a powerful means to determine this absolute configuration, but its application rests on the comparison between experimental and computed spectra for which the inherent uncertainty in conformational Boltzmann factors has proven to be extremely hard to tackle. Here we present a novel approach that overcomes this issue by combining a genetic algorithm that identifies the relevant conformers by accounting for the uncertainties in DFT relative energies, and a hierarchical clustering algorithm that analyses the trends in the spectra of the considered conformers and identifies on-the-fly when a given chiroptical technique is not able to make reliable predictions. The effectiveness of this approach is demonstrated by considering the challenging cases of papuamine and haliclonadiamine, two bis-indane natural products with eight chiral centres and considerable conformational heterogeneity that could not be assigned unambiguously with current approaches.

5.
J Am Chem Soc ; 143(21): 8056-8068, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34028251

RESUMO

Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, "graspetides" (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a "hairpin topology" with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.


Assuntos
Trifosfato de Adenosina/metabolismo , Produtos Biológicos/metabolismo , Ligases/metabolismo , Peptídeos/metabolismo , Trifosfato de Adenosina/química , Amidas/química , Amidas/metabolismo , Produtos Biológicos/química , Ésteres/química , Ésteres/metabolismo , Ligases/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Conformação Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional
6.
Molecules ; 26(23)2021 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-34885778

RESUMO

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.


Assuntos
Actinomycetales/química , Pirazinas/isolamento & purificação , Microbiologia do Solo , Vias Biossintéticas/genética , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Genoma Bacteriano , Família Multigênica , Peptídeo Sintases/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
7.
J Am Chem Soc ; 142(38): 16161-16166, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32866011

RESUMO

Chrysophaentin A is an antimicrobial natural product isolated from the marine alga C. taylori in milligram quantity. Structurally, chrysophaentin A features a macrocyclic biaryl ether core incorporating two trisubstituted chloroalkenes at its periphery. A concise synthesis of iso- and 9-dechlorochrysophaentin A enabled by a Z-selective ring-closing metathesis (RCM) cyclization followed by an oxygen to carbon ring contraction is described. Fluorescent microscopy studies revealed 9-dechlorochrysophaentins leads to inhibition of bacterial cell wall biosynthesis by disassembly of key divisome proteins, the cornerstone to bacterial cell wall biosynthesis and division.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Produtos Biológicos/farmacologia , Parede Celular/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Parede Celular/metabolismo , Eucariotos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fenótipo , Estereoisomerismo
8.
J Am Chem Soc ; 142(6): 2755-2759, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31986017

RESUMO

Haliclonadiamine and papuamine are bis-indane marine natural products isolated from the marine sponge Haliclona sp. Their relative structures were previously reported to differ by inversion at only one of their eight shared stereocenters. Here X-ray crystallography shows the opposite to be true: papuamine has a 1R,3S,8R,9S,14S,15R,20S,22R configuration, while haliclonadiamine has a 1S,3R,8S,9R,14R,15S,20R,22R configuration. Paradoxically the ECD of each structure displays a negative Cotton effect. X-ray crystallography reveals the two structures adopt similar conformations of their 13-membered macrocyclic core that comprises a configurationally relevant diene. B97x-D/Def2-TZVPP-(MeOH)-calculated ECD supports the diene configuration with the macrocycle dominating the ECD Cotton effect for haliclonadiamine and papuamine. Additional crystallographic and chiroptical analyses of three sponge samples from geographically distant locations indicate this pair of natural products always exists as a configurationally related couple. The co-discovery of a biosynthetic precursor, halichondriamine C, present in these same Haliclona samples must be considered when discussing any biosynthetic pathway. Taken together, this work justifies a reassignment of haliclonadiamine's structure and opens the question of how this complex stereochemical relationship between haliclonadiamine and palauamine arises biosynthetically.


Assuntos
Alcaloides/química , Cristalografia por Raios X/métodos , Óptica e Fotônica/métodos , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular
9.
Artigo em Inglês | MEDLINE | ID: mdl-33077647

RESUMO

WR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite's dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.


Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Antimaláricos/farmacologia , Resistência a Medicamentos , Antagonistas do Ácido Fólico/farmacologia , Humanos , Plasmodium falciparum/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase , Triazinas
10.
J Nat Prod ; 83(4): 1288-1294, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32191460

RESUMO

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.


Assuntos
Depsipeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Theonella/química , Proteínas ras/antagonistas & inibidores , Aminoácidos/química , Animais , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Papua Nova Guiné , Poríferos/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo
11.
J Nat Prod ; 82(1): 148-153, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30623657

RESUMO

A laboratory culture of the colonial marine alga Chrysophaeum taylorii NIES-1699 yielded a set of new bioactive chrysophaentin analogs, and their structures were determined by HRESIMS and NMR spectroscopy. Differences in the metabolites identified between cultured C. taylorii NIES-1699 and field-collected strains from the U.S. Virgin Islands revealed additional structure-activity relationships for the Gram-positive antibiotic activity of the chrysophaentins. The presence of new hemichrysophaentins and a C-C linked biphenyl analog suggest novel features of their biosynthetic pathway. Bayesian analysis of the alignment of the 18S rRNA gene places the microalga C. taylorii in the pelagophyte clade.


Assuntos
Antibacterianos/isolamento & purificação , Compostos de Bifenilo/isolamento & purificação , Microalgas/metabolismo , Antibacterianos/farmacologia , Teorema de Bayes , Compostos de Benzil , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Éteres Cíclicos , Biologia Marinha , Relação Estrutura-Atividade
12.
PLoS Pathog ; 12(4): e1005537, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27064278

RESUMO

The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-2(7312A). This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy.


Assuntos
Produtos do Gene env/imunologia , Anticorpos Anti-HIV/imunologia , Vírus da Imunodeficiência Símia/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Epitopos/imunologia , Anticorpos Anti-HIV/isolamento & purificação , Humanos , Testes de Neutralização/métodos
14.
Artigo em Inglês | MEDLINE | ID: mdl-28373194

RESUMO

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico/genética , Transporte Biológico/fisiologia , Genes MDR/genética , Genes MDR/fisiologia , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nucleosídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade
15.
Chembiochem ; 18(8): 764-771, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28166380

RESUMO

Man9 GlcNAc2 (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D 13 C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of 13 C,15 N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.


Assuntos
Proteínas de Bactérias/química , Moléculas de Adesão Celular/química , Lectinas Tipo C/química , Espectroscopia de Ressonância Magnética , Mananas/química , Lectina de Ligação a Manose/química , Receptores de Superfície Celular/química , Células A549 , Configuração de Carboidratos , Radioisótopos de Carbono , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Mananas/biossíntese , Microcystis , Simulação de Dinâmica Molecular , Radioisótopos de Nitrogênio
16.
Mol Pharm ; 14(8): 2681-2689, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28494151

RESUMO

HIV/AIDS continues to pose an enormous burden on global health. Current HIV therapeutics include inhibitors that target the enzymes HIV protease, reverse transcriptase, and integrase, along with viral entry inhibitors that block the initial steps of HIV infection by preventing membrane fusion or virus-coreceptor interactions. With regard to the latter, peptides derived from the HIV coreceptor CCR5 were previously shown to modestly inhibit entry of CCR5-tropic HIV strains, with a peptide containing residues 178-191 of the second extracellular loop (peptide 2C) showing the strongest inhibition. Here we use an iterative approach of amino acid scanning at positions shown to be important for binding the HIV envelope, and recombining favorable substitutions to greatly improve the potency of 2C. The most potent candidate peptides gain neutralization breadth and inhibit CXCR4 and CXCR4/CCR5-using viruses, rather than CCR5-tropic strains only. We found that gains in potency in the absence of toxicity were highly dependent on amino acid position and residue type. Using virion capture assays we show that 2C and the new peptides inhibit capture of CD4-bound HIV-1 particles by antibodies whose epitopes are located in or around variable loop 3 (V3) on gp120. Analysis of antibody binding data indicates that interactions between CCR5 ECL2-derived peptides and gp120 are localized around the base and stem of V3 more than the tip. In the absence of a high-resolution structure of gp120 bound to coreceptor CCR5, these findings may facilitate structural studies of CCR5 surrogates, design of peptidomimetics with increased potency, or use as functional probes for further study of HIV-1 gp120-coreceptor interactions.


