During the course of our optimization

During the course of our optimization of the diarylsulfonamide chemotype as FFA4 agonists, we identified xanthene as a potent and selective antagonist of FFA4 (). Xanthene was able to block the agonist induced intracellular Ca response elicited by both linoleic bifonazole synthesis and compound in competition studies run in FFA4 expressing U2OS cells (). While competitive antagonism maybe expected given the closely related structures of agonist and antagonist , further studies would be needed to confirm the precise nature of antagonism due to the potential for insurmountable antagonism in these studies. Pharmacological profiling of the responses of the selective FFA4 agonist and antagonist in both murine and human cell lines was next explored. Previous studies have shown that free fatty acids such as linoleic acid can produce a glucose-dependent increase in insulin secretion from the mouse insulinoma cell line MIN6. As our data demonstrate the presence of FFA4 gene expression in the MIN6 cell line, consistent with findings reported in the literature, we chose to examine the effects of compound on glucose-stimulated insulin secretion (GSIS) in MIN6 cells., , Compound produced a concentration-dependent increase in glucose stimulated insulin secretion () under high glucose conditions (25mM). The magnitude of the potentiation of insulin secretion observed under conditions of high glucose is noteworthy and is similar to that demonstrated in our laboratories with other insulin secretagogues including glibenclamide, linoleic acid, and FFA1 agonists. The enhancement in glucose-stimulated insulin secretion by was abolished in the presence of the selective FFA4 antagonist . Finally, effects on GLP-1 secretion utilizing compounds and were studied in the human intestinal cell line NCI-H716 (). While literature reports vary on the levels of FFA4 expression in the NCI-H716 cell line,, our in-house data was supportive of the presence of the receptor. Treatment with agonist agonist provided a modest increase in GLP-1 secretion which could be blocked with the addition of the antagonist . In summary, the SAR of a series of diarylsulfonamide FFA4 agonists was explored which resulted in the identification of a selective FFA4 agonist GSK137647A) and a selective FFA4 antagonist . While the tool compounds did not have appropriate properties for utilization in an in vivo setting, experiments in secretory cell lines detail the utility of this highly synthetically accessible class of low molecular weight diarylsulfonamides for the in vitro exploration of FFA4-mediated biology. Further improvements in physiochemical properties for this series will need to be realized in order to access tool compounds suitable for in vivo experimentation.
Introduction Zinc is the second-most abundant d-block metal in the body (after iron). It is considered essential for life in both eukaryotic and prokaryotic organisms, since it is required for development and function of all cells [[1], [2], [3], [4], [5]]. Zn2+ ions are estimated to be present in 10% of all mammalian proteins [6,7], where they can play structural (most notably in zinc finger motifs), catalytic or regulatory roles. Most recently, Zn2+ has been recognised as a signalling agent [2,8,9] and plays critical roles in determining the activity and fate of cells [10]. This may also at least partially account for the surprisingly high toxicity of uncomplexed (=free) Zn2+ [[11], [12], [13]]. For these reasons, Zn2+ homeostasis in all organisms including humans is tightly regulated [14]. Dietary zinc deficiency (hypozincemia) is commonly associated with impaired growth, infertility, and immunodeficiency [2], but an increasing number of genetic and other disorders are characterised by defective zinc transport into particular cells or their sub-compartments [4,15]. A particularly drastic example of genetically encoded zinc dyshomeostasis is the ‘lethal-milk syndrome’ in mice. This arises from a non-sense mutation in the gene for the Zn2+ transporter ZnT4, resulting in defective secretion of Zn2+ into breast milk [16]. In humans, a Gly-to-Arg mutation in the functionally analogous ZnT2 transporter leads to transient neonatal zinc deficiency [17]. Zinc dyshomeostasis may also be a hallmark of ageing and several neurological disorders [[18], [19], [20]]. Direct and indirect effects of zinc on hormonal signalling are also pervasive [8,21,22]. Furthermore, zinc is known to be crucial for immune system function in multiple ways [[23], [24], [25]]. Apart from the requirement of zinc for normal cellular functions [24], zinc also acts as a signalling agent to modulate (immune) cell activity [25]. Moreover, both zinc sequestration and zinc toxicosis are used as strategies by the human host during bacterial and fungal infections [26].