The Antidiabetic Drug Lobeglitazone Has the Potential to Inhibit PTP1B T Activity ⁎ Ruth F

Bioorganic Chemistry 100 (2020) 103927 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg The antidiabetic drug lobeglitazone has the potential to inhibit PTP1B T activity ⁎ Ruth F. Rochaa, Tiago Rodriguesc, Angela C.O. Menegattia,b, , Gonçalo J.L. Bernardesc,d, Hernán Terenzia a Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Campus Trindade, 88040-900 Florianópolis, SC, Brazil b Universidade Federal do Piauí, CPCE, 64900-000 Bom Jesus, PI, Brazil c Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisbon, Portugal d Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK ARTICLE INFO ABSTRACT Keywords: Protein tyrosine phosphatase 1B (PTP1B) is considered a potential therapeutic target for the treatment of type 2 Thiazolidinediones diabetes mellitus (T2DM), since this enzyme plays a significant role to down-regulate insulin and leptin sig- Lobeglitazone nalling and its over expression has been implicated in the development of insulin resistance, T2DM and obesity. PPAR-γ Some thiazolidinediones (TZD) derivatives have been reported as promising PTP1B inhibitors with anti hy- PTP1B perglycemic effects. Recently, lobeglitazone, a new TZD, was described as an antidiabetic drug that targetsthe Non-competitive inhibitors PPAR-γ (peroxisome γ proliferator-activated receptor) pathway, but no information on its effects on PTP1B have been reported to date. We investigated the effects of lobeglitazone on PTP1B activity in vitro. Surprisingly, lobeglitazone led to moderate inhibition on PTP1B (IC50 42.8 ± 3.8 µM) activity and to a non-competitive reversible mechanism of action. As lobeglitazone inhibits PTP1B activity in vitro, we speculate that it could also target PTP1B signalling pathway in vivo and thus contribute to potentiate its antidiabetic effects. 1. Introduction as a negative regulator of both signalling pathways, where their inhibition would restore a normal hormonal response [18]. Protein tyrosine phosphatases (PTPs) as potential therapeutic tar- PTP1B is one such target that counteracts tyrosine phosphorylation gets have been an important topic in biomedical science for the last two events by promoting insulin signalling and simultaneously interrupts decades, because a large number of PTPs dysfunctions are associated glycaemic control. Hence, PTP1B inhibition has been validated as a with several human diseases, such as cancer, diabetes, autoimmune and potential therapeutic strategy to enhance insulin sensitivity, and in- neurological diseases [1–6]. Several years of study revealed that protein hibitors of this enzyme are being suggested as potent antidiabetic drugs tyrosine phosphatase 1B (PTP1B), which is a negative regulator of in- [19]. Numerous PTP1B inhibitors have been reported so far, including sulin and leptin signalling pathway [5–14], plays an important role in natural products, synthetic heterocyclic or hybrid compounds, but only the development of many diseases, including diabetes and obesity. a few have reached clinical trials. However, none of them have reached Knockout studies in mice models show that animals that do not express the market yet [20]. PTP1B present insulin hypersensitivity and low blood glucose levels There are currently several treatment options available to control [14,15]. Furthermore, the gene encoding PTP1B is located in chromo- T2DM, in particular antidiabetic agents that act as hypoglycemic drugs, some 20q13, a genomic region associated with type 2 diabetes mellitus such as thiazolidinediones (TZDs), which are synthetic peroxisome γ (T2DM) [16]. Also, PTP1B overexpression contributes to the develop- proliferator-activated receptor (PPAR-γ) agonists [21–23]. Activation of ment of insulin resistance, T2DM and obesity [17]. Insulin exerts its PPAR-γ increases glucose uptake and use in peripheral organs, in ad- effects when it binds to insulin receptor protein tyrosine kinase, which dition to fatty acid storage in adipocytes, insulin signalling and de- initiates signalling pathways, particularly those that induce phosphor- creases liver gluconeogenesis, thereby improving insulin sensitivity ylation. Diabetes is a disease, related to insulin resistance, in which [21,24,25]. TZDs also affect a range of activities, including cell pro- signalling responses to hormones are attenuated [13]. According to liferation, apoptosis, inflammation, and responses to oxidative stress Krishnan et al. an ideal therapeutic target would be one that functions [26,27]. Although effective, TZDs, such as rosiglitazone and ⁎ Corresponding author. E-mail address: [email protected] (A.C.O. Menegatti). https://doi.org/10.1016/j.bioorg.2020.103927 Received 7 January 