Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors.

The X-ray crystal structure of the adduct between the zinc metalloenzyme carbonic anhydrase II (CA, EC 4.2.1.1) with the recently discovered natural product coumarin derivative 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one showed the coumarin hydrolysis product, a cis-2-hydroxy-cinnamic acid derivative, and not the parent coumarin, bound within the enzyme active site. The bound inhibitor exhibits an extended, two-arm conformation that effectively plugs the entrance to the enzyme active site with no interactions with the catalytically crucial zinc ion. The inhibitor is sandwiched between Phe131, with which it makes an edge-to-face stacking, and Asn67/Glu238sym, with which it makes several polar and hydrogen bonding interactions. This unusual binding mode, with no interactions between the inhibitor molecule and the active site metal ion is previously unobserved for this enzyme class and presents a new opportunity for future drug design campaigns to target a mode of inhibition that differs substantially from classical inhibitors such as the clinically used sulfonamides and sulfamates. Several structurally simple coumarin scaffolds were also shown to inhibit all 13 catalytically active mammalian CA isoforms, with inhibition constants ranging from nanomolar to millimolar. The inhibition is time dependent, with maximum inhibition being observed after 6 h.

[1]  C. Supuran,et al.  Carbonic anhydrase inhibitors. Interaction of indapamide and related diuretics with 12 mammalian isozymes and X-ray crystallographic studies for the indapamide-isozyme II adduct. , 2008, Bioorganic & medicinal chemistry letters.

[2]  Richard D. Smith,et al.  Using Electrospray Ionization FTICR Mass Spectrometry To Study Competitive Binding of Inhibitors to Carbonic Anhydrase , 1995 .

[3]  R. Khalifah,et al.  The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. , 1971, The Journal of biological chemistry.

[4]  S. Sethna,et al.  The Chemistry of Coumarins. , 1945 .

[5]  Andrea Scozzafava,et al.  Carbonic anhydrase inhibitors: stacking with Phe131 determines active site binding region of inhibitors as exemplified by the X-ray crystal structure of a membrane-impermeant antitumor sulfonamide complexed with isozyme II. , 2005, Journal of medicinal chemistry.

[6]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[7]  Andrea Scozzafava,et al.  Carbonic anhydrase IX: A new druggable target for the design of antitumor agents , 2008, Medicinal research reviews.

[8]  Olgun Guvench,et al.  Tryptophan side chain electrostatic interactions determine edge-to-face vs parallel-displaced tryptophan side chain geometries in the designed beta-hairpin "trpzip2". , 2005, Journal of the American Chemical Society.

[9]  R. Quinn,et al.  Direct Screening of Natural Product Extracts Using Mass Spectrometry , 2008, Journal of biomolecular screening.

[10]  C. Supuran,et al.  Carbonic anhydrase inhibitor coated gold nanoparticles selectively inhibit the tumor-associated isoform IX over the cytosolic isozymes I and II. , 2008, Journal of the American Chemical Society.

[11]  C. Supuran,et al.  Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates. , 2008, Bioorganic & medicinal chemistry letters.

[12]  C. Supuran,et al.  Hypoxia activates the capacity of tumor‐associated carbonic anhydrase IX to acidify extracellular pH , 2004, FEBS letters.

[13]  C. Supuran,et al.  Carbonic anhydrase inhibitors: interactions of phenols with the 12 catalytically active mammalian isoforms (CA I-XIV). , 2008, Bioorganic & medicinal chemistry letters.

[14]  C. Supuran,et al.  Are carbonic anhydrase inhibitors suitable for obtaining antiobesity drugs? , 2008, Current pharmaceutical design.

[15]  C. Supuran,et al.  Carbonic anhydrase inhibitors as emerging drugs for the treatment of obesity , 2008, Expert opinion on emerging drugs.

[16]  Adam R. Urbach,et al.  Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. , 2008, Chemical reviews.

[17]  J. Malone,et al.  Stereodynamics and edge-to-face CH-pi aromatic interactions in o-phenethyl-substituted biaryls. , 2006, The Journal of organic chemistry.

[18]  C. Supuran,et al.  Carbonic anhydrase inhibitors: Inhibition of the new membrane-associated isoform XV with phenols. , 2008, Bioorganic & medicinal chemistry letters.

[19]  C. Supuran,et al.  Anticonvulsant sulfonamides/sulfamates/sulfamides with carbonic anhydrase inhibitory activity: drug design and mechanism of action. , 2008, Current pharmaceutical design.

[20]  C. Supuran,et al.  The α and β Classes Carbonic Anhydrases from Helicobacter pylori as Novel Drug Targets , 2008 .

[21]  Claudiu T. Supuran,et al.  Carbonic anhydrases: novel therapeutic applications for inhibitors and activators , 2008, Nature Reviews Drug Discovery.

[22]  C. Supuran.  Diuretics: from classical carbonic anhydrase inhibitors to novel applications of the sulfonamides. , 2008, Current pharmaceutical design.

[23]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[24]  C. Supuran,et al.  Carbonic anhydrase inhibitors: X-ray and molecular modeling study for the interaction of a fluorescent antitumor sulfonamide with isozyme II and IX. , 2006, Journal of the American Chemical Society.

[25]  G. Whitesides,et al.  Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry. , 1996, Journal of medicinal chemistry.

[26]  C. Supuran,et al.  Targeting tumor-associated carbonic anhydrase IX in cancer therapy. , 2006, Trends in pharmacological sciences.

[27]  G. Klebe,et al.  Saccharin inhibits carbonic anhydrases: possible explanation for its unpleasant metallic aftertaste. , 2007, Angewandte Chemie.

[28]  J. Pastorek,et al.  Cancer-associated carbonic anhydrases and their inhibition. , 2008, Current pharmaceutical design.

[29]  C. Supuran,et al.  Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates. , 2005, Bioorganic & medicinal chemistry letters.

[30]  S. Nair,et al.  Two-Site Binding of Phenol in the Active Site of Human Carbonic Anhydrase II: Structural Implications for Substrate Association , 1994 .

[31]  Carbonic anhydrase inhibitors: design of spin-labeled sulfonamides incorporating TEMPO moieties as probes for cytosolic or transmembrane isozymes. , 2008, Bioorganic & medicinal chemistry letters.

[32]  A. Scaloni,et al.  Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide , 2009, Proteins.

[33]  D. Christianson,et al.  Structure of a 129Xe-cryptophane biosensor complexed with human carbonic anhydrase II. , 2008, Journal of the American Chemical Society.

[34]  D. Silverman,et al.  Inhibition of carbonic anhydrase II by thioxolone: a mechanistic and structural study. , 2008, Biochemistry.