Is RK-682 a promiscuous enzyme inhibitor? Synthesis and in vitro evaluation of protein tyrosine phosphatase inhibition of racemic RK-682 and analogues.
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.
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, 13084-971 Campinas, SP, Brazil.
Vânia M T
VM
Carneiro
National Center for Research in Energy and Material, Brazilian Biosciences National Laboratory, 13083-100 Campinas, SP, Brazil.
Daniela B B
DB
Trivella
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, 13084-971 Campinas, SP, Brazil.
Valéria
V
Scorsato
National Center for Research in Energy and Material, Brazilian Biosciences National Laboratory, 13083-100 Campinas, SP, Brazil.
Viviane L
VL
Beraldo
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, 13084-971 Campinas, SP, Brazil.
Mariana P
MP
Dias
National Center for Research in Energy and Material, Brazilian Biosciences National Laboratory, 13083-100 Campinas, SP, Brazil.
Tiago J P
TJ
Sobreira
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, 13084-971 Campinas, SP, Brazil.
Ricardo
R
Aparicio
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, 13084-971 Campinas, SP, Brazil. Electronic address: pilli@iqm.unicamp.br.
Ronaldo A
RA
Pilli
D001665:Binding Sites; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D013237:Stereoisomerism
D004791:Enzyme Inhibitors; C094097:RK 682; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases
Enzyme inhibition; Meldrum's acid derivatives; Natural 3-acyl-5-hydroxymethyltetronic acid; Promiscuous enzyme inhibition; RK-682; Tyrosine phosphatases inhibitors