title: Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1.
abstract: (1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.
authors.affiliation: Unità di Farmacologia, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università di Bari, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Liantonio
authors.forename: Annamaria
authors.initials: A
authors.lastname: De Luca
authors.forename: Sabata
authors.initials: S
authors.lastname: Pierno
authors.forename: Maria Paola
authors.initials: MP
authors.lastname: Didonna
authors.forename: Fulvio
authors.initials: F
authors.lastname: Loiodice
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Fracchiolla
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortorella
authors.forename: Laghezza
authors.initials: L
authors.lastname: Antonio
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Bonerba
authors.forename: Sonia
authors.initials: S
authors.lastname: Traverso
authors.forename: Laura
authors.initials: L
authors.lastname: Elia
authors.forename: Alessandra
authors.initials: A
authors.lastname: Picollo
authors.forename: Michael
authors.initials: M
authors.lastname: Pusch
authors.forename: Diana Conte
authors.initials: DC
authors.lastname: Camerino
mesh_terms: D008456:2-Methyl-4-chlorophenoxyacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D018118:Chloride Channels / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008297:Male; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D021281:Quantitative Structure-Activity Relationship; D051381:Rats; D017208:Rats, Wistar; D013237:Stereoisomerism; D014982:Xenopus laevis
chemical_list: D018118:Chloride Channels; C048213:2-(4-chlorophenoxy)propionic acid; D008456:2-Methyl-4-chlorophenoxyacetic Acid