RESUMO
Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.
Assuntos
Antiprotozoários , Apicomplexa/crescimento & desenvolvimento , Bivalves/microbiologia , Gammaproteobacteria/metabolismo , Simbiose , Animais , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Camundongos , Infecções por Protozoários/tratamento farmacológicoRESUMO
Methane-oxidizing bacteria, aerobes that utilize methane as their sole carbon and energy source, are being increasingly studied for their environmentally significant ability to remove methane from the atmosphere. Their genomes indicate that they also have a robust and unusual secondary metabolism. Bioinformatic analysis of the Methylobacter tundripaludum genome identified biosynthetic gene clusters for several intriguing metabolites, and this report discloses the structural and genetic characterization of tundrenone, one of these metabolites. Tundrenone is a highly oxidized metabolite that incorporates both a modified bicyclic chorismate-derived fragment and a modified lipid tail bearing a ß,γ-unsaturated α-hydroxy ketone. Tundrenone has been genetically linked to its biosynthetic gene cluster, and quorum sensing activates its production. M. tundripaludum's genome and tundrenone's discovery support the idea that additional studies of methane-oxidizing bacteria will reveal new naturally occurring molecular scaffolds and the biosynthetic pathways that produce them.
Assuntos
Vias Biossintéticas , Hidroxiácidos/metabolismo , Indenos/metabolismo , Methylobacteriaceae/metabolismo , Metabolismo Secundário , Biologia Computacional , Genoma Bacteriano , Hidroxiácidos/química , Indenos/química , Metano/metabolismo , Methylobacteriaceae/genética , Família MultigênicaRESUMO
Small molecules produced by Actinobacteria have played a prominent role in both drug discovery and organic chemistry. As part of a larger study of the actinobacterial symbionts of fungus-growing ants, we discovered a small family of three previously unreported piperazic acid-containing cyclic depsipeptides, gerumycins A-C. The gerumycins are slightly smaller versions of dentigerumycin, a cyclic depsipeptide that selectively inhibits a common fungal pathogen, Escovopsis. We had previously identified this molecule from a Pseudonocardia associated with Apterostigma dentigerum, and now we report the molecule from an associate of the more highly derived ant Trachymyrmex cornetzi. The three previously unidentified compounds, gerumycins A-C, have essentially identical structures and were produced by two different symbiotic Pseudonocardia spp. from ants in the genus Apterostigma found in both Panama and Costa Rica. To understand the similarities and differences in the biosynthetic pathways that produced these closely related molecules, the genomes of the three producing Pseudonocardia were sequenced and the biosynthetic gene clusters identified. This analysis revealed that dramatically different biosynthetic architectures, including genomic islands, a plasmid, and the use of spatially separated genetic loci, can lead to molecules with virtually identical core structures. A plausible evolutionary model that unifies these disparate architectures is presented.
Assuntos
Actinobacteria/fisiologia , Formigas/fisiologia , Fungos/crescimento & desenvolvimento , Simbiose , Actinobacteria/genética , Animais , Genes Bacterianos , Dados de Sequência MolecularRESUMO
A strategy that leverages solvent effects to noncovalently trap solid and unstable liquid organic compounds within a crystalline sponge ({[(ZnI2)3(tris(4-pyridyl)-1,3,5-triazine)2]·x(CHCl3)}n) in a simple, mild, and efficient fashion for target molecule structure determination via X-ray diffraction is disclosed. Host-guest structures were obtained using third-generation synchrotron radiation, and new beamline hardware allowed rapid data collection in ~5-24 minutes. This is 40-90% faster than previously reported crystalline sponge synchrotron datasets collected by us, and approximately a 150-720-fold decrease in time versus using a typical in-house diffractometer, effectively enabling the potential for high-throughput analysis. The new target molecule inclusion method using methyl tert-butyl ether (MTBE) solvent was demonstrated by trapping (E)-stilbene, vanillin, 4-(trifluoromethyl)phenyl azide, and (+)-artemisinin (an antimalarial drug). The potential of guests to maximize intermolecular interactions with the crystalline sponge framework at the expense of attenuating intramolecular interactions (e.g., π-conjugation) was observed for (E)-stilbene. Trapping of vanillin and (+)-artemisinin elicited single-crystal-to-single-crystal transformations where space group symmetry reduced from C2/c to P1Ì and C2, respectively, and the absolute configuration of (+)-artemisinin was determined through anomalous dispersion.
