Alessandro Cecchi

Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH

Acidic extracellular pH (pHe) is a typical attribute of a tumor microenviroment, which has an impact on cancer development and treatment outcome. It was believed to result from an accumulation of lactic acid excessively produced by glycolysis. However, metabolic profiles of glycolysis‐impaired tumors have revealed that CO2 is a significant source of acidity, thereby indicating a contribution of carbonic anhydrase (CA). The tumor‐associated CA IX isoform is the best candidate, because its extracellular enzyme domain is highly active, expression is induced by hypoxia and correlates with poor prognosis. This study provides the first evidence for the role of CA IX in the control of pHe. We show that CA IX can acidify the pH of the culture medium in hypoxia but not in normoxia. This acidification can be perturbed by deletion of the enzyme active site and inhibited by CA IX‐selective sulfonamides, which bind only to hypoxic cells containing CA IX. Our findings suggest that hypoxia regulates both expression and activity of CA IX in order to enhance the extracellular acidification, which may have important implications for tumor progression.

Carbonic Anhydrase Inhibitor Coated Gold Nanoparticles Selectively Inhibit the Tumor-Associated Isoform IX over the Cytosolic Isozymes I and II

An approach for the synthesis of carbonic anhydrase (CA, EC 4.2.1.1) inhibitor coated gold nanoparticles is reported. This nanomaterial selectively inhibited the tumor-associated isoform CA IX overexpressed in hypoxic cancers over the ubiquitous, cytosolic housekeeping isozymes CA I and II and was membrane impermeant. As CA IX has an extracellular active site, the new nanomaterial which is confined to the extracellular space may be useful for imaging and treatment of hypoxic tumors.

Nitric oxide-donating carbonic anhydrase inhibitors for the treatment of open-angle glaucoma.

Novel bi-functional compounds with a nitric oxide (NO)-releasing moiety bound to a dorzolamide scaffold were investigated. Several compounds were synthesized and their activity as selective carbonic anhydrase inhibitors (CAI) evaluated in vitro on recombinant hCA type I, II and IV enzyme isoforms where they showed different degrees of potency and selectivity to hCA II. A high resolution X-ray crystal structure for the CA II adduct with 8 confirmed the high affinity of this class of compounds for the enzyme. Compounds 4, 6, and 8 showed highly potent and efficacious NO-mediated properties as assessed by their vascular relaxant effect on methoxamine-precontracted rabbit aortic rings. Finally, compounds 4 and 6 exerted potent intraocular pressure (IOP) lowering effects in vivo in normotensive rabbits thereby anticipating their potential for the treatment of hypertensive glaucoma.

Carbonic anhydrase inhibitors. Interaction of indapamide and related diuretics with 12 mammalian isozymes and X-ray crystallographic studies for the indapamide-isozyme II adduct.

Diuretics such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide, and bumetanide containing primary sulfamoyl moieties were reevaluated as inhibitors of 12 human carbonic anhydrases (hCAs, EC 4.2.1.1). These drugs considerably inhibit (low nanomolar range) some CA isozymes involved in critical physiologic processes, among the 16 present in vertebrates, for example, metolazone against CA VII, XII, and XIII, chlorthalidone against CA VB, VII, IX, XII, and XIII, indapamide against CA VII, IX, XII, and XIII, furosemide against CA I, II, and XIV, and bumetanide against CA IX and XII. The X-ray crystal structure of the hCA II-indapamide adduct was also resolved at high resolution.

Ureido-substituted sulfamates show potent carbonic anhydrase IX inhibitory and antiproliferative activities against breast cancer cell lines

A series of 50 sulfamates were obtained by reacting 4-aminophenol with isocyanates followed by sulfamoylation. Most of the new compounds were nanomolar inhibitors of the tumor-associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII, whereas they inhibited less cytosolic offtarget isoforms CA I and II. Some of these sulfamates showed significant antiproliferative activity in several breast cancer cell lines, such as SKBR3, MCF10A, ZR75/1, MDA-MB-361 and MCF7, constituting interesting anticancer leads.

