Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
1.
J Am Chem Soc ; 146(13): 8877-8886, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38503564

RESUMO

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-regulated α subunit and a constitutively expressed ß subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription and reprogramming cells to enable adaptation to a hypoxic microenvironment. Given the central role played by HIF in the survival and growth of tumors in hypoxia, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimization, and characterization of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1ß. These compounds are shown to bind to HIF-α and disrupt the protein-protein interaction between the α and ß subunits of the transcription factor, resulting in disruption of hypoxia-response signaling by our lead molecule in several cancer cell lines.


Assuntos
Fator 1 Induzível por Hipóxia , Neoplasias , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/metabolismo , Hipóxia , Transdução de Sinais , Oxigênio/metabolismo , Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/tratamento farmacológico
2.
J Biol Chem ; 295(28): 9551-9566, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32439803

RESUMO

The purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand.


Assuntos
Hidroximetil e Formil Transferases/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Complexos Multienzimáticos/metabolismo , Nucleotídeo Desaminases/metabolismo , Purinas/biossíntese , Hipóxia Celular , Células HeLa , Humanos , Hidroximetil e Formil Transferases/genética , Fator 1 Induzível por Hipóxia/genética , Complexos Multienzimáticos/genética , Nucleotídeo Desaminases/genética
3.
Nat Chem Biol ; 14(4): 375-380, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29507389

RESUMO

In this article we describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.


Assuntos
Proteínas de Ligação a DNA/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Escherichia coli/metabolismo , Peptídeo Sintases/química , Peptídeos/química , Fatores de Transcrição/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Cristalografia por Raios X , Receptores ErbB/metabolismo , Proteínas de Escherichia coli/química , Etilmaleimida/química , Biblioteca Gênica , Células HEK293 , HIV , Células HeLa , Humanos , Concentração Inibidora 50 , Iodoacetamida/química , Peptídeo Hidrolases/química , Plasmídeos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Especificidade por Substrato
4.
Anal Chem ; 86(21): 10524-30, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25268573

RESUMO

Since noncovalent protein macrocomplexes are implicated in many cellular functions, their characterization is essential to understand how they drive several biological processes. Over the past 20 years, because of its high sensitivity, mass spectrometry has been described as a powerful tool for both the protein identification in macrocomplexes and the understanding of the macrocomplexes organization. Nonetheless, stabilizing these protein macrocomplexes, by introducing covalent bonds, is a prerequisite before their analysis by the denaturing mass spectrometry technique. In this study, using the Hsp90/Aha1 macrocomplex as a model (where Hsp denotes a heat shock protein), we optimized a double cross-linking protocol with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). This protocol takes place in a two-step process: initially, a cross-linking is performed according to a previously optimized protocol, and then a second cross-linking is performed by increasing the EDC concentration, counterbalanced by a high dilution of sample and, thus, protein macrocomplexes. Using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we verified the efficiency of our optimized protocol by submitting (or not submitting) samples to the K200 MALDI MS analysis kit containing N-succinimidyl iodo-acetate, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester), suberic acid bis(N-hydroxysuccinimide ester), disuccinimidyl tartrate, and dithiobis(succinimidyl) propionate, developed by the CovalX Company. Results obtained show that our optimized cross-linking protocol allows a complete stabilization of protein macrocomplexes and appears to be very accurate. Indeed, contrary to other cross-linkers, the "zero-length" feature of the EDC reagent prevents overdetermination of the mass of complexes, because EDC does not remain as part of the linkage.


Assuntos
Reagentes de Ligações Cruzadas/química , Etildimetilaminopropil Carbodi-Imida/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Humanos , Estabilidade Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
5.
ACS Chem Biol ; 14(9): 1874-1878, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31411851

RESUMO

The budding of HIV from infected cells is driven by the protein-protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 µM by binding to UEV with a Kd of 11.9 ± 2.8 µM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of ∼2 µM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peptídeos Cíclicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desenvolvimento de Medicamentos , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Escherichia coli/genética , Células HEK293 , HIV/química , HIV/efeitos dos fármacos , Células HeLa , Humanos , Peptídeos Cíclicos/toxicidade , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Liberação de Vírus/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA