A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase.
HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.
Department of Pharmacology, Nagoya University School of Medicine, Japan.
N H
NH
Win
T
T
Ishikawa
N
N
Saito
M
M
Kato
H
H
Yokokura
Y
Y
Watanabe
Y
Y
Iida
H
H
Hidaka
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001557:Benzenesulfonates / Q000494:pharmacology*; D001596:Benzylamines / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D004730:Endothelium, Vascular / Q000187:drug effects*; D066298:In Vitro Techniques; D008297:Male; D009126:Muscle Relaxation; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology
D001557:Benzenesulfonates; D001596:Benzylamines; D002147:Calmodulin; D013449:Sulfonamides; D014665:Vasodilator Agents; C100821:2-(N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl))amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; C017967:W 7; D019001:Nitric Oxide Synthase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000109:Acetylcholine