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1.
J Nat Prod ; 81(7): 1666-1672, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29979591

RESUMO

Six new macrophilone-type pyrroloiminoquines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton-deficient and heteroatom-rich structures of macrophilones B-G (2-7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1-7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macrophilones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal transduction pathway.


Assuntos
Hidrozoários/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pirroliminoquinonas/isolamento & purificação , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Pirroliminoquinonas/farmacologia , Sumoilação/efeitos dos fármacos
2.
Nucleic Acids Res ; 46(6): 2722-2732, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29481610

RESUMO

Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.


Assuntos
Anti-Infecciosos Locais/metabolismo , Clorexidina/metabolismo , Quadruplex G , Bibliotecas de Moléculas Pequenas/metabolismo , Anti-Infecciosos Locais/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clorexidina/farmacologia , DNA/genética , DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ressonância de Plasmônio de Superfície
3.
Org Lett ; 19(7): 1726-1729, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28345939

RESUMO

A previously uncharacterized pyrroloiminoquinone natural product, macrophilone A, was isolated from the stinging hydroid Macrorhynchia philippina. The structure was assigned utilizing long-range NMR couplings and DFT calculations and proved by a concise, five-step total synthesis. Macrophilone A and a synthetic analogue displayed potent biological activity, including increased intracellular reactive oxygen species levels and submicromolar cytotoxicity toward lung adenocarcinoma cells.


Assuntos
Quinonas/química , Produtos Biológicos , Estrutura Molecular , Espécies Reativas de Oxigênio
4.
SLAS Discov ; 22(6): 760-766, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28346086

RESUMO

E2 enzymes in ubiquitin-like conjugation pathways are important, highly challenging pharmacological targets, and despite significant efforts, few noncovalent modulators have been discovered. Small-molecule microarray (SMM)-based screening was employed to identify an inhibitor of the "undruggable" small ubiquitin-like modifier (SUMO) E2 enzyme Ubc9. The inhibitor, a degradation product from a commercial screening collection, was chemically synthesized and evaluated in biochemical, mechanistic, and structure-activity relationship studies. Binding to Ubc9 was confirmed through the use of ligand-detected nuclear magnetic resonance, and inhibition of sumoylation in a reconstituted enzymatic cascade was found to occur with an IC50 of 75 µM. This work establishes the utility of the SMM approach for identifying inhibitors of E2 enzymes, targets with few known small-molecule modulators.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Cromatografia Líquida , Descoberta de Drogas/métodos , Humanos , Espectrometria de Massas , Estrutura Molecular , Transdução de Sinais , Relação Estrutura-Atividade , Sumoilação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo
5.
Angew Chem Int Ed Engl ; 55(19): 5703-7, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27038327

RESUMO

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.


Assuntos
Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade por Substrato , Sumoilação , Ressonância de Plasmônio de Superfície , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética
6.
ACS Chem Neurosci ; 6(4): 542-50, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25615693

RESUMO

The natural product vinaxanthone has demonstrated a remarkable capability to promote nerve growth following injury or transplantation. In rats following total transection of the spinal cord delivery of vinaxanthone enhanced axonal regeneration, remyelination and angiogenesis at the site of injury all leading to an improved reinstatement of motor function. Through the development of a new ynone coupling reaction, chemically edited derivatives of vinaxanthone have been prepared and studied for improved activity. The coupling reaction allows rapid access to new derivatives, wherein n ynone precursors provide n(2) vinaxanthone analogues. These compounds have been tested for their ability to promote neuronal regrowth using laser axotomy, severing axonal connections in Caenorhabditis elegans. This precise microsurgery using C. elegans allows a new in vivo approach for medicinal chemistry based optimization of neuronal growth promoting compounds.


Assuntos
Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Xantonas/síntese química , Xantonas/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Caenorhabditis elegans , Cromonas/síntese química , Cromonas/química , Dimerização , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Lasers , Microcirurgia , Estrutura Molecular , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/química , Água/química , Xantonas/química
7.
Chembiochem ; 14(17): 2338-44, 2013 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-24123757

RESUMO

A general protocol for exogenous small-molecule pull-down experiments with Caenorhabditis elegans is described; it provides a link between small-molecule screens in worms and existing mutant and RNAi technologies, thereby enabling organismal mechanism of action studies for the natural product clovanemagnolol. Forward chemical genetic screens followed by mechanism of action studies with C. elegans, when coupled with genetic validation of identified targets to reproduce the small molecule's phenotypic effects, provide a unique platform for discovering the biological targets of compounds that affect multicellular processes. First, the use of an immobilized FK506 derivative and soluble competition experiments with optimally prepared soluble C. elegans proteome successfully identified interactions with FK506 binding proteins 1 to 6. This approach was used to determine an unknown mechanism of action for clovanemagnolol, a small molecule that promotes axonal branching in both primary neuronal cultures and in vivo in C. elegans. Following the synthesis of an appropriately functionalized solid-phase reagent bearing a clovanemagnolol analogue pull-down experiments employing soluble competition identified kinesin light chain-1 (KLC-1), a protein involved in axonal cargo transport, as a putative target. This was corroborated through the use of mutant worms lacking klc-1 and possessing GFP neuronal labeling, reproducing the axonal branching phenotype induced by the small molecule clovanemagnolol.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Fenóis/metabolismo , Sesquiterpenos/metabolismo , Animais , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Cinesinas/genética , Mutação , Fenóis/química , Sesquiterpenos/química , Tacrolimo/química , Tacrolimo/metabolismo
8.
Chembiochem ; 14(3): 307-10, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23362121

RESUMO

An in vivo system for monitoring small-molecule-mediated neuronal branching has been developed by using C. elegans. Growth-promoting compounds can be detected by visual inspection of GFPlabeled cholinergic neurons, as axonal branching occurs following treatment with neurotrophic agents. Investigation of the structure-activity relationship of the neurotrophic natural product clovanemagnolol (1) led us to a comparable chemically edited derivative.


Assuntos
Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Bibliotecas de Moléculas Pequenas/química , Animais , Axônios/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fenóis/química , Fenóis/farmacologia , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
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