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Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach.
Assuntos
Apoptose/efeitos dos fármacos , Diarileptanoides/farmacologia , Inibidores Enzimáticos/farmacologia , Janus Quinase 2/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Janus Quinase 2/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A's formation by this path, a six-electron oxidation, could be a response to oxidative stress triggered by bafilomycin C1.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Policetídeos/química , Policetídeos/metabolismo , Trichoderma/química , Proteínas Virais/química , Proteínas Virais/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Trichoderma/metabolismo , Regulação para CimaRESUMO
During the course of exploring crystallization conditions in generating metal-organic frameworks (MOFs) for use in the crystalline sponge method, two discrete metal-organic complexes, namely, aqua[2,4,6-tris(pyridin-4-yl)-1,3,5-triazine]zinc(II) bromide, [Zn(C18H12N6)(H2O)]Br2, and aqua[2,4,6-tris(pyridin-4-yl)-1,3,5-triazine]zinc(II) chloride, [Zn(C18H12N6)(H2O)]Cl2, were encountered. Structures in the orthorhombic space group Pnma (No. 62) for the bromide congener at 299â K and the chloride congener at 100â K were obtained. A phase transition for the bromide congener occurred upon cooling from 299 to 100â K, yielding a crystal polymorph with four domains that exhibits monoclinic P21/m space-group symmetry (No. 11), which arises from conformational changes. The main intramolecular contacts that contribute to the crystal packing in all observed structures are H...H, Halide...H/H...Halide, C...H/H...C, and N...H/H...N. Intramolecular hydrogen bonding between the Zn-bound water and non-Zn-bound pyridyl N atoms is a prominent feature within the three-dimensional networks. Aromatic π-stacking between the non-Zn-bound pyridine rings and contacts involving the halide ligands further stabilize the crystal packing.
RESUMO
The double allylboration of nitriles and acid anhydrides to form bis-allyl amines and esters, respectively, can be achieved through the use of potassium allyltrifluoroborate in the presence of boron trifluoride etherate at room temperature. The method described is relatively mild, exhibits chemoselectivity to other electrophiles present, avoids the use of metals, and features the use of an operationally stable and robust potassium organotrifluoroborate reagent.
RESUMO
While single-crystal X-ray diffraction (SC-XRD) is one of the most powerful structural determination techniques for organic molecules, the requirement of obtaining a suitable crystal for analysis limits its applicability, particularly for liquids and amorphous solids. The emergent use of preformed porous crystalline matrices that can absorb organic compounds and stabilize them via host-guest interactions for observation via SC-XRD offers a way to overcome this hindrance. A topical and current discussion of SC-XRD in organic chemistry and the use of preformed matrices for the in crystallo analysis of organic compounds, with a particular focus on the absolute structure determination of chiral molecules, is presented. Preformed crystalline matrices that are covered include metal-organic frameworks (MOFs) as used in the crystalline sponge method, metal-organic polyhedra (MOPs, coordination cages), porous organic materials (POMs)/porous organic molecular crystals (POMCs), and biological scaffolds. An outlook and perspective on the current technology and on its future directions is provided.
RESUMO
New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.
Assuntos
Antiparasitários/farmacologia , Resistência a Medicamentos/genética , Lactonas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/genética , Criptosporidiose , Cryptosporidium , Cryptosporidium parvum , Humanos , SarcocystisRESUMO
The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23-65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression.
Assuntos
Bactérias/metabolismo , Bacteroides/metabolismo , Fezes/microbiologia , Microbioma Gastrointestinal , Ácido gama-Aminobutírico/metabolismo , Adulto , Idoso , Bactérias/classificação , Bacteroides/genética , Encéfalo/diagnóstico por imagem , Estudos de Coortes , Depressão/microbiologia , Transtorno Depressivo Maior/microbiologia , Feminino , Trato Gastrointestinal/microbiologia , Perfilação da Expressão Gênica , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
The tendency for carbocyclic analogues of penicillins to undergo hydrate and hemiketal formation is central to their ability to function as beta-lactamase inhibitors. 2-Thiabicyclo[3.2.0]heptan-6-one-4-carboxylates with alkoxy functionality at C3 have been prepared through two complementary diastereoselective substitution reactions following a highly stereoselective chlorination with sulfuryl chloride. We have found that carbocyclic analogues with 3beta substituents favor an endo envelope conformation in solution, the solid state, and the gas phase, whereas those with 3alpha substituents adopt an exo envelope. Evidence from X-ray crystal structures and ab initio calculations suggests that an anomeric effect contributes to the large conformational preference of the tetrahydrothiophene ring that favors the C3 substituent in an axial orientation. In addition, the envelope conformation of the bicycle, which is determined by the stereochemistry of the C3 substituent, has a dramatic effect on the ability of the cyclobutanone to undergo hemiketal formation in methanol-d4.
