Discovery of a novel series of potent carbonic anhydrase inhibitors with selective affinity for µ Opioid receptor for Safer and long-lasting analgesia.

In this study, we investigated the development of dual-targeted ligands that bind to both µ-opioid receptor (MOR) and carbonic anhydrase (CA) enzymes, using fentanyl structure as a template. We synthesized and evaluated 21 novel compounds with dual-targeted affinity identifying the lead candidate compound 8, showing selective affinity for MOR and potent inhibition of several cytosolic CA isoforms. By means of repeated treatment of 3 daily administrations for 17 days, fentanyl (0.1 mg/kg, subcutaneously) led to tolerance development, pain threshold alterations and withdrawal symptoms in CD-1 mice, as well as astrocyte and microglia activation in the dorsal horn of the lumbar spinal cord. In contrast, compound 8 (0.32 mg/kg s.c.) maintained stable during days its analgesic effect at the higher dose tested with fewer withdrawal symptoms, allodynia development and glial cells activation. Our results suggest that targeting both MOR and CA enzymes can lead to the development of new class of potent analgesic agents with fewer side effects and reduced tolerance development. Further studies are needed to explore the potential mechanisms underlying these effects and to further optimize the therapeutic potential of these compounds.

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.

Biochemical, structural, and computational studies of a γ-carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei.

Melioidosis is a severe disease caused by the highly pathogenic gram-negative bacterium Burkholderia pseudomallei. Several studies have highlighted the broad resistance of this pathogen to many antibiotics and pointed out the pivotal importance of improving the pharmacological arsenal against it. Since γ-carbonic anhydrases (γ-CAs) have been recently introduced as potential and novel antibacterial drug targets, in this paper, we report a detailed characterization of BpsγCA, a γ-CA from B. pseudomallei by a multidisciplinary approach. In particular, the enzyme was recombinantly produced and biochemically characterized. Its catalytic activity at different pH values was measured, the crystal structure was determined and theoretical pKa calculations were carried out. Results provided a snapshot of the enzyme active site and dissected the role of residues involved in the catalytic mechanism and ligand recognition. These findings are an important starting point for developing new anti-melioidosis drugs targeting BpsγCA.

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.

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.

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.

Inhibition of the newly discovered ß­carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with inorganic anions and small molecules.

The protozoan pathogen Trichomonas vaginalis encodes two carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the ß-class. One of these enzymes, T. vaginalis carbonic anhydrase 1 (TvaCA1), was recently cloned and characterized by our group, and its X-ray crystal structure reported. No inhibitors of this enzyme were reported up until now. Here we investigated the inhibition of TvaCA1 with inorganic anions and small molecules and observed that thiocyanate, cyanide, selenite, selenocyanate and divanadate are sub-millimolar inