Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.
A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.
Blasko
Eric
E
Cardiovascular Research, Berlex Biosciences, Richmond, California 94804-0099, USA.
Glaser
Charles B
CB
Devlin
James J
JJ
Xia
Wei
W
Feldman
Richard I
RI
Polokoff
Mark A
MA
Phillips
Gary B
GB
Whitlow
Marc
M
Auld
Douglas S
DS
McMillan
Kirk
K
Ghosh
Sanjay
S
Stuehr
Dennis J
DJ
Parkinson
John F
JF
D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II; D011743:Pyrimidines / Q000494:pharmacology; D011869:Radioligand Assay
D004791:Enzyme Inhibitors; D007093:Imidazoles; D011743:Pyrimidines; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II