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1.
ChemMedChem ; 16(23): 3576-3587, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34524728

RESUMO

The NRF2 transcription factor is a key regulator in cellular oxidative stress response, and acts as a tumor suppressor. Aberrant activation of NRF2 has been implicated in promoting chemo-resistance, tumor growth, and metastasis by activating its downstream target genes. Hence, inhibition of NRF2 promises to be an attractive therapeutic strategy to suppress cell proliferation and enhance cell apoptosis in cancer. Direct targeting of NRF2 with small-molecules to discover protein-DNA interaction inhibitors is challenging as it is a largely intrinsically disordered protein. To discover molecules that bind to NRF2 at the DNA binding interface, we performed an NMR-based fragment screen against its DNA-binding domain. We discovered several weakly binding fragment hits that bind to a region overlapping with the DNA binding site. Using SAR by catalogue we developed an initial structure-activity relationship for the most interesting initial hit series. By combining NMR chemical shift perturbations and data-driven docking, binding poses which agreed with NMR information and the observed SAR were elucidated. The herein discovered NRF2 hits and proposed binding modes form the basis for future structure-based optimization campaigns on this important but to date 'undrugged' cancer driver.


Assuntos
DNA/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , DNA/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Relação Estrutura-Atividade
3.
Acta Neuropathol ; 136(1): 69-87, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29934874

RESUMO

Alzheimer's disease (AD) is the most common form of dementia in the elderly affecting more than 5 million people in the U.S. AD is characterized by the accumulation of ß-amyloid (Aß) and Tau in the brain, and is manifested by severe impairments in memory and cognition. Therefore, removing tau pathology has become one of the main therapeutic goals for the treatment of AD. Tau (tubulin-associated unit) is a major neuronal cytoskeletal protein found in the CNS encoded by the gene MAPT. Alternative splicing generates two major isoforms of tau containing either 3 or 4 repeat (R) segments. These 3R or 4RTau species are differentially expressed in neurodegenerative diseases. Previous studies have been focused on reducing Tau accumulation with antibodies against total Tau, 4RTau or phosphorylated isoforms. Here, we developed a brain penetrating, single chain antibody that specifically recognizes a pathogenic 3RTau. This single chain antibody was modified by the addition of a fragment of the apoB protein to facilitate trafficking into the brain, once in the CNS these antibody fragments reduced the accumulation of 3RTau and related deficits in a transgenic mouse model of tauopathy. NMR studies showed that the single chain antibody recognized an epitope at aa 40-62 of 3RTau. This single chain antibody reduced 3RTau transmission and facilitated the clearance of Tau via the endosomal-lysosomal pathway. Together, these results suggest that targeting 3RTau with highly specific, brain penetrating, single chain antibodies might be of potential value for the treatment of tauopathies such as Pick's Disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Expansão das Repetições de DNA/genética , Doença de Pick/tratamento farmacológico , Anticorpos de Cadeia Única/uso terapêutico , Proteínas tau/genética , Proteínas tau/imunologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Apolipoproteínas B/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Transformada , Técnicas de Cocultura , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Neuroblastoma/patologia , Fosforilação , Doença de Pick/genética , Doença de Pick/patologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas tau/metabolismo
4.
Nat Struct Mol Biol ; 22(8): 636-41, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26167881

RESUMO

The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters, which have two-fold direct or inverted quasisymmetry, AAC has apparent three-fold rotational symmetry. Further, its transport rate is relatively fast for transporters that carry large solutes. Here, we study the yeast AAC carrier 3 by obtaining comprehensive NMR relaxation dispersion measurements, which provide residue-specific information on the protein's conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a sparsely populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP substantially increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that although the transporter catalyzes the translocation of substrate the substrate also facilitates interconversion between alternating states, and this interconversion may be relevant to the transport function.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Translocases Mitocondriais de ADP e ATP/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Algoritmos , Sítios de Ligação , Cinética , Translocases Mitocondriais de ADP e ATP/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Termodinâmica
5.
Structure ; 22(2): 209-17, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24332718

