RESUMO
The histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) is frequently dysregulated in cancers, and gain-of-function (GOF) EZH2 mutations have been identified in non-Hodgkin lymphomas. Small-molecule inhibitors against EZH2 demonstrated anti-tumor activity in EZH2-mutated lymphomas and entered clinical trials. Here, we developed models of acquired resistance to EZH2 inhibitor EI1 with EZH2-mutated lymphoma cells. Resistance was generated by secondary mutations in both wild-type (WT) and GOF Y641N EZH2 alleles. These EZH2 mutants retained the substrate specificity of their predecessor complexes but became refractory to biochemical inhibition by EZH2 inhibitors. Resistant cells were able to maintain a high level of H3K27Me3 in the presence of inhibitors. Interestingly, mutation of EZH2 WT alone generated an intermediate resistance phenotype, which is consistent with a previously proposed model of cooperation between EZH2 WT and Y641N mutants to promote tumorigenesis. In addition, the findings presented here have implications for the clinical translation of EZH2 inhibitors and underscore the need to develop novel EZH2 inhibitors to target potential resistance emerging in clinical settings.
Assuntos
Alelos , Antineoplásicos/farmacologia , Linfoma/tratamento farmacológico , Linfoma/genética , Mutação , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Linfoma/patologiaRESUMO
We describe the complete purification of aromatic aminotransferase I, the enzyme responsible for the ability of Klebsiella aerogenes to use tryptophan and phenylalanine as sole sources of nitrogen, as well as the partial purification of aromatic aminotransferase IV. An examination of the properties of these enzymes revealed that aminotransferase I had much greater affinity for the aromatic amino acids than aminotransferase IV, explaining the essential role of aminotransferase I in the utilization of exogenously supplied aromatic amino acids. The properties of aminotransferase IV suggest that this enzyme is actually an aspartate aminotransferase (EC 2.6.1.1), corresponding to the product of the aspC gene of Escherichia coli.
Assuntos
Klebsiella pneumoniae/enzimologia , Transaminases/isolamento & purificação , Aminoácidos/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Fenilalanina/metabolismo , Temperatura , Triptofano/metabolismoRESUMO
Klebsiella aerogenes utilized aromatic amino acids as sole sources of nitrogen but not as sole sources of carbon. K. aerogenes abstracted the alpha-amino group of these compounds by transamination and excreted the arylpyruvate portions into the medium. When tryptophan was utilized as the sole source of nitrogen by K. aerogenes, indolepyruvate was excreted into the medium, where it polymerized non-enzymatically to form a brick red pigment. At least four separate aromatic aminotransferase activities were found in K. aerogenes. One activity (aromatic aminotransferase I) appeared to be solely responsible for the aminotransferase reaction necessary for the growth of K. aerogenes when tryptophan was the source of nitrogen; the loss of this activity by mutation (tut) prevented the growth of cells on media containing this and other aromatic amino acids. None of the other aminotransferase activities in the cells could substitute for aromatic aminotransferase in this regard. Tryptophan-dependent pigment formation in K. aerogenes was positively controlled by the intracellular level of glutamine synthetase. Nevertheless, the aromatic aminotransferase activity in cells varied less than 2-fold in response to 10-fold or greater changes in the levels of glutamine synthetase. Glutamine synthetase affected the ability of the cells to take up tryptophan from the medium.
Assuntos
Klebsiella pneumoniae/metabolismo , Transaminases/metabolismo , Triptofano/metabolismo , Aminoácidos/metabolismo , Glutamato-Amônia Ligase/metabolismo , Pigmentos Biológicos/biossínteseRESUMO
The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1' pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2' and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.
Assuntos
Ácidos Hidroxâmicos , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/química , Pirazinas , Sítios de Ligação , Colagenases/metabolismo , Simulação por Computador , Fibroblastos/enzimologia , Humanos , Espectroscopia de Ressonância Magnética , Metaloproteinase 3 da Matriz/metabolismo , Modelos Moleculares , Inibidores de Proteases/metabolismo , Ligação Proteica , Conformação Proteica , SulfonamidasRESUMO
The conformations of cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-) in dimethyl sulfoxide-d6 (DMSO-d6) and water were determined using two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Comparisons were made between conformations of the cyclic pentapeptide in both solvents. The NMR study revealed that, while the backbone remained relatively unchanged in both solvents, the side-chains adopted distinctly different orientations in DMSO-d6 vs. H2O. A modeling study, minus NOE constraints, produced a set of low-energy conformers possessing agreement in backbone conformation with the NMR-derived structures; however, lowest-energy conformers did not have this agreement. These results show that different solvents can significantly affect the preferred side-chain conformation of small cyclic peptides in solution. This finding will impact the selection of solvent when determining structures for use as templates in rational drug design.
Assuntos
Dimetil Sulfóxido/química , Modelos Moleculares , Peptídeos Cíclicos/química , Conformação Proteica , Água/química , Sequência de Aminoácidos , Fenômenos Químicos , Físico-Química , Antagonistas dos Receptores de Endotelina , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Soluções , Solventes/química , TermodinâmicaRESUMO
The transferred nuclear Overhauser effect has been used to determine the biologically active conformations of two stromelysin inhibitors. Both inhibitors used in this study were hydroxamic acids generated via chemical synthesis. These structures, representing the conformation of each inhibitor bound to stromelysin, superimposed with excellent agreement. The study also provided information on the shape and orientation of the S2' and S1' pockets of the enzyme relative to thermolysin. Comparisons were made between stromelysin and thermolysin inhibitors to critically examine thermolysin as a template for stromelysin-inhibitor design. The enzyme-bound conformations of these stromelysin inhibitors were determined for use as a template in conformationally restricted drug design.