Polyoxometalates—Potent and selective ecto-nucleotidase inhibitors
Graphical abstract
Introduction
Polyoxometalates (POMs) are negatively charged inorganic compounds which contain early transition metal ions such as tungsten (W), molybdenum (Mo), niobium (Nb), antimony (Sb) or vanadium (V), surrounded by oxygen atoms [1], [2], [3]. In POMs transition-metal ions are typically in their highest oxidation state [4]. Those anionic complexes are relatively stable, some of them are even highly stable in aqueous solutions at physiological pH values [4]. The diversity in structure and composition of POMs allows for a wide versatility in terms of shape, polarity, redox potential, surface charge distribution, and acidity, resulting in many possible applications in the fields of catalysis, electronics, magnetic materials and nanotechnology [5], [6]. Furthermore, POMs have been shown to exhibit biological activities in vitro as well as in vivo, including anticancer [7], [8], antibacterial [9], [10], antiprotozoal [11], antiviral[12], [13], and antidiabetic activities [1], [14]. However, their biological mechanisms of action at the molecular level are not well understood. Due to their anionic character and their high negative charge at physiologic pH values, POMs will hardly be able to penetrate cells [15]. Therefore, it has been speculated that POMs are likely to act extracellularly inhibiting several different enzyme families such as phosphatases [11], kinases [15], sulfotransferases [9], sialyltransferases [9], and ecto-nucleotidases [16], [17], [18], [19], [20], which are mostly located on the plasma membrane and display extracellular binding sites [21].
Ecto-nucleotidases regulate extracellular levels of nucleotides by catalyzing their hydrolysis, eventually leading to the formation of the respective nucleosides and inorganic phosphates [22], [23], [24], [25]. Since extracellular nucleosides and nucleotides act as signaling molecules in almost all tissues and organs by activating P1 and P2 purinergic receptors, respectively, ecto-nucleotidases have recently gained considerable interest due to their important roles in modulating purinergic signal transduction [26], [27], [28]. At least four protein families display ecto-nucleoside activity: ecto-nucleoside triphosphate diphosphohydrolases (NTPDases, EC 3.6.1.5), ecto-5′-nucleotidase (eN, EC 3.1.3.5), ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs, EC 3.1.4.1, and EC 3.6.1.9), and alkaline phosphatases (APs, EC. 3.1.3.1) [26], [27], [29]. Nucleoside 5′-triphosphates and 5′-diphosphates are hydrolyzed by members of the E-NTPDase family, the E-NPP family, and by APs (e.g. ATP or ADP to AMP), whereas nucleoside 5′-monophosphates are hydrolyzed by APs and eN (e.g. AMP to adenosine) [26], [30], [31].
Many cancer cells show an overexpression of NTPDases and eN leading to increased extracellular adenosine levels. Since adenosine promotes angiogenesis, tumor growth, and immunosuppression, inhibitors of eN and NTPDases have considerable potential for the treatment of various diseases which are associated with elevated adenosine concentrations, e.g. cancer or immunodeficiency disorders [17], [31], [32]. Beside mammalian NTPDases, microbial NTPDases have been found to be expressed by some important pathogens (e.g. Legionella pneumophila) and reported to contribute to their virulence. Therefore NTPDase inhibitors have been further proposed as novel anti-bacterial therapeutics [16], [26], [33], [34], [35].
NPPs have been involved in various biological processes including bone mineralization, blood coagulation and regulation of insulin receptor signaling. Moreover, expression of NPPs in cancer cells has been demonstrated to promote angiogenesis, lymphocyte trafficking and tumor growth [29], [36], [37], [38], [39]. Thus inhibition of NPPs has been proposed as a new potential therapeutic strategy, e.g., for the treatment of diabetes and cancer.
Tissue-nonspecific alkaline phosphatase (TNAP) ensures normal bone mineralization by hydrolyzing extracellular diphosphate (PPi), a potent inhibitor of hydroxyapatite formation [40], [41], [42], [43]. TNAP levels rise when bones are growing due to bone fractures or tumors [44]. TNAP is further involved in pathophysiological abnormalities, which can lead to ankylosis, vascular calcification, and osteoarthritis [45], [46]. Therefore TNAP inhibitors may be therapeutically useful for the treatment of diseases such as arterial calcification, ankylosis and cancer. Furthermore potent and selective inhibitors are required as pharmacological tools to further investigate the (patho)physiological roles of ecto-nucleotidases in various tissues and pathological contexts.
