Post-translational modifications in tumor-associated carbonic anhydrases.

Human carbonic anhydrases IX (hCA IX) and XII (hCA XII) are two proteins associated with tumor formation and development. These enzymes have been largely investigated both from a biochemical and a functional point of view. However, limited data are currently available on the characterization of their post-translational modifications (PTMs) and the functional implication of these structural changes in the tumor environment. In this review, we summarize existing literature data on PTMs of hCA IX and hCA XII, such as disulphide bond formation, phosphorylation, O-/N-linked glycosylation, acetylation and ubiquitination, highlighting, when possible, their specific role in cancer pathological processes.

Intrinsically disordered features of carbonic anhydrase IX proteoglycan-like domain.

hCA IX is a multi-domain protein belonging to the family of hCAs which are ubiquitous zinc enzymes that catalyze the reversible hydration of CO2 to HCO3- and H+. hCA IX is a tumor-associated enzyme with a limited distribution in normal tissues, but over-expressed in many tumors, and is a promising drug target. Although many studies concerning the CA IX catalytic domain were performed, little is known about the proteoglycan-like (PG-like) domain of hCA IX which has been poorly investigated so far. Here we attempt to fill this gap by providing an overview on the functional, structural and therapeutic studies of the PG-like domain of hCA IX which represents a unique feature within the CA family. The main studies and recent advances concerning PG role in modulating hCA IX catalytic activity as well as in tumor spreading and migration are here reported. Special attention has been paid to the newly discovered disordered features of the PG domain which open new perspectives about its molecular mechanisms of action under physiological and pathological conditions, since disorder is likely involved in mediating interactions with partner proteins. The emerged disordered features of PG domain will be explored for putative diagnostic and therapeutic applications involving CA IX targeting in tumors.

Biochemical and Structural Insights into the Winged Helix Domain of P150, the Largest Subunit of the Chromatin Assembly Factor 1.

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.

α-CAs from Photosynthetic Organisms.

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible carbon dioxide hydration reaction. Among the eight different CA classes existing in nature, the α-class is the largest one being present in animals, bacteria, protozoa, fungi, and photosynthetic organisms. Although many studies have been reported on these enzymes, few functional, biochemical, and structural data are currently available on α-CAs isolated from photosynthetic organisms. Here, we give an overview of the most recent literature on the topic. In higher plants, these enzymes are engaged in both supplying CO2 at the Rubisco and determining proton concentration in PSII membranes, while in algae and cyanobacteria they are involved in carbon-concentrating mechanism (CCM), photosynthetic reactions and in detecting or signaling changes in the CO2 level in the environment. Crystal structures are only available for three algal α-CAs, thus not allowing to associate specific structural features to cellular localizations or physiological roles. Therefore, further studies on α-CAs from photosynthetic organisms are strongly needed to provide insights into their structure-function relationship.

Benzyl alcohol inhibits carbonic anhydrases by anchoring to the zinc coordinated water molecule.

Up to date alcohols have been scarcely investigated as carbonic anhydrase (CA) inhibitors. To get more insights into the CA inhibition properties of this class of molecules, in this paper, by means of inhibition assays and X-ray crystallographic studies we report a detailed characterization of the CA inhibition properties and the binding mode to human CA II of benzyl alcohol. Results show that, although possessing a very simple scaffold, this molecule acts as a micromolar CA II inhibitor, which anchors to the enzyme active site by means of an H-bond interaction with the zinc bound solvent molecule. Taken together our results clearly indicate primary alcohols as a class of CA inhibitors that deserve to be more investigated.

Surface plasmon resonance study for a reliable determination of the affinity constant of multivalent grafted beads.

In this study, galactose-grafted beads were prepared using the main design principle of the cluster effect. Galactose was chosen as the sugar for investigation because it acts as the main building block of long glycan chains and because a simple and fast protocol is still required for its immobilization. For the analysis, the lectin, ligand of the galactose, was immobilized on a gold plasmonic substrate. After preliminary characterization of the galactose-grafted beads, the investigation of the surface plasmon surface behavior of the system was carried out, for studying the affinity constant of the multivalent beads. The results of steady-state and of the kinetics analysis evidenced a higher affinity of the galactose-grafted beads over the beadless galactose solution. For the association kinetics analysis, a Langmuir isotherm was applied to the data. The analysis of the rate of dissociation evidenced the most important differences between the two samples, based on the more difficult release of the galactose-grafted beads during washing. To confirm the influence of the glycoside cluster effect, a low-density lectin substrate was tested, and the results evidenced that the characteristic size of the molecules determines a threshold for the cluster density. The calculated detection limit and dissociation constants were 3.5 µM and 40.2 µM, respectively. Considering those results, the evaluation of the affinities toward the receptors depends on the cluster density and then, it should be designed for mimicking the biological samples.

Design, synthesis and biochemical evaluation of novel carbonic anhydrase inhibitors triggered by structural knowledge on hCA VII.

To tackle the challenge of isoform selectivity, we explored the entrance of the cavity for selected druggable human Carbonic Anhydrases (hCAs). Based on X-ray crystallographic studies on the 4-(4-(2-chlorobenzoyl)piperazine-1-carbonyl)benzenesulfonamide in complex with the brain expressed hCA VII (PDB code: 7NC4), a series of 4-(4(hetero)aroylpiperazine-1-carbonyl)benzene-1-sulfonamides has been developed. To evaluate their capability to fit the hCA VII catalytic cavity, the newer benzenesulfonamides were preliminary investigated by means of docking simulations. Then, this series of thirteen benzenesulfonamides was synthesized and tested against selected druggable hCAs. Among them, the 4-(4-(furan-2-carbonyl)piperazine-1-carbonyl)benzenesulfonamide showed remarkable affinity towards hCA VII (Ki: 4.3 nM) and good selectivity over the physiologically widespread hCA I when compared to Topiramate (TPM).

Inhibition of the ß-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides.

Sulphonamides and their isosteres are classical inhibitors of the carbonic anhydrase (CAs, EC 4.2.1.1) metalloenzymes. The protozoan pathogen Trichomonas vaginalis encodes two such enzymes belonging to the ß-class, TvaCA1 and TvaCA2. Here we report the first sulphonamide inhibition study of TvaCA1, with a series of simple aromatic/heterocyclic primary sulphonamides as well as with clinically approved/investigational drugs for a range of pathologies (diuretics, antiglaucoma, antiepileptic, antiobesity, and antitumor drugs). TvaCA1 was effectively inhibited by acetazolamide and ethoxzolamide, with KIs of 391 and 283 nM, respectively, whereas many other simple or clinically used sulphonamides were micromolar inhibitors or did not efficiently inhibit the enzyme. Finding more effective TvaCA1 inhibitors may constitute an innovative approach for fighting trichomoniasis, a sexually transmitted infection, caused by T. vaginalis.

Reducing salt intake by urine chloride self-measurement in non-compliant patients with chronic kidney disease followed in nephrology clinics: a randomized trial.

BACKGROUND: Adherence to low salt diets and control of hypertension remain unmet clinical needs in chronic kidney disease (CKD) patients. METHODS: We performed a 6-month multicentre randomized trial in non-compliant patients with CKD followed in nephrology clinics testing the effect of self-measurement of urinary chloride (69 patients) as compared with standard care (69 patients) on two primary outcome measures, adherence to a low sodium (Na) diet (<100 mmol/day) as measured by 24-h urine Na (UNa) excretion and 24-h ambulatory blood pressure (ABPM) monitoring. RESULTS: In the whole sample (N = 138), baseline UNa and 24-h ABPM were143 ± 64 mmol/24 h and 131 ± 18/72 ± 10 mmHg, respectively, and did not differ between the two study arms. Patients in the active arm of the trial used >80% of the chloride strips provided to them at the baseline visit and at follow-up visits. At the third month, UNa was 35 mmol/24 h (95% CI 10.8-58.8 mmol/24 h; P = 0.005) lower in the active arm than the control arm, whereas at 6 months the between-arms difference in UNa decreased and was no longer significant [23 mmol/24 h (95% CI -5.6-50.7); P = 0.11]. The 24-h ABPM changes as well as daytime and night-time BP changes at 3 and 6 months were similar in the two study arms (Month 3, P = 0.69-0.99; Month 6, P = 0.73-0.91). Office BP, the use of antihypertensive drugs, estimated Glomerular Filtration Rate (eGFR) and proteinuria remained unchanged across the trial. CONCLUSIONS: The application of self-measurement of urinary chloride to guide adherence to a low salt diet had a modest effect on 24-h UNa and no significant effect on 24-h ABPM.