RESUMO

The mitochondrial carriers play essential roles in energy metabolism. The short Ca²âº-binding mitochondrial carrier (SCaMC) transports ATP-Mg in exchange for Pi and is important for activities that depend on adenine nucleotides. SCaMC adopts, in addition to the transmembrane domain (TMD) that transports solutes, an extramembrane N-terminal domain (NTD) that regulates solute transport in a Ca²âº-dependent manner. Crystal structure of the Ca²âº-bound NTD reveals a compact architecture in which the functional EF hands are sequestered by an endogenous helical segment. Nuclear magnetic resonance (NMR) relaxation rates indicated that removal of Ca²âº from NTD results in a major conformational switch from the rigid and compact Ca²âº-bound state to the dynamic and loose apo state. Finally, we showed using surface plasmon resonance and NMR titration experiments that free apo NTDs could specifically interact with liposome-incorporated TMD, but that Ca²âº binding drastically weakened the interaction. Our results together provide a molecular explanation for Ca²âº-dependent ATP-Mg flux in mitochondria.


Assuntos
Antiporters/química , Antiporters/fisiologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/química , Calmodulina/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/fisiologia , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Magnésio/química , Espectroscopia de Ressonância Magnética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Fosfatos/química , Estrutura Terciária de Proteína , Proteolipídeos/química , Homologia de Sequência de Aminoácidos
6.
ACS Chem Biol ; 8(7): 1600-10, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23651431

RESUMO

The KIX domain of the transcriptional coactivator CREB binding protein (CBP) co-operatively mediates interactions between transcription factors. Binding of the transcription factor mixed-lineage leukemia (MLL) induces the formation of a low-populated conformer of KIX that resembles the conformation of the KIX domain in the presence of a second transcription factor molecule. NMR spin relaxation studies have previously shown that allosteric coupling proceeds through a network of hydrophobic core residues that bridge the two binding sites. Here we describe high-resolution NMR solution structures of the binary complex of KIX with MLL and the ternary complex of KIX formed with MLL and phosphorylated kinase inducible domain of CREB (pKID) as a second ligand. We show that binding of pKID to the binary complex of KIX with MLL is accompanied by a defined repacking of the allosteric network in the hydrophobic core of the protein. Rotamer populations derived from methyl group (13)C chemical shifts reveal a dynamic contribution to the repacking process that is not captured by the structural coordinates and exemplify the dynamic nature of allosteric communication in the KIX domain.


Assuntos
Proteína de Ligação a CREB/fisiologia , Estrutura Terciária de Proteína , Regulação Alostérica , Complexos de Coordenação/química , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Conformação Molecular , Estrutura Terciária de Proteína/fisiologia
8.
J Am Chem Soc ; 131(8): 3063-8, 2009 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-19203263

RESUMO

Allosteric regulation is an effective mechanism of control in biological processes. In allosteric proteins a signal originating at one site in the molecule is communicated through the protein structure to trigger a specific response at a remote site. Using NMR relaxation dispersion techniques we directly observe the dynamic process through which the KIX domain of CREB binding protein communicates allosteric information between binding sites. KIX mediates cooperativity between pairs of transcription factors through binding to two distinct interaction surfaces in an allosteric manner. We show that binding the activation domain of the mixed lineage leukemia (MLL) transcription factor to KIX induces a redistribution of the relative populations of KIX conformations toward a high-energy state in which the allosterically activated second binding site is already preformed, consistent with the Monod-Wyman-Changeux (WMC) model of allostery. The structural rearrangement process that links the two conformers and by which allosteric information is communicated occurs with a time constant of 3 ms at 27 degrees C. Our dynamic NMR data reveal that an evolutionarily conserved network of hydrophobic amino acids constitutes the pathway through which information is transmitted.


Assuntos
Proteína de Ligação a CREB/química , Proteína de Leucina Linfoide-Mieloide/química , Sítios de Ligação , Proteína de Ligação a CREB/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/metabolismo , Transdução de Sinais
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