Inhibition of protein tyrosine phosphatase 1B by flavonoids: A structure - activity relationship study
Graphical abstract
Introduction
According to the International Diabetes Federation (IDF) (International Diabetes Federation, 2015), Diabetes mellitus (DM) is an important public health problem that affected 415 million people worldwide in 2015, and it is estimated that this number will increase to 642 million people in 2040. Type 2 DM (T2DM) represents the much more prevalent form of DM, accounting for 90–95% of the diabetes cases, and it is characterized by the combination of insulin action resistance and an inadequate compensatory insulin secretory response (American Diabetes Association, 2011). The development of new effective antidiabetic agents is therefore a permanent concern for the scientific community.
The classical non-transmembrane protein tyrosine phosphatase 1B (PTP1B) has emerged as a key negative regulator of insulin signaling pathways that leads to insulin resistance, turning this enzyme into a promising therapeutic target in the management of T2DM (Kennedy and Ramachandran, 2000, Feldhammer et al., 2013, Panzhinskiy et al., 2013, Qian et al., 2016). In brief, the binding of insulin to the α-subunits of its receptor leads to autophosphorylation of the β-subunits and the tyrosine phosphorylation of insulin receptor substrates (IRS). Phosphorylated IRS proteins serve as docking proteins for other secondary messengers, leading to the activation of protein kinase B (PKB), also known as Akt. The net effect of this pathway is to produce a translocation of the glucose transporter 4 (GLUT4) from cytoplasmic vesicles to the cell membrane in order to facilitate glucose transport (Fig. 1) (Youngren, 2007, Boucher et al., 2014, Bakke and Haj, 2015, Wang et al., 2015a). PTP1B is involved in the dephosphorylation and concomitantly inactivation of the insulin receptor (IR) and IRS, leading to the attenuation of the insulin signal. Thus, any changes in the expression levels or activity of this enzyme relative to IR can affect insulin signaling and contribute to the insulin resistance observed in T2DM (Kennedy and Ramachandran, 2000, Feldhammer et al., 2013, Panzhinskiy et al., 2013, Tamrakar et al., 2011-2014, Wang et al., 2015b, Sun et al., 2016). It was also described that PTP1B overexpression is closely related to a large number of diseases: liver disorders (Chen et al., 2015), obesity, cardiovascular complications, inflammation, endoplasmic reticulum (ER) stress and prostate and breast cancer (Feldhammer et al., 2013). Taking the above rationale into account, inhibition of PTP1B may modulate T2DM (and be potentially useful in the treatment of PTP1B related diseases), being an outstanding target for the treatment of this epidemic disease (Zhang and Lee, 2003, Zhang and Zhang, 2007). A large number of PTP1B inhibitors have been studied, the development of novel highly effective, selective and safe compounds still remains a challenge.
Flavonoids are phenolic compounds widely distributed in the Plant Kingdom and are important components of the human diet. The basic structural feature of flavonoids is a 2-phenyl-benzo-γ-pyrane nucleus consisting of two benzene rings (A and B rings) linked by a pyran heterocyclic ring (C ring) (Ribeiro et al., 2015a). Some flavonoids have already been described as inhibitors of PTP1B activity, such as morin (Paoli et al., 2013), quercetin (Jiang et al., 2015), luteolin (Choi et al., 2014a), apigenin (Choi et al., 2014b) and kaempferol (Paoli et al., 2013, Na et al., 2016). The main aim of the present work is to test a diversified panel of flavonoids (Fig. 2), with different types of substituents in different positions, including flavonoids that were never studied in what concerns their PTP1B inhibitory activity, establishing an accurate structure - activity relationship. For that purpose, an in vitro microanalysis screening system was used to test the panel of flavonoids. The type of inhibition of the most active flavonoids was also evaluated.
Section snippets
Chemicals
The following reagents were purchased from Sigma-Aldrich Co. LLC (St. Louis, USA): PTP1B (human recombinant), p-nitrophenyl phosphate (pNPP), dimethylsulfoxide (DMSO), citric acid, dithiotheritol (DTT), ethylenediaminetetraacetic acid (EDTA), compounds D3 (baicalein), D4 (apigenin), D5 (kaempferol), D6 (luteolin), D7 (quercetin), D8 (morin), D9 (acacetin), D10 (rutin), E1 (naringenin), E2 (eriodictyol), E3 (taxifolin) and ursolic acid. Compounds A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, C1, C2, C3
In vitro inhibition assay for PTP1B activity
The studied flavonoids were divided into five groups (A - E) (Table 1), according to their structures.
In group A, based on the structure of flavone (A1), the studied flavonoids presented no or low activity up to the highest tested concentration (200 μM). From the tested flavonoids in group B, all presenting a 5-OH group in A ring, it was observed that the presence of a 4′-OH group in B ring slightly improved PTP1B inhibition. In accordance, flavonoid B3 was the most potent (IC50 = 184 ± 13 μM).
Discussion
In the physiological environment, phosphorylation states are orchestrated by two dynamic and opposing groups of enzymes: protein tyrosine kinases (PTKs), which catalyse tyrosine phosphorylation, and protein tyrosine phosphatases (PTPs), which are responsible for dephosphorylation (Feldhammer et al., 2013, Zhang and Lee, 2003). The mechanism of phosphorylation/dephosphorylation of protein tyrosine residues, named protein tyrosine phosphorylation, is implicated in the control of fundamental
Conclusion
In this work some promising flavonoids scaffolds were found as potential effective PTP1B inhibitors for the treatment of T2DM. The obtained results clearly show that the nature, position, and number of substituents present in the flavonoid scaffold are determinant for the inhibition of PTP1B activity. The majority of the flavonoids were here studied for the first time, including the most effective one, flavonoid C13 (Fig. 4) that presented a mixed type of inhibition. These preliminary results
Conflicts of interest
The authors have declared no conflicts of interest.
Acknowledgments
The authors acknowledge the financial support from National funds [Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência (FCT/MEC)] and European Union funds [Fundo Europeu de Desenvolvimento Regional (FEDER)] under the program PT2020 (PT2020 UID/MULTI/04378/2013 - POCI/01/0145/FEDER/007728; QOPNA research unit FCT UID/QUI/00062/2013), the framework of QREN (NORTE-01-0145-FEDER-000024), and Programa Operacional Competitividade e Internacionalização (COMPETE) (
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