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1.
J Med Chem ; 62(3): 1626-1642, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30657666

RESUMO

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ß1c, ß1i, ß5c, and ß5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ß2c or ß2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the ß2c- and ß2i-selective compounds LU-002c (4; IC50 ß2c: 8 nM, IC50 ß2i/ß2c: 40-fold) and LU-002i (5; IC50 ß2i: 220 nM, IC50 ß2c/ß2i: 45-fold), respectively. Co-crystal structures with ß2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of ß2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.


Assuntos
Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Camundongos , Mutação , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Biblioteca de Peptídeos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/metabolismo , Ligação Proteica , Engenharia de Proteínas , Subunidades Proteicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Estereoisomerismo
2.
Cell Chem Biol ; 24(2): 218-230, 2017 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-28132893

RESUMO

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the ß5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of ß1 and ß2 to demonstrate that inhibiting a second site of the proteasome, particularly the ß2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both ß5 and ß2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual ß5 and ß2 inhibitors for the treatment of solid tumors.


Assuntos
Antineoplásicos/farmacologia , Fator 1 Nuclear Respiratório/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Fator 1 Nuclear Respiratório/metabolismo , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas
3.
Haematologica ; 100(10): 1350-60, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26069288

RESUMO

Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.


Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Org Chem ; 79(12): 5664-72, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24916888

RESUMO

Phlorins bearing different substituents at the sp(3)-hybridized meso-position were investigated. The extent to which different substituents at this unique position can influence phlorin spectroscopic properties, structure, and stability is of interest given that such substituents are not in direct conjugation with the phlorin macrocycle. While the effect of various substituents at the sp(2)-hybridized positions has been the subject of prior investigations, the impact of different substituents at the saturated carbon atom has not been systematically examined. In this study, phlorins with different combinations of geminal methyl and phenyl substituents were prepared in yields of 24-49% via dipyrromethane + dipyrromethanedicarbinol routes, and their NMR spectra, UV-vis spectra, X-ray crystal structures, and stability toward light and air were compared. The nature of the substituents at the sp(3)-hybridized position was found to impact spectroscopic properties, structure, and stability to varying degrees. Thus, the choice of substituents at the sp(3)-hybridized meso-position provides a further option for altering phlorin properties.


Assuntos
Porfirinas/química , Pirróis/química , Carbono/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares
5.
Anal Biochem ; 451: 1-3, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24486333

RESUMO

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.


Assuntos
Medições Luminescentes , Complexo de Endopeptidases do Proteassoma/análise , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Especificidade por Substrato , Tripsina/análise , Tripsina/metabolismo
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