Assuntos
Ciclobutanos/química , Penicilinas/química , Simulação por Computador , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Penicilinas/síntese química , Teoria Quântica , Padrões de Referência , EstereoisomerismoRESUMO
The growing importance of solid-phase peptide synthesis (SPPS) has necessitated the development of spectroscopic experiments that can be used to obtain structural and conformational information on resin-bound peptides. Despite the utility of two-dimensional high-resolution magic angle spinning (HRMAS) NMR experiments that provide homonuclear shift correlations, experiments that provide heteronuclear shift correlations are necessary for complex conformational and structural elucidatory problems. Here we report the optimization and implementation of non-gradient inverse NMR experiments for acquiring the 1H-13C shift correlations of resin-bound peptides. The use of non-gradient experiments is advantageous as many magic angle spinning (MAS) probes do not possess gradient coils. An HRMAS BIRD-HMQC experiment with a reduced 1JCH constant has proven very suitable for obtaining one-bond correlations. Long-range correlations can be interpolated by using a non-gradient HRMAS CT-HMBC-1 experiment where the resulting data is processed with forward linear prediction. It has been shown that removing the effects of 1H-1H J-modulation is crucial in order to view cross peaks that correspond to long-range correlations. Additionally, both experiments prove extremely useful over routine one-dimensional 13C HRMAS experiments for extracting carbon chemical shift data. The non-gradient HRMAS BIRD-HMQC and CT-HMBC-1 experiments can be used to assist in conformational analysis and to identify and deconvolute situations where accidental equivalence and seemingly correlated isochronous signals arise.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Oligopeptídeos/síntese química , Carbono , Hidrogênio , Oligopeptídeos/química , Transição de Fase , Conformação ProteicaRESUMO
Bioassay-guided metabolomic analyses led to the characterization of four new 20-membered glycosylated polyketide macrolactams, macrotermycins A-D, from a termite-associated actinomycete, Amycolatopsis sp. M39. M39's sequenced genome revealed the macrotermycin's putative biosynthetic gene cluster. Macrotermycins A and C had antibacterial activity against human-pathogenic Staphylococcus aureus and, of greater ecological relevance, they also had selective antifungal activity against a fungal parasite of the termite fungal garden.
Assuntos
Actinomycetales , Animais , Antibacterianos , Glicosilação , Isópteros , Estrutura MolecularRESUMO
Bromide and chloride analogs of the commonly used zinc iodide-based metal organic framework for the crystalline sponge method were synthesized and evaluated. Inclusion of (1R)-(-)-menthyl acetate into these MOFs was analysed using third-generation synchrotron radiation, and the effects and potential benefits of varying the MOF terminal ligand are discussed.
Assuntos
Compostos Organometálicos/química , Zinco/química , Cristalização , Ligantes , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese químicaRESUMO
A detailed set of synthetic and crystallographic guidelines for the crystalline sponge method based upon the analysis of expediently synthesized crystal sponges using third-generation synchrotron radiation are reported. The procedure for the synthesis of the zinc-based metal-organic framework used in initial crystal sponge reports has been modified to yield competent crystals in 3 days instead of 2 weeks. These crystal sponges were tested on some small molecules, with two being unexpectedly difficult cases for analysis with in-house diffractometers in regard to data quality and proper space-group determination. These issues were easily resolved by the use of synchrotron radiation using data-collection times of less than an hour. One of these guests induced a single-crystal-to-single-crystal transformation to create a larger unit cell with over 500 non-H atoms in the asymmetric unit. This led to a non-trivial refinement scenario that afforded the best Flack x absolute stereochemical determination parameter to date for these systems. The structures did not require the use of PLATON/SQUEEZE or other solvent-masking programs, and are the highest-quality crystalline sponge systems reported to date where the results are strongly supported by the data. A set of guidelines for the entire crystallographic process were developed through these studies. In particular, the refinement guidelines include strategies to refine the host framework, locate guests and determine occupancies, discussion of the proper use of geometric and anisotropic displacement parameter restraints and constraints, and whether to perform solvent squeezing/masking. The single-crystal-to-single-crystal transformation process for the crystal sponges is also discussed. The presented general guidelines will be invaluable for researchers interested in using the crystalline sponge method at in-house diffraction or synchrotron facilities, will facilitate the collection and analysis of reliable high-quality data, and will allow construction of chemically and physically sensible models for guest structural determination.
