title: The Ca2+-ATPase (SERCA1) is inhibited by 4-aminoquinoline derivatives through interference with catalytic activation by Ca2+, whereas the ATPase E2 state remains functional.
abstract: Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to μm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the μm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.3 and 8.0 μm as demonstrated by measurements of steady state ATPase activity as well as single cycle charge transfer. Characterization of sequential reactions comprising the ATPase catalytic and transport cycle then demonstrated that NF1058, and similarly CLT, interferes with the mechanism of Ca(2+) binding and Ca(2+)-dependent enzyme activation (E(2) to E(1)·Ca(2) transition) required for formation of phosphorylated intermediate by ATP utilization. On the other hand, Ca(2+) independent phosphoenzyme formation by utilization of P(i) (i.e. reverse of the hydrolytic reaction in the absence of Ca(2+)) was not inhibited by NF1058 or CLT. Comparative experiments showed that the high affinity inhibitor thapsigargin interferes not only with Ca(2+) binding and phosphoenzyme formation with ATP but also with phosphoenzyme formation by utilization of P(i) even though this reaction does not require Ca(2+). It is concluded that NF1058 and CLT inhibit SERCA by stabilization of an E(2) state that, as opposed to that obtained with thapsigargin, retains the functional ability to form E(2)-P by reacting with P(i).
authors.affiliation: Department of Chemistry "Ugo Schiff," University of Florence, 50019 Sesto Fiorentino, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bartolommei
authors.affiliation: Department of Chemistry "Ugo Schiff," University of Florence, 50019 Sesto Fiorentino, Italy. Electronic address: francesco.tadini@unifi.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tadini-Buoninsegni
authors.affiliation: Department of Chemistry "Ugo Schiff," University of Florence, 50019 Sesto Fiorentino, Italy.
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Moncelli
authors.affiliation: European Research Centre for Drug Discovery and Development and Department of Pharmaceutical and Applied Chemistry, University of Siena, 53100 Siena, Italy.
authors.forename: Sandra
authors.initials: S
authors.lastname: Gemma
authors.affiliation: European Research Centre for Drug Discovery and Development and Department of Pharmaceutical and Applied Chemistry, University of Siena, 53100 Siena, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Camodeca
authors.affiliation: European Research Centre for Drug Discovery and Development and Department of Pharmaceutical and Applied Chemistry, University of Siena, 53100 Siena, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Butini
authors.affiliation: European Research Centre for Drug Discovery and Development and Department of Pharmaceutical and Applied Chemistry, University of Siena, 53100 Siena, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Campiani
authors.affiliation: California Pacific Medical Center Research Institute, San Francisco, California 94107.
authors.forename: David
authors.initials: D
authors.lastname: Lewis
authors.affiliation: California Pacific Medical Center Research Institute, San Francisco, California 94107.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Inesi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000634:Aminoquinolines / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003022:Clotrimazole / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D008956:Models, Chemical; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D003022:Clotrimazole; C001920:4-aminoquinoline; D002118:Calcium