Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1.
(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.
Liantonio
Antonella
A
Unità di Farmacologia, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università di Bari, Italy.
De Luca
Annamaria
A
Pierno
Sabata
S
Didonna
Maria Paola
MP
Loiodice
Fulvio
F
Fracchiolla
Giuseppe
G
Tortorella
Paolo
P
Antonio
Laghezza
L
Bonerba
Elisabetta
E
Traverso
Sonia
S
Elia
Laura
L
Picollo
Alessandra
A
Pusch
Michael
M
Camerino
Diana Conte
DC
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
D018118:Chloride Channels; C048213:2-(4-chlorophenoxy)propionic acid; D008456:2-Methyl-4-chlorophenoxyacetic Acid