Assuntos
Peptídeos/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Peptídeos/química , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Proteínas Virais/química , Proteínas Virais/farmacologia , Vírion/química
17.
J Nat Prod ; 80(9): 2556-2560, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28837335

RESUMO

Antibacterial-guided fractionation of an extract of a deep-water Topsentia sp. marine sponge led to the isolation of two new indole alkaloids, tulongicin A (1) and dihydrospongotine C (2), along with two known analogues, spongotine C (3) and dibromodeoxytopsentin (4). Their planar structures were determined by NMR spectroscopy. Their absolute configurations were determined through a combination of experimental and computational analyses. Tulongicin (1) is the first natural product to contain a di(6-Br-1H-indol-3-yl)methyl group linked to an imidazole core. The coexistence of tri-indole 1 and bis-indole alcohol 2 suggests a possible route to 1. All of the compounds showed strong antimicrobial activity against Staphylococcus aureus.


Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Imidazóis/química , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/farmacologia , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Alcaloides Indólicos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Poríferos , Água
18.
Nature ; 480(7377): 336-43, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22113616

RESUMO

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded ß-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.


Assuntos
Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/química , HIV-1/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Afinidade de Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Sítios de Ligação de Anticorpos/imunologia , Sequência Conservada , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Anticorpos Anti-HIV/química , Ligação de Hidrogênio , Evasão da Resposta Imune , Modelos Moleculares , Dados de Sequência Molecular , Polissacarídeos/química , Polissacarídeos/imunologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
19.
Bioorg Med Chem Lett ; 26(24): 5863-5866, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27876320

RESUMO

Novel approaches that do not rely upon developing microbicidal compounds are sorely needed to combat multidrug resistant (MDR) bacteria. The potential of marine secondary metabolites to serve as a source of non-traditional anti-bacterial agents is demonstrated by showing that pyrrole-imidazole alkaloids inhibit biofilm formation and suppress antibiotic resistance.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Alcaloides/farmacologia , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Alcaloides/química , Alcaloides/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Relação Dose-Resposta a Droga , Imidazóis/química , Imidazóis/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Poríferos , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade
20.
Environ Sci Technol ; 50(17): 9652-60, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27443216

RESUMO

Fluorochloridone (FLC) is a herbicide used worldwide that is thought to be safe. However, due to its potential genotoxicity, cytotoxicity, and even systematic toxicity, there are increasing concerns about human exposure to this compound. Thus, the metabolism and bioactivation of FLC was investigated. After oral administration to mice, 27 metabolites were identified by ultrahigh performance liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry and with further structural identification by nuclear magnetic resonance spectroscopy. Hydroxylation and oxidative dechlorination were the major phase I pathways, while glutathione (GSH) and N-acetylcysteine conjugations were two major phase II pathways, indicating the formation of a reactive intermediate. In vitro microsomal and cytosolic studies revealed that a GSH conjugate (M13) was the predominant metabolite of FLC formed through a nucleophilic SN2 substitution of 3-Cl by GSH; this pathway is NADPH independent and accelerated by glutathione S-transferase (GST). Further, a kinetic study showed that M13 formation in both human liver microsomes and cytosols obeyed typical Michaelis-Menten kinetics. The maximum clearance (Vmax/Km) of GSH conjugation in human liver microsomes was approximately 5.5-fold higher than human liver cytosol, thus implying that microsomal GST was mainly responsible for M13 formation. These findings are important for understanding the potential hazard of human exposure to FLC.


Assuntos
Microssomos Hepáticos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Animais , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Herbicidas/metabolismo , Humanos , Camundongos
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