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Is RK-682 a promiscuous enzyme inhibitor? Synthesis and in vitro evaluation of protein tyrosine phosphatase inhibition of racemic RK-682 and analogues

Texto completo
Carneiro, Vania M. T. [1] ; Trivella, Daniela B. B. [2] ; Scorsato, Valeria [1] ; Beraldo, Viviane L. [2] ; Dias, Mariana P. [1] ; Sobreira, Tiago J. P. [2] ; Aparicio, Ricardo [1] ; Pilli, Ronaldo A. [1]
Número total de Autores: 8
Afiliação do(s) autor(es):
[1] Univ Estadual Campinas, Inst Chem, UNICAMP, BR-13084971 Campinas, SP - Brazil
[2] Brazilian Biosci Natl Lab, Natl Ctr Res Energy & Mat, BR-13083100 Campinas, SP - Brazil
Número total de Afiliações: 2
Tipo de documento: Artigo Científico
Citações Web of Science: 4

RK-682 (1) is a natural product known to selectively inhibit protein tyrosine phosphatases (PTPases) and is used commercially as a positive control for phosphatase inhibition in in vitro assays. Protein phosphatases are involved in several human diseases including diabetes, cancer and inflammation, and are considered important targets for pharmaceutical development. Here we report the synthesis of racemic RK-682 (rac-1) and a focused set of compounds, including racemic analogues of I, dihydropyranones and C-acylated Meldrum's acid derivatives, the later obtained in one synthetic step from commercially available starting material. We further characterized the behavior of some representative compounds in aqueous solution and evaluated their in vitro PTPase binding and inhibition. Our data reveal that rac-1 and some derivatives are able to form large aggregates in solution, in which the aggregation capacity is dependent on the acyl side chain size. However, compound aggregation per se is not able to promote PTPase inhibition. Our data disclose a novel family of PTPase inhibitors (C-acylated Meldrum's acid derivatives) and that rac-1 and derivatives with an exposed latent negatively charged substructure (e.g.: the tetronic acid core of 1) can bind to the PTPase binding site, as well promiscuously to protein surfaces. The combined capacity of compounds to bind to proteins together with their intrinsic capacity to aggregate in solution seems essential to promote enzyme aggregation and thus, its inhibition. We also observed that divalent cations, such as magnesium frequently used in enzyme buffer solutions, can deplete the inhibitory activity of rac-1, thus influencing the enzyme inhibition experiment. Overall, these data help to characterize the mechanism of PTPase inhibition by rac-1 and derivatives, revealing that enzyme inhibition is not solely dependent on compound binding to the PTPase catalytic site as generally accepted in the literature. In addition, our results point to promiscuous mechanisms that influence significantly the in vitro evaluation of enzyme inhibition by rac-1. Therefore, we recommend caution when using natural or synthetic RK-682 (1) as an internal control for evaluating PTPase inhibition and selectivity, since many events can modulate the apparent enzyme inhibition. (C) 2015 Elsevier Masson SAS. All rights reserved. (AU)

Processo FAPESP: 13/07607-8 - CMPO - Centro Multidisciplinar de Pesquisa em Obesidade e Doenças Associadas
Beneficiário:Licio Augusto Velloso
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
Processo FAPESP: 11/00457-5 - Síntese de diidropiranonas, avaliação da atividade inibidora de CDC25, da citotoxicidade em células tumorais e estudos estruturais
Beneficiário:Vânia Maria Teixeira Carneiro
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 09/51602-5 - Biologia química: novos alvos moleculares naturais e sintéticos contra o câncer, estudos estruturais, avaliação biológica e modo de ação
Beneficiário:Ronaldo Aloise Pilli
Linha de fomento: Auxílio à Pesquisa - Temático
Processo FAPESP: 10/17544-5 - Bases moleculares e estruturais do reconhecimento de inibidores pelas proteínas fosfatases humanas CDC-25 e LMW-PTP envolvidas em câncer
Beneficiário:Daniela Barretto Barbosa Trivella
Linha de fomento: Bolsas no Brasil - Pós-Doutorado