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1.
Bioorg Med Chem Lett ; 29(2): 204-211, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30528168

RESUMO

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.


Assuntos
Carbonatos/farmacologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Carbonatos/síntese química , Carbonatos/química , Enzimas Desubiquitinantes/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Ubiquitinação/efeitos dos fármacos
2.
J Biol Chem ; 280(6): 4188-94, 2005 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-15556932

RESUMO

Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50-67 of the beta-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an alpha-helical conformation. In the folded, peptide-loaded DR1 structure, the beta-chain residues 50-67 contain a kinked alpha-helical segment spanning Glu-52-Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50-67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (k off). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 beta-chain.


Assuntos
Anticorpos Monoclonais/química , Epitopos/química , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Alanina/química , Animais , Técnicas Biossensoriais , Biotinilação , Mapeamento de Epitopos/métodos , Glicina/química , Hibridomas/imunologia , Cinética , Ligantes , Camundongos , Modelos Químicos , Modelos Moleculares , Mutação , Peptídeos/química , Prolina/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Fatores de Tempo
3.
J Biol Chem ; 279(16): 16561-70, 2004 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-14757758

RESUMO

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.


Assuntos
Anticorpos Monoclonais/imunologia , Antígeno HLA-DR1/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Mapeamento de Epitopos , Epitopos , Antígeno HLA-DR1/química , Antígeno HLA-DR1/genética , Humanos , Dados de Sequência Molecular , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica
4.
J Mol Recognit ; 15(3): 126-34, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12203838

RESUMO

The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we determined the peptide sequences recognized by the trypsin active site, and by the anti-c-Myc antibody, 9E10. Here we describe the use of two peptide libraries of different diversities, one a constrained library based on the trypsin inhibitor EETI-II, where only the six residues in the first loop were randomized (6.4 x 10(7) possible sequences, 6.0 x 10(11) sequences in the library), the other a linear-peptide library with 27 randomized amino acids (1.3 x 10(35) possible sequences, 2 x 10(13) sequences in the library). The constrained library was screened against the natural target of wild-type EETI, bovine trypsin, and the linear library was screened against the anti-c-myc antibody, 9E10. The analysis of selected sequences revealed minimal consensus sequences of PR(I,L,V)L for the first loop of EETI-II and LISE for the 9E10 epitope. The wild-type sequences, PRILMR for the first loop of EETI-II and QKLISE for the 9E10 epitope, were selected with the highest frequency, and in each case the complete wild-type epitope was selected from the library.


Assuntos
Sequência Consenso , Epitopos/análise , Biblioteca de Peptídeos , Proteínas de Plantas , RNA Mensageiro/metabolismo , Tripsina/química , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Bovinos , Sequência Conservada , Epitopos/imunologia , Dados de Sequência Molecular , Oligonucleotídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/imunologia , Puromicina/química , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tripsina/genética , Tripsina/imunologia , Inibidores da Tripsina
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