Carbonic anhydrase inhibitors. Comparison of chlorthalidone, indapamide, trichloromethiazide, and furosemide X-ray crystal structures in adducts with isozyme II, when several water molecules make the difference.

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K(I)s of 65-138 nM), whereas indapamide is a much weaker one (K(I) of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2-3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.

Carbonic Anhydrase Inhibitors. Comparison of Chlorthalidone and Indapamide X-ray Crystal Structures in Adducts with Isozyme II: When Three Water Molecules and the Keto−Enol Tautomerism Make the Difference†

Thiazide diuretics inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a different profile as compared to classical inhibitors. Acting as moderate-weak inhibitors of CA II and CA I, chlorthalidone and indapamide considerably inhibit other isozymes among the 16 CAs present in vertebrates. These compounds show a different behavior against CAs I and II, with chlorthalidone being 18.3 times more potent against CA II and 150 times more potent against CA I, as compared to indapamide. In the X-ray crystal structures of the CA II-chlorthalidone adduct three active site water molecules interacting with the inhibitor scaffold were observed that lack in the corresponding indapamide adduct. Chlorthalidone bound within the active site is in an enolic tautomeric form, with the OH moiety participating in two strong hydrogen bonds with Asn67 and a water molecule. This binding mode may be exploited for designing better CA II inhibitors.

Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO2 hydration reaction at 20°C and pH 7.5: kcat of 1.3 × 104 s− 1 and kcat/KM of 2.5.105 M− 1 s− 1. The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (KIs of 21–78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed KIs around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed KIs in the range of 10–90 μM. It is difficult to explain the inhibitory activity of carbonate (KI of 10 μM) against hCA III, also considering the fact that this ion has an affinity of 15–73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (KI of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).

Synthesis and crystallographic analysis of new sulfonamides incorporating NO-donating moieties with potent antiglaucoma action

Several aromatic/heterocyclic sulfonamide scaffolds have been used to synthesize compounds incorporating NO-donating moieties of the nitrate ester type, which have been investigated for the inhibition of five physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms: hCA I (offtarget), II, IV and XII (antiglaucoma targets) and IX (antitumor target). Some of the new compounds showed effective in vitro inhibition of the target isoforms involved in glaucoma, and the X-ray crystal structure of one of them revealed factors associated with the marked inhibitory activity. In an animal model of ocular hypertension, one of the new compounds was twice more effective than dorzolamide in reducing elevated intraocular pressure characteristic of this disease, anticipating their potential for the treatment of glaucoma.

Carbonic Anhydrase Inhibitors: Design of Membrane‐Impermeant Copper(II) Complexes of DTPA‐, DOTA‐, and TETA‐Tailed Sulfonamides Targeting the Tumor‐Associated Transmembrane Isoform IX

The synthesis and carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of two series of aromatic sulfonamides and their CuII derivatives, incorporating metal‐complexing moieties of the DTPA, DOTA, and TETA type are reported. The new compounds were designed in such a way as to possess high affinity for CuII ions, exploiting four pendant carboxylate moieties in the DTPA derivatives, as well as the cyclen/cyclam macrocyles, and three pendant acetate moieties in the DOTA and TETA derivatives. The new derivatives showed modest inhibition of the cytosolic isoform CA I (KI values in the range of 66–2130 nM), were better CA II inhibitors (KI values in the range of 21–360 nM), and excellent inhibitors of the tumor‐associated isoform CA IX (KI values in the range of 4.1–110 nM), with selectivity ratios for the inhibition of the tumor (CA IX) over the cytosolic (CA II) isozyme in the range of 10.74–20.88 for the best derivatives. Copper complexes were more inhibitory than the corresponding ligand sulfonamides, and showed membrane impermeability, thus, having the possibility to specifically target the transmembrane CA IX that has an extracellular active site. Incorporation of radioactive copper isotopes in this type of CA inhibitor may lead to interesting diagnostic/therapeutic applications for such compounds.