Human carbonic anhydrases and post-translational modifications: a hidden world possibly affecting protein properties and functions.

Human carbonic anhydrases (CAs) have become a well-recognized target for the design of inhibitors and activators with biomedical applications. Accordingly, an enormous amount of literature is available on their biochemical, functional and structural aspects. Nevertheless post-translational modifications (PTMs) occurring on these enzymes and their functional implications have been poorly investigated so far. To fill this gap, in this review we have analysed all PTMs occurring on human CAs, as deriving from the search in dedicated databases, showing a widespread occurrence of modification events in this enzyme family. By combining these data with sequence alignments, inspection of 3 D structures and available literature, we have summarised the possible functional implications of these PTMs. Although in some cases a clear correlation between a specific PTM and the CA function has been highlighted, many modification events still deserve further dedicated studies.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis.

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (ß-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, ß, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only ß- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The ß-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of ß-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

A Novel Inhibitor of Carbonic Anhydrases Prevents Hypoxia-Induced TNBC Cell Plasticity.

Cell plasticity is the ability that cells have to modify their phenotype, adapting to the environment. Cancer progression is under the strict control of the the tumor microenvironment that strongly determines its success by regulating the behavioral changes of tumor cells. The cross-talk between cancer and stromal cells and the interactions with the extracellular matrix, hypoxia and acidosis contribute to trigger a new tumor cell identity and to enhance tumor heterogeneity and metastatic spread. In highly aggressive triple-negative breast cancer, tumor cells show a significant capability to change their phenotype under the pressure of the hypoxic microenvironment. In this study, we investigated whether targeting the hypoxia-induced protein carbonic anhydrase IX (CA IX) could reduce triple-negative breast cancer (TNBC) cell phenotypic switching involved in processes associated with poor prognosis such as vascular mimicry (VM) and cancer stem cells (CSCs). The treatment of two TNBC cell lines (BT-549 and MDA-MB-231) with a specific CA IX siRNA or with a novel inhibitor of carbonic anhydrases (RC44) severely impaired their ability to form a vascular-like network and mammospheres and reduced their metastatic potential. In addition, the RC44 inhibitor was able to hamper the signal pathways involved in triggering VM and CSC formation. These results demonstrate that targeting hypoxia-induced cell plasticity through CA IX inhibition could be a new opportunity to selectively reduce VM and CSCs, thus improving the efficiency of existing therapies in TNBC.

Biochemical, biophysical and molecular dynamics studies on the proteoglycan-like domain of carbonic anhydrase IX.

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.

Exploration of the residues modulating the catalytic features of human carbonic anhydrase XIII by a site-specific mutagenesis approach.

Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. In humans there are 15 isoforms among which only 12 are catalytically active. Since active human (h) CAs show different efficiency, the understanding of the molecular determinants affecting it is a matter of debate. Here we investigated, by a site-specific mutagenesis approach, residues modulating the catalytic features of one of the least investigated cytosolic isoform, i.e. hCA XIII. Results showed that residues assisting the formation of an ordered solvent network within the catalytic site as well as those forming a histidine cluster on the protein surface are important to guarantee an efficient proton transfer.

Exploring benzoxaborole derivatives as carbonic anhydrase inhibitors: a structural and computational analysis reveals their conformational variability as a tool to increase enzyme selectivity.

Recent studies identified the benzoxaborole moiety as a new zinc-binding group able to interact with carbonic anhydrase (CA) active site. Here, we report a structural analysis of benzoxaboroles containing urea/thiourea groups, showing that these molecules are very versatile since they can bind the enzyme assuming different binding conformations and coordination geometries of the catalytic zinc ion. In addition, theoretical calculations of binding free energy were performed highlighting the key role of specific residues for protein-inhibitor recognition. Overall, these data are very useful for the development of new inhibitors with higher selectivity and efficacy for various CAs.

Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and cancer stem cells: Three for the price of one.

Human carbonic anhydrase (CA) IX is a tumor-associated protein, since it is scarcely present in normal tissues, but highly overexpressed in a large number of solid tumors, where it actively contributes to survival and metastatic spread of tumor cells. Due to these features, the characterization of its biochemical, structural, and functional features for drug design purposes has been extensively carried out, with consequent development of several highly selective small molecule inhibitors and monoclonal antibodies to be used for different purposes. Aim of this review is to provide a comprehensive state-of-the-art of studies performed on this enzyme, regarding structural, functional, and biomedical aspects, as well as the development of molecules with diagnostic and therapeutic applications for cancer treatment. A brief description of additional pharmacologic applications for CA IX inhibition in other diseases, such as arthritis and ischemia, is also provided.

Crystal structure of the human carbonic anhydrase II adduct with 1-(4-sulfamoylphenyl-ethyl)-2,4,6-triphenylpyridinium perchlorate, a membrane-impermeant, isoform selective inhibitor.

Pyridinium containing sulfonamides have been largely investigated as carbonic anhydrase inhibitors (CAIs), showing interesting selectivity features. Nevertheless, only few structural studies are so far available on adducts that these compounds form with diverse CA isoforms. In this paper, we report the structural characterization of the adduct that a triphenylpyridinium derivative forms with hCA II, showing that the substitution of the pyridinium ring plays a key role in determining the conformation of the inhibitor in the active site and consequently the binding affinity to the enzyme. These findings open new perspectives on the basic structural requirements for designing sulfonamide CAIs with a selective inhibition profile.

The Crystal Structure of a hCA VII Variant Provides Insights into the Molecular Determinants Responsible for Its Catalytic Behavior.

Although important progress has been achieved in understanding the catalytic mechanism of Carbonic Anhydrases, a detailed picture of all factors influencing the catalytic efficiency of the various human isoforms is still missing. In this paper we report a detailed structural study and theoretical pKa calculations on a hCA VII variant. The obtained data were compared with those already known for another thoroughly investigated cytosolic isoform, hCA II. Our structural studies show that in hCA VII the network of ordered water molecules, which connects the zinc bound solvent molecule to the proton shuttle His64, is altered compared to hCA II, causing a reduction of the catalytic efficiency. Theoretical calculations suggest that changes in solvent network are related to the difference in pKa of the proton shuttle in the two enzymes. The residue that plays a major role in determining the diverse pKa values of the proton shuttle is the one in position four, namely His for hCA II and Gly for hCA VII. This residue is located on the protein surface, outside of the active site cavity. These findings are in agreement with our previous studies that highlighted the importance of histidines on the protein surface of hCA II (among which His4) as crucial residues for the high catalytic efficiency of this isoform.

Stochastic phenotypic interconversion in tumors can generate heterogeneity.

Phenotype variations define heterogeneity in biological and molecular systems, and play a crucial mechanistic role, and heterogeneity has been demonstrated in tumor cells. In this work, cells from blood of patients affected by colon cancer were analyzed and sorted using a microfluidic assay based on galactose-active moieties and incubated for culturing in severe combined immunodeficiency (SCID) mice. Based on the results of these experiments, a model based on Markov theory is implemented and discussed to explain the equilibrium existing between phenotypes of cell subpopulations sorted using the microfluidic assay. In combination with the experimental results, the model has many implications for tumor heterogeneity; For example, it displays interconversion of phenotypes, confirming the experiments. Such interconversion generates metastatic cells and implies that targeting circulating tumor cells (CTC) will not be an efficient method for prevention of tumor recurrence. Most importantly, understanding the transitions between cell phenotypes in the cell population can improve understanding of tumor generation and growth.

Insights into the role of reactive sulfhydryl groups of Carbonic Anhydrase III and VII during oxidative damage.

Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO2 hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.