A novel high affinity class of Ca2+ channel blockers.
Benzolactams (HOE 166 and analogs) form a new class of molecules acting on the 1,4-dihydropyridine-sensitive L-type Ca2+ channels. The main binding properties of HOE 166 and analogs to rabbit skeletal muscle membranes are as follows. (i) The compounds have a specific binding site to which they associate with a high affinity (0.25 nM for HOE 166). (ii) Unlabeled HOE 166 and analogs completely inhibit 1,4-dihydropyridine binding [(+)-[3H]PN 200-110] in a competitive way. (iii) Affinity values measured for HOE 166 inhibition of (+)-[3H]PN 200-110 (K0.5 = 0.25 nM and K1 = 0.55 nM) and of [3H]HOE 166 binding (K0.5 = 0.5 nM) are in good agreement. They also fit with results from direct binding experiments with tritiated HOE 166 (Kd = 0.27 nM) and from kinetic experiments (Kd = 0.39 nM). (iv) HOE 166 completely inhibits the specific binding of other classes of Ca2+ channel antagonists such as phenylalkylamines [(-)[3H] desmethoxyverapamil], benzothiazepines (d-cis-[3H]diltiazem), diphenylbutylpiperidines ([3H]fluspirilene), and [3H]bepridil. In all these cases the binding inhibition is of a noncompetitive nature. (v) The maximum binding capacity for [3H]HOE 166 binding to transverse tubule membranes, 65 pmol/mg of protein, is the same as that found for other classes of Ca2+ channel antagonists. 45Ca2+ uptake experiments performed with the rat aortic cell line A7r5 and the insulin-secreting cell line RINm5F demonstrate that HOE 166 and analogs fully inhibit the 1,4-dihydropyridine-sensitive 45Ca2+ influx elicited by depolarization. There is a good correlation between inhibitory potencies of compounds in the HOE 166 series measured on (+)-[3H]PN 200-110 binding to A7r5 membranes and on the activity of Ca2+ channels followed by 45Ca2+ fluxes with the same cells. Structure-function relationships of HOE 166 and analogs for Ca2+ channel blockade in A7r5 and RINm5F cells were also in good correlation. Finally, voltage-clamp experiments confirmed that voltage-dependent L-type Ca2+ channels are completely blocked by 100 nM HOE 166 even at a membrane potential held at -80 mV.
Qar
J
J
Centre de Biochimie du Centre National de la Recherche Scientifique, Parc Valrose, Nice, France.
Barhanin
J
J
Romey
G
G
Henning
R
R
Lerch
U
U
Oekonomopulos
R
R
Urbach
H
H
Lazdunski
M
M
D000818:Animals; D015764:Bepridil; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002460:Cell Line; D004095:Dihydropyridines / Q000378:metabolism; D005484:Fluspirilene / Q000378:metabolism; D017275:Isradipine; D007700:Kinetics; D009132:Muscles / Q000187:drug effects / Q000378:metabolism; D010069:Oxadiazoles / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology*; D011759:Pyrrolidines / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship; D013843:Thiazines / Q000494:pharmacology*; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism
D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D010069:Oxadiazoles; D010880:Piperidines; D011759:Pyrrolidines; D013843:Thiazines; C055625:Hoe 166; D015764:Bepridil; C038806:1,4-dihydropyridine; C043184:4-desmethoxyverapamil; D005484:Fluspirilene; D014700:Verapamil; D017275:Isradipine