Identification of novel inhibitors of human SPCA2.
To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 μM and 3.8 μM, respectively.
Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan. Electronic address: s_yamamoto@ks.kyorin-u.ac.jp.
Sachiko
S
Yamamoto
Department of Materials Science, The University of Shiga Prefecture, Hassaka, Hikone 522-8533, Japan.
Munenori
M
Takehara
Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Yoshiki
Y
Kabashima
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Toshiyuki
T
Fukutomi
Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Makoto
M
Ushimaru
D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D009829:Oleic Acids / Q000737:chemistry*; D011485:Protein Binding; D019284:Thapsigargin / Q000737:chemistry*
D004791:Enzyme Inhibitors; D009829:Oleic Acids; D019284:Thapsigargin; C452183:ATP2C2 protein, human; D000252:Calcium-Transporting ATPases
Ca(2+)/Mn(2+) pump; Inhibitor; Primary fatty acid amide; Secretary pathway Ca(2+)-ATPase 2; Signaling lipid