Carbonic anhydrase inhibitors. Zonisamide is an effective inhibitor of the cytosolic isozyme II and mitochondrial isozyme V: solution and X-ray crystallographic studies.
The antiepileptic drug zonisamide was considered to act as a weak inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) (with a K(I) of 4.3 microM against the cytosolic isozyme II). Here we prove that this is not true. Indeed, testing zonisamide in the classical assay conditions of the CO2 hydrase activity of hCA II, with incubation times of enzyme and inhibitor solution of 15 min, a K(I) of 10.3 microM has been obtained. However, when the incubation between enzyme and inhibitor was prolonged to 1 h, the obtained K(I) was of 35.2 nM, of the same order of magnitude as that of the clinically used sulfonamides/sulfamates acetazolamide, methazolamide, ethoxzolamide and topiramate (K(I)s in the range of 5.4-15.4 nM). The inhibition of the human mitochondrial isozyme hCA V with these compounds has been also tested by means of a dansylamide competition binding assay, which showed zonisamide and topiramate to be effective inhibitors, with K(I)s in the range of 20.6-25.4 nM. The X-ray crystal structure of the adduct of hCA II with zonisamide has also been solved at a resolution of 1.70 A, showing that the sulfonamide moiety participates in the classical interactions with the Zn(II) ion and the residues Thr199 and Glu106, whereas the benzisoxazole ring is oriented toward the hydrophobic half of the active site, establishing a large number of strong van der Waals interactions (<4.5 A) with residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202.
De Simone
Giuseppina
G
Dipartimento di Chimica Biologica-Sezione Biostrutture and Istituto di Biostrutture e Bioimmagini-CNR, University of Naples Federico II, via Mezzocannone 16, 80134 Naples, Italy. gmg@chemistry.unina.it
Di Fiore
Anna
A
Menchise
Valeria
V
Pedone
Carlo
C
Antel
Jochen
J
Casini
Angela
A
Scozzafava
Andrea
A
Wurl
Michael
M
Supuran
Claudiu T
CT
D001665:Binding Sites; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000378:metabolism*; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D024404:Carbonic Anhydrase V / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D011487:Protein Conformation; D000078305:Zonisamide
D002257:Carbonic Anhydrase Inhibitors; D007555:Isoxazoles; D000078305:Zonisamide; D024402:Carbonic Anhydrase II; D024404:Carbonic Anhydrase V