We previously identified several polyoxotungstates as potent inhibitors of rat NTPDases with submicromolar potency [16]. Subsequently, POMs have been applied in a number of biological studies to inhibit NTPDases [17], [18], [19], [20]. The compound [H2W12O40]6−, designated POM1, has been used in physiological studies, where it was found to increase infarct sizes and abolish the protective effects of cardiac and renal ischemia preconditioning by NTPDase1 inhibition [17]. This compound has been subsequently tested in rat cerebellar and hippocampal slice preparations and was found to be more effective in blocking ATP breakdown than the standard NTPDase1 inhibitor (ARL 67156) [18]. Additionally, the inhibition of NTPDase1 activity by POM1 significantly inhibited hepatic metastatic colonic tumour growth in mice [19]. Recently, PV4 (or POM4, [TiW11CoO40]8−), which was found to be a particularly potent inhibitor of rat NTPDase2 [16], was shown to inhibit the hydrolysis of the extracellular ATP under physiological and ischemic conditions in vivo in the rat striatum [20]. Also, crystallographic studies of two different POMs (POM1 and (NH4)6[Mo7O24]) demonstrated that the negatively charged POMs bind electrostatically to the positively charged domain of rat NTPDase1 [47]. Inhibition of enzyme function by POMs was thereby proposed to be induced by changes in the Y409 substrate-binding loop structure restricting enzyme flexibility [47]. Very recently, POM1, a potent inhibitor of rat NTPDase1, and POM6 ([NaSb9W21O86]18−), a selective inhibitor of rat NTPDase2 and -3 [16], have been investigated in plasma and blood cell samples from human volunteers, and the ADPase activity in hematocytes was shown to be primarily blocked by POM1, while its activity in plasma was potently inhibited by POM6. This suggests the presence of NTPDase1 on cell membranes of hematocytes and the presence of NTPDase2 or -3 in blood plasma [48].
Although a number of reports have documented the inhibitory effect of POMs on NTPDases, their effect on the other ecto-nucleotidases have scarcely been investigated so far. Moreover, the previous enzyme inhibition studies were performed only on rat, but not on human NTPDases. In the present study, we investigated the inhibitory potency of an extended series of 16 POMs and structurally related chalcogenide hexarhenium cluster complexes on human NTPDases. In addition, their inhibitory activity on further ecto-nucleotidases, including human NPP1, -2, and -3, TNAP, and rat ecto-5′-nucleotidase (eN) was investigated, and selectivity profiles of the compounds were established. This led to the discovery of the first POM inhibitor of NPP1 and the most potent and selective NPP1 inhibitors known to date. Finally, we analyzed their mechanism of inhibition of the various ecto-nucleotidases.
Section snippets
Materials
2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), levamisole hydrochloride and tris(hydroxymethyl)aminomethane (Tris) were purchased from Applichem (Darmstadt, Germany). pAcGP67-A and pAcG2T baculovirus expression vectors were purchased from BD BaculoGold (Heidelberg, Germany). The mouse monoclonal ADP antibody (3 mg/mL), the monoclonal AMP/GMP antibody (1.2 mg/mL), the ADP AlexaFluor® 633 tracer (400 nM), the AMP/GMP AlexaFluor® 633
Results and discussion
In the present study we investigated the inhibitory potency of a series of 12 different polyoxometalates (POMs) and of four chalcogenide hexarhenium cluster compounds on a broad range of ecto-nucleotidases. Table 1 lists the investigated POMs including twelve polyoxotungstates (1–12) and four rhenium clusters (13–16). Compounds 1–4 and 7 are Keggin structure complexes, 5 is a trivacant Keggin-derived sandwich complex, 6 features a crypt-like structure, 8 a Preyssler-structure, 9 is a
Acknowledgments
J.S. was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP-102472, MOP-93683) and he was also a recipient of a “Chercheur National” award from the Fonds de recherche du Québec–Santé (FRQS).
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2022, Coordination Chemistry ReviewsCitation Excerpt :In the last 30 years, the number of studies of polyoxidometalates (POMs, previously named polyoxometalates) and polyoxidovanadates (POVs, previously named polyoxovanadates) associated with enzymatic inhibition [1–5] and diseases, including insulin enhancement agents [6–9] for diabetes mellitus, and inhibitors of the aggregation of amyloid β-peptides associated with Alzheimer's disease have clearly increased [10,11].
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Both authors contributed equally to this study.