Carneiro Vânia M T, Trivella Daniela B B, Scorsato Valéria, Beraldo Viviane L, Dias Mariana P, Sobreira Tiago J P, Aparicio Ricardo, Pilli Ronaldo A
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, 13084-971 Campinas, SP, Brazil.
National Center for Research in Energy and Material, Brazilian Biosciences National Laboratory, 13083-100 Campinas, SP, Brazil.
Eur J Med Chem. 2015 Jun 5;97:42-54. doi: 10.1016/j.ejmech.2015.04.036. Epub 2015 Apr 20.
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 1, 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.
RK - 682 (1)是一种天然产物，已知其能选择性抑制蛋白酪氨酸磷酸酶（PTPases），并在体外试验中作为磷酸酶抑制的商业阳性对照使用。蛋白磷酸酶与包括糖尿病、癌症和炎症在内的多种人类疾病相关，被认为是药物开发的重要靶点。在此，我们报道了外消旋RK - 682（rac - 1）及一组重点化合物的合成，包括1的外消旋类似物、二氢吡喃酮和C - 酰化丙二酸亚异丙酯衍生物，后者可从市售起始原料通过一步合成得到。我们进一步表征了一些代表性化合物在水溶液中的行为，并评估了它们与PTPase的结合及抑制作用。我们的数据表明，rac - 1和一些衍生物能够在溶液中形成大聚集体，其聚集能力取决于酰基侧链大小。然而，化合物聚集本身并不能促进PTPase抑制。我们的数据揭示了一类新型的PTPase抑制剂（C - 酰化丙二酸亚异丙酯衍生物），并且rac - 1和具有暴露的潜在负电荷亚结构（例如：1的特窗酸核心）的衍生物能够结合到PTPase结合位点，也能杂乱地结合到蛋白质表面。化合物结合蛋白质的能力及其在溶液中聚集的内在能力似乎对于促进酶聚集从而抑制酶活性至关重要。我们还观察到，酶缓冲溶液中常用的二价阳离子，如镁，会消耗rac - 1的抑制活性，从而影响酶抑制实验。总体而言，这些数据有助于表征rac - 1及其衍生物抑制PTPase的机制，揭示酶抑制并非如文献中普遍接受的那样仅依赖于化合物与PTPase催化位点的结合。此外，我们的结果指出了一些杂乱的机制，这些机制显著影响了rac - 1对酶抑制的体外评估。因此，我们建议在使用天然或合成的RK - 682 (1)作为评估PTPase抑制和选择性的内部对照时要谨慎，因为许多因素可能会调节表观酶抑制作用。