2020; Received in revised form 3 April 2020; Accepted 8 May 2020 Available online 11 May 2020 0045-2068/ © 2020 Elsevier Inc. All rights reserved. R.F. Rocha, et al. Bioorganic Chemistry 100 (2020) 103927 Fig. 1. Structure of Lobeglitazone. pioglitazone, are associated with weight gain, increased risk of heart failure, and bladder cancer among others [28–30]. Recently, a new PPAR-γ agonist was developed with a TZD moiety and substituted pyrimidines, lobeglitazone (Duvie®; Fig. 1) is structu- rally similar to rosiglitazone and pioglitazone, two well-known TZD drugs [31–36]. Phase III trials have shown that glycated haemoglobin Fig. 2. Michaelis–Menten chart that represents the inhibition mechanism for levels were decreased following lobeglitazone monotherapy and as a lobeglitazone against PTP1B. complement to metformin therapy. This drug was approved by the Ministry of Food and Pharmaceutical Safety (Korea) in 2013 and has PTP-based drug discovery [18]. Thus highlighting the importance of the since been used as a daily treatment of T2DM [37,38]. As PTP1B in- search for non-competitive inhibitors of these enzymes, mainly because hibition improves the sensitivity to insulin and some TZD analogues their active sites are well conserved among the family [48,49]. have been reported as PTP1B inhibitors [39–45], we sought to de- Besides, different types of inhibitions are described for TZD in- termine the effect of lobeglitazone on PTP1B activity in vitro. Lobegli- hibitors of PTP1B, which includes non-competitive mechanisms, but all tazone is used as an antidiabetic drug that targets PPAR-γ pathway, but the reports indicate reversible inhibition [40–42,46]. Thus, to de- no information regarding its effects on PTP1B is reported. termine whether the inhibition was caused by the formation of a reversible or irreversible enzyme-inhibitor complex, we investigated the 2. Results and discussion recovery of the phosphatase activity by a pre-incubation and dilution assay [50]. The results demonstrated that the recovery of activity for Initially, in vitro inhibitory activity of lobeglitazone against PTP1B lobeglitazone was 93.0 ± 5.0%, which means it behaves as a re- was assessed spectrophotometrically at 40 µM using pNPP as substrate. versible inhibitor of PTP1B (Fig. 3). This also potentially excludes the Lobeglitazone reduced 41.5 ± 2.7% of enzymatic activity with an IC50 formation of colloidal aggregates by lobeglitazone that could lead to value of 42.8 ± 3.8 µM, which means it is a moderate inhibitor of local enzyme denaturation and false positive readouts [59]. PTP1B. To corroborate the results of the kinetic analysis, we conducted a Several TZD derivatives have been reported as PTP1B inhibitors limited proteolysis assay to understand the availability of the active site with antihyperglycemic and antiobesity effects [39–45]. Liu et al. dis- of the enzyme for proteolysis. First, we analyzed the PTP1B peptide covered and optimized a series of new PTP1B inhibitors that contain mass fingerprint (PMF) in the presence and absence of lobeglitazone. TZD-substituted biphenyl scaffold and further evaluated the inhibitory Approximately 86.3% of the PTP1B sequence was covered in the PMF effects of these compounds in vitro and in vivo [44]. Among them, analysis in the presence and 76.3% in the absence of lobeglitazone. By compound (Z)-5-((4′-(benzyloxy)biphenyl-4-yl)methylene)-2-thiox- comparing the PMF spectra, we observed that peptides of the PTP1B othiazolidin4-one (7Fb), derived from 4 oxothiazolidine-2-thione, ex- active site were not protected by the presence of the compound, the hibited an IC50 value of 0.69 ± 0.07 μM for PTP1B. The compound peaks 2175 and 2430 m/z (Fig. S1 and S2 Supplementary material) significantly decreased postprandial and fasting glucose levels, and remain the same, which confirms that lobeglitazone is a non-competi- blood glucose levels decreased more rapidly than in mice treated with tive inhibitor, however further studies are needed to determine the metformin, which demonstrates its potency as an antidiabetic agent. A binding site of this inhibitor on the enzyme. number of new 5-benzene lidene-2,4-thiazolidinedione derivatives have Our results reveal that lobeglitazone has an inhibitory activity of been designed and synthesized by Wang et al. based on their previous PTP1B in vitro, thus despite its weaker PTP1B activity, this partial in- studies [45]. Compound (Z)-5-(5-bromo-4-((3-methylbut-2-en-1-yl) hibition could contribute positively to the effects observed with its oxy)-2-methoxybenzylidene)-3-isopropylthiazolidine-2, 4-dione (7e) primary target PPAR-γ. Some studies have shown the effect of insulin exhibited

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