RESUMO
Patients with melanoma with activating BRAF mutations (BRAF V600E/K) initially respond to combination therapy of BRAF and MEK inhibitors. However, their clinical efficacy is limited by acquired resistance, in some cases driven by amplification of the mutant BRAF gene and subsequent reactivation of the MAPK pathway. DS03090629 is a novel and orally available MEK inhibitor that inhibits MEK in an ATP-competitive manner. In both in vitro and in vivo settings, potent inhibition of MEK by DS03090629 or its combination with the BRAF inhibitor dabrafenib was demonstrated in a mutant BRAF-overexpressing melanoma cell line model that exhibited a higher MEK phosphorylation level than the parental cell line and then became resistant to dabrafenib and the MEK inhibitor trametinib. DS03090629 also exhibited superior efficacy against a melanoma cell line-expressing mutant MEK1 protein compared with dabrafenib and trametinib. Biophysical analysis revealed that DS03090629 retained its affinity for the MEK protein regardless of its phosphorylation status, whereas the affinity of trametinib declined when the MEK protein was phosphorylated. These results suggest that DS03090629 may be a novel therapeutic option for patients who acquire resistance to the current BRAF- and MEK-targeting therapies.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Humanos , Trifosfato de Adenosina , MAP Quinase Quinase 1/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Oximas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
P2X4 receptor is known to be involved in neuropathic pain. In order to detect the expression of P2X4 receptor on microglia at the time of onset of neuropathic pain, one approach consists on the preparation of the monoclonal antibodies with both selective binding and high affinity. We have recently established a monoclonal antibody (named 12-10H) which had high affinity to rat P2X4 receptor expressed in 1321N1 cells. The dissociation constants of the complex between the monoclonal antibodies obtained so far and the head domain (HD) in the rat P2X4 receptor were in the nanomolar range. To improve the affinity by rational mutations, we need to know the precious location of the binding site in these monoclonal antibodies. Here, we have analysed and identified the binding residues in the monoclonal antibody (12-10H) with high affinity for the HD of the rat P2X4 receptor by site-directed mutagenesis.
Assuntos
Anticorpos Monoclonais Murinos/química , Afinidade de Anticorpos , Receptores Purinérgicos P2X4/química , Animais , Anticorpos Monoclonais Murinos/genética , Domínios Proteicos , Ratos , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/imunologiaRESUMO
P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4's head domain (rHD, Gln111-Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.
