Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.
Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.
McMillan
K
K
Pharmacopeia, Inc., Princeton, NJ 08512, USA.
Adler
M
M
Auld
D S
DS
Baldwin
J J
JJ
Blasko
E
E
Browne
L J
LJ
Chelsky
D
D
Davey
D
D
Dolle
R E
RE
Eagen
K A
KA
Erickson
S
S
Feldman
R I
RI
Glaser
C B
CB
Mallari
C
C
Morrissey
M M
MM
Ohlmeyer
M H
MH
Pan
G
G
Parkinson
J F
JF
Phillips
G B
GB
Polokoff
M A
MA
Sigal
N H
NH
Vergona
R
R
Whitlow
M
M
Young
T A
TA
Devlin
J J
JJ
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding; D051381:Rats
D004791:Enzyme Inhibitors; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat