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1.
J Pharmacol Exp Ther ; 356(3): 574-86, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26669426

RESUMO

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies.


Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Proteína Relacionada a TNFR Induzida por Glucocorticoide/agonistas , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
2.
Biosci Rep ; 34(6): e00168, 2014 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-25423174

RESUMO

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.


Assuntos
Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA Mensageiro/química , Timidilato Sintase/química , Sequência de Bases , Sítios de Ligação/genética , Códon de Iniciação/genética , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Mutagênese , Motivos de Nucleotídeos/genética , Ligação Proteica/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Raios Ultravioleta
3.
J Med Chem ; 57(5): 1694-707, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24138284

RESUMO

The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5' UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide, and small molecule ligands. Emphasis will be given to the IRES subdomain IIa, which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.


Assuntos
Hepacivirus/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Regiões 5' não Traduzidas , Hepacivirus/genética , Modelos Moleculares
4.
Bioorg Med Chem ; 21(20): 6139-44, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23602522

RESUMO

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.


Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Benzoxazóis/farmacologia , Hepacivirus/genética , RNA Viral/antagonistas & inibidores , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Sequência de Bases , Transferência Ressonante de Energia de Fluorescência , Genoma Viral , Hepacivirus/química , Programas de Rastreamento , Modelos Moleculares , Dados de Sequência Molecular , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/metabolismo , Transdução de Sinais
5.
Chembiochem ; 13(18): 2738-44, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23143777

RESUMO

Thymidylate synthase (TS) is a key enzyme in the biosynthesis of thymidine. The use of TS inhibitors in cancer chemotherapy suffers from resistance development in tumors through upregulation of TS expression. Autoregulatory translation control has been implicated with TS overexpression. TS binding at its own mRNA, which leads to sequestration of the start codon, is abolished when the enzyme forms an inhibitor complex, thereby relieving translation suppression. We have used the protein-binding site from the TS mRNA in the context of a bicistronic expression system to validate targeting the regulatory motif with stabilizing ligands that prevent ribosomal initiation. Stabilization of the RNA by mutations, which were studied as surrogates of ligand binding, suppresses translation of the TS protein. Compounds that stabilize the TS-binding RNA motif and thereby inhibit ribosomal initiation might be used in combination with existing TS enzyme-targeting drugs to overcome resistance development during chemotherapy.


Assuntos
Sequências Reguladoras de Ácido Ribonucleico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Timidilato Sintase/genética , Sequência de Bases , Genes Reporter/genética , Humanos , Sequências Repetidas Invertidas/efeitos dos fármacos , Ligantes , Motivos de Nucleotídeos/efeitos dos fármacos , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo
6.
Proc Natl Acad Sci U S A ; 109(14): 5223-8, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22431596

RESUMO

The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution. Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.


Assuntos
Hepacivirus/genética , RNA Viral/química , Riboswitch , Benzimidazóis/farmacologia , Transferência Ressonante de Energia de Fluorescência , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas/efeitos dos fármacos
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