Sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I–XIV T. Abdülkadir Coban a ,S ßükrü Beydemir b, * , _ Ilhami Gücin b , Deniz Ekinci b , Alessio Innocenti c , Daniela Vullo c , Claudiu T. Supuran c, * a Erzincan University, Educational Faculty, Department of Chemistry Education-24030, Erzincan, Turkey b Atatürk University Faculty of Science, Department of Chemistry-25240, Erzurum, Turkey c Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Universita‘ degli, Studi di Firenze, Italy article info Article history: Received 9 June 2009 Revised 7 July 2009 Accepted 13 July 2009 Available online 17 July 2009 Keywords: Carbonic anhydrase Isozymes I–XIV Sildenafil citrate Enzyme activator Piperazine Proton shuttling abstract Sildenafil citrate, a phosphodiesterase-5 (PDE5) inhibitor widely used for the treatment of erectile dys- function was investigated for its interaction with the zinc-enzyme carbonic anhydrase (CA, EC 4.2.1.1), as it has in its molecule a piperazine moiety also found in some CA activators (CAAs). Sildenafil was a potent, low micromolar activator of several CA isozymes, such as CA I, VA and VI (K A s in the range of 1.08–6.54 lM), and activated slightly less the isoforms CA III, IV and VA (K A s of 13.4–16.8 lM). CA iso- zymes II, IX, XIII and XIV showed activation constants in the range of 27.5–34.0 lM, whereas the least activated isoforms were CA VII and XII (K A s of 72.9–73.0 lM). Sildenafil citrate was also given orally to Sprague-Dawley rats at 1 mg/kg body weight. Red blood cell CA activity was inhibited in the treated ani- mals at 3–5 h post-administration (in the range of 60–85%), probably due to NO/nitrite formed by PDE5 inhibition or by another, unknown mechanism. Whether CA activation by sildenafil has clinical conse- quences in humans is beyond the scope of the present work and warrants further studies. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Sildenafil citrate—Viagra TM (1-[4-ethoxy-3-(6,7-dihydro-1-methyl- 7-oxo-3-propyl-1Hpyrazolo[4,3-d]pyrimidin-5-yl)phenylsulphonyl]- 4-methylpiperazine citrate) is a widely used drug for the treatment of erectile dysfunction (ED). 1–3 The discovery that inhibition of phospho- diesterase-5 (PDE5) reduces the degradation of cGMP, allowing erec- tile function to occur by relaxation of penile smooth muscle, represents a revolutionary approach for the treatment of ED, with sev- eral drugs available to date, such as sildenafil 1, tadalafil 2, and varde- nafil 3. 1–3 Similarly to all PDE5 inhibitors known so far, sildenafil binds within the active site of the enzyme, preventing thus the hydroly- sis of the cGMP substrate. The active site of PDE5 is located at the center of a C-terminal helical bundle, with the substrate pocket being approximately 10 Å deep, being composed of: (i) a metal binding subsite (denominated the M site); (ii) a core pocket (the Q pocket); (iii) a hydrophobic pocket (the H pocket); and (iv) a lid region (the L region). 2a,3 As shown in Figure 1, the M pocket contains two metal ions: the catalytically critical Zn(II) ion, together with a second metal ion which presumably is Mg(II). The Zn(II) ion is coordinated by the side chains of His617, His653, Asp654, Asp764 and two water mol- ecules, in an octahedral geometry. Mg(II) has the same octahedral geometry, being coordinated by the bridging ligand Asp654 and a water molecule (shared with the Zn(II) ion) as well as to four other water molecules. 2a,3 The zinc-bound water is probably the nucleo- phile (as neutral water molecule or as hydroxide ion) attacking the HN N N N O O S N O O N HN N N N O O S N O O N N N N H O O O O 1 Sildenafil 3 Vardenafil 2 Tadalafil 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.07.019 * Corresponding authors. Tel.: +90 442 2314444; fax: +90 442 2360948 (S.B.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.). E-mail addresses: beydemir@atauni.edu.tr (S ß. Beydemir), claudiu.supuran@ unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry 17 (2009) 5791–5795 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc