In our H K ATPase assay

In our H+, K+-ATPase assay (Fig. 1 and Supplementary Fig. 1), myricetin (IC50 = 0.58μM) was more potent than acid-activated omeprazole (IC50 = 1.50μM). In contrast, in oral administration in mice (Fig. 3), omeprazole (20mg/kg) more effectively inhibited gastric ApoBrdU DNA Fragmentation Assay Kit secretion compared to myricetin (50mg/kg). The discrepancy between in vitro and in vivo assessments may reflect unfavorable pharmacokinetic features of natural flavonoids. The oral bioavailability of myricetin has been reported to be 9.6% in rat (Dang et al., 2014), which is likely due to its instability in the gastrointestinal tract, which harbors bacterial flora (Xiang et al., 2017). Additionally, myricetin is easily converted into various derivatives after being taken up by the body (Lin et al., 2012). In contrast, the reported bioavailability of omeprazole is 9.6–70% in human and experimental animals (Larsson et al., 1983, Stedman and Barclay, 2000, Watanabe et al., 1994). Proton pump inhibitors, including omeprazole, are widely used for the clinical treatment of gastric acid-related disorders (Schubert, 2010), but recent studies indicate that these inhibitors increase the risk of other diseases, including cardiovascular events (Cardoso et al., 2015), bone fracture (Leontiadis and Moayyedi, 2014), infection (Sultan et al., 2008), chronic kidney disease (Klatte et al., 2017), and dementia (Goldstein et al., 2017). To develop new proton pump inhibitors with less adverse effects, myricetin could serve as a useful seed compound. It may be valuable to attempt to improve the bioavailability of myricetin, as well as its potency in proton pump inhibition, by chemical modification. In conclusion, the natural flavonoid myricetin inhibited H+, K+-ATPase and attenuated gastric acid secretion. Based on this study, myricetin might provide a promising backbone structure for developing new drugs and supplements for alleviating gastric acid-related diseases.
Acknowledgements This work was supported in part by the MEXT/JSPS KAKENHI Grant Number 15H04676, 15H05652, Platform for drug discovery, informatics and structural life science, the Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Salt Science Research Foundation. We thank members of Drug Discovery Initiative, The University of Tokyo for their useful suggestions about chemical screening. This research is partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research funded by Japan Agency for Medical Research and Development (AMED).
Introduction All cells have an array of sophisticated membrane-bound enzymatic systems which perform different essential processes for life. These processes range from regulation of the intracellular milieu to the genesis of information transfer and communication between cells. ATPases (EC 3.6.1.3) which are categorized as one of the primary active transporters are a main functional class of these membrane-bound enzymes. They catalyze the transport of molecules against an electrochemical potential by reactions directly linked to the hydrolysis of ATP (Chow and Forte, 1995). The ATPases which actively transport cations have been classified into three distinct families: F-type ATPases (F-ATPases), V-type ATPases (V-ATPases) and P-type ATPases (P-ATPases) (Pedersen and Carafoli, 1987). Each group members share common structure and characteristics that can prove shared evolutionary ancestry (Chow and Forte, 1995). There is a difference among P-ATPase with the others, so that, the formation of a phosphorylated intermediate throughout the catalytic cycle is a characteristic of P-ATPases and differentiates them from V-ATPases and F-ATPases (Pedersen and Carafoli, 1987). The P-ATPase family is an enormous, physiologically key family of membrane proteins existing in all living cells, which can be divided into two main groups, P1 and P2, based on cation specificity. Members of the P1 group transport heavy metals, like Cu2+, Cd2+, and Hg2+, whereas the members of the P2 group transport an extensive range of monovalent and divalent cations, such as H+, Na+, K+, Mg2+ and Ca2+ (Magalhães et al., 2005). Consequently, P-ATPases are a huge superfamily of ion pumps which use the energy of ATP hydrolysis to fuel the transmembrane transport of charged substrates across biological membranes. The “P” stands for phosporylation, since a hallmark of the more than 300 members of this family is the reversible formation of an acyl-intermediate in which the g-phosphate of the enzymatically hydrolyses ATP is covalently linked to a highly conserved Asp side-chain. This Asp residue is part of the DKTGTLT-sequence motif which is located in the so-called “P-domain” and describes the membership of the P-ATPase family. Two other conserved motifs related to the phosphorylation and subsequent dephosphorylation reaction are the GDGXNDXP motif, present exactly after the ATP binding domain (N-domain, for “nucleotide-binding domain”) while the TGES sequence situated to the so-called actuator-domain (A-domain) (Dürr, 2009). Members of P-ATPase family are characterized via the formation of a phosphorylated intermediate during the enzymatic cycle, by a common membrane topology and domain organization, and by conserved sequence motifs implicated in ATP hydrolysis and phosphorylation (Kühlbrandt, 2004, Møller et al., 1996).