Anna Di Fiore

Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX.

Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the α-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical α-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO2 hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated α-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

Biochemical characterization of CA IX: one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys119-Cys299) and a unique N-linked glycosylation site (Asn309) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr78) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.

Are Carbonic Anhydrase Inhibitors Suitable for Obtaining Antiobesity Drugs

Obesity is widespread disease both in the developed and developing world, which currently affects over 300 million individuals worldwide and is associated with premature mortality and chronic morbidity. Although diet, physical activity and behavioral modifications should theoretically help in controlling this condition, very often these strategies are insufficient to normalize the multiple risks associated with this condition. Thus, pharmacological interventions for the treatment of this disease are essential. Paradoxically, the currently available drugs for the treatment of obesity are very few, their mechanism of action is hardly understood and their side effects are generally quite serious. Therefore, novel effective anti-obesity drugs possessing different mechanisms of action are needed. In this review we describe in detail a possible new approach for the treatment and prophylaxis of this disease based on the inhibition of Carbonic Anhydrases (CAs, EC 4.2.1.1), enzymes involved in several steps of de novo lipogenesis. In particular, we summarize here a series of kinetic and structural studies recently reported on Topiramate (TPM) and Zonisamide (ZNS), two antiepileptic drugs showing strong CA inhibitory properties, that were shown to induce persistent weight loss in obese patients. On the basis of the reviewed studies we suggest that the use of TPM and ZNS as lead molecules for the design of CA inhibitors targeting isozymes involved in lipogenesis could represent the beginning of a very promising approach for the treatment of obesity.

Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide.

The cytosolic isoform XIII is a recently discovered member of the human carbonic anhydrase (hCA, EC 4.2.1.1) family. It is selectively expressed among other tissues in the reproductive organs, where it may control pH and ion balance regulation, ensuring thus proper fertilization conditions. The authors report here the X‐ray crystallographic structure of this isozyme in the unbound state and in complex with a classical sulfonamide inhibitor, namely acetazolamide. A detailed comparison of the obtained structural data with those already reported for other CA isozymes provides novel insights into the catalytic properties of the members of this protein family. On the basis of the inhibitory properties of acetazolamide against various cytosolic/transmembrane isoforms and the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme‐specific CA inhibitors are here proposed. Proteins 2009.

The zinc coordination pattern in the η-carbonic anhydrase from Plasmodium falciparum is different from all other carbonic anhydrase genetic families

In this Letter we reinvestigate the sequence analysis and report a homology model of the carbonic anhydrase (CA, EC 4.2.1.1) from the protozoan parasite Plasmodium falciparum, recently reported by us to belong to a new genetic family, the η-CA class. Our findings show that the metal ion coordination pattern of this CA is unique among all five other genetic families encoding for such enzymes, comprising two His and one Gln residues, in addition to the water molecule/hydroxide ion acting as nucleophile in the catalytic cycle. Although the η- and α-CAs present the same 3D fold, strongly suggesting the first ones to be evolutionary derived from the last, there are significant differences between the two families to allow optimism for the drug design of selective inhibitors for the parasite over the host enzymes. The preliminary studies reported here are relevant for drug design campaigns of anti-plasmodium CA inhibitors but further work by X-ray crystallography should validate the proposed model.

Structure-activity relationships of C-17 cyano-substituted estratrienes as anticancer agents

The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as anticancer agents are discussed. 2-Methoxy-17beta-cyanomethylestra-1,3,5(10)-trien-3-ol ( 14), but not the related 2-ethyl derivative 7, and the related 3- O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7 GI 50 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was investigated by protein crystallography. The potential of these multimechanism anticancer agents was confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-MB-231 human breast cancer xenograft model.

Carbonic anhydrase inhibitors: X-ray crystallographic studies for the binding of N-substituted benzenesulfonamides to human isoform II

N-substituted benzenesulfonamides, incorporating the N-amino-, N-hydroxy- and N-methoxy-moieties at the sulfonamide zinc binding group, have been investigated as CAIs by means of inhibition and structural studies, unraveling interesting aspects related to their inhibition mechanism.

Carbonic anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity for sulfonamide inhibitors

Human carbonic anhydrase (CA, EC 4.2.1.1) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Here we report an unexpected S-glutathionylation of hCA VII which has also been observed earlier in vivo for hCA III, another cytosolic isoform. Cys183 and Cys217 were found to be the residues involved in reaction with glutathione for hCA VII. The two reactive cysteines were then mutated and the corresponding variant (C183S/C217S) expressed. The native enzyme, the variant and the S-glutathionylated adduct (sgCA VII) as well as hCA III were fully characterized for their CO(2) hydration, esterase/phosphatase activities, and inhibition with sulfonamides. Our findings suggest that hCA VII could use the in vivo S-glutathionylation to function as an oxygen radical scavenger for protecting cells from oxidative damage, as the activity and affinity for inhibitors of the modified enzyme are similar to those of the wild type.

Anticancer steroid sulfatase inhibitors: synthesis of a potent fluorinated second-generation agent, in vitro and in vivo activities, molecular modeling, and protein crystallography

An improved steroid sulfatase inhibitor was prepared by replacing the N-propyl group of the second-generation steroid-like inhibitor (2) with a N-3,3,3-trifluoropropyl group to give (10). This compound is 5-fold more potent in vitro, completely inhibits rat liver steroid sulfatase activity after a single oral dose of 0.5 mg/kg, and exhibits a significantly longer duration of inhibition over (2). These biological properties are attributed to the increased lipophilicity and metabolic stability of (10) rendered by its trifluoropropyl group and also the potential H-bonding between its fluorine atom(s) and Arg98 in the active site of human steroid sulfatase. Like other sulfamates, (10) is expected to be sequestered, and transported by, erythrocytes in vivo because it inhibits human carbonic anhydrase II (hCAII) potently (IC50, 3 nmol/L). A congener (4), which possesses a N-(pyridin-3-ylmethyl) substituent, is even more active (IC50, 0.1 nmol/L). To rationalize this, the hCAII-(4) adduct, obtained by cocrystallization, reveals not only the sulfamate group and the backbone of (4) interacting with the catalytic site and the associated hydrophobic pocket, respectively, but also the potential H-bonding between the N-(pyridin-3-ylmethyl) group and Nε2 of Gln136. Like (2), both (10) and its phenolic precursor (9) are non-estrogenic using a uterine weight gain assay. In summary, a highly potent, long-acting, and nonestrogenic steroid sulfatase inhibitor was designed with hCAII inhibitory properties that should positively influence in vivo behavior. Compound (10) and other related inhibitors of this structural class further expand the armory of steroid sulfatase inhibitors against hormone-dependent breast cancer. [Mol Cancer Ther 2008;7(8):2435–44]

The role of the hinge loop in domain-swapping: The special case of Bovine seminal Ribonuclease

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16–22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.