Assuntos
Cristalografia/métodos , Síncrotrons , Anisotropia , Cristalografia por Raios X , Coleta de Dados , Modelos Moleculares , Porosidade , Solventes , TermodinâmicaRESUMO
TP53 is the most frequently mutated gene in human cancer, and small-molecule reactivation of mutant p53 function represents an important anticancer strategy. A cell-based, high-throughput small-molecule screen identified chetomin (CTM) as a mutant p53 R175H reactivator. CTM enabled p53 to transactivate target genes, restored MDM2 negative regulation, and selectively inhibited the growth of cancer cells harboring mutant p53 R175H in vitro and in vivo. We found that CTM binds to Hsp40 and increases the binding capacity of Hsp40 to the p53 R175H mutant protein, causing a potential conformational change to a wild-type-like p53. Thus, CTM acts as a specific reactivator of the p53 R175H mutant form through Hsp40. These results provide new insights into the mechanism of reactivation of this specific p53 mutant.
Assuntos
Antineoplásicos/farmacologia , Dissulfetos/farmacologia , Proteínas de Choque Térmico HSP40/metabolismo , Alcaloides Indólicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Dissulfetos/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Ensaios de Triagem em Larga Escala , Humanos , Alcaloides Indólicos/química , Camundongos , Camundongos Nus , Mutação , Bibliotecas de Moléculas Pequenas/química , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As increased antibiotic resistance erodes the efficacy of currently used drugs, the need for new candidates with therapeutic potential grows. Although the majority of antibiotics in clinical use originated from natural products, mostly from environmental actinomycetes, high rediscovery rates, among other factors, have diminished the enthusiasm for continued exploration of this historically important source. Several well-studied insect agricultural systems have bacterial symbionts that have evolved to produce small molecules that suppress environmental pathogens. These molecules represent an underexplored reservoir of potentially useful antibiotics. This report describes the multilateral symbioses common to insect agricultural systems, the general strategy used for antibiotic discovery and pertinent examples from three farming systems: fungus-farming ants, southern pine beetles (SPBs) and fungus-growing termites.
Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Simbiose , Agricultura , Animais , Formigas/microbiologia , Produtos Biológicos/farmacologia , Besouros/microbiologia , Farmacorresistência Bacteriana , Fungos/fisiologia , Humanos , Isópteros/microbiologiaRESUMO
We report a preliminary functional and complete structural characterization of a highly unusual geldanamycin analog, natalamycin A, that was isolated from Streptomyces strain M56 recovered from a South African nest of Macrotermes natalensis termites. Bioassay-guided fractionation based on antifungal activity led to the isolation of natalamycin A, and a combination of NMR spectroscopy and X-ray crystallographic analysis, including highly-accurate quantum-chemical NMR calculations on the largest and most conformationally-flexible system to date, revealed natalamycin A's three-dimensional solid- and solution-state structure. This structure along with the structures of related compounds isolated from the same source suggest a geldanamycin-like biosynthetic pathway with unusual post-PKS modifications.
RESUMO
A domino [3,3]-sigmatropic aryl-Claisen rearrangement of cyclic and acyclic bisaryloxy-substituted alkenes can be performed in high yield by using Ln(fod)(3) catalysis to obtain bisphenolic products incorporating two contiguous aryl-C(sp(3)) bonds. Stereospecific rearrangement was observed for cyclic substrates. The precursor diaryl ethers were typically synthesized from the corresponding diols by double arylation procedures using either copper catalyzed coupling of aryltrifluoroborate salts or by S(N)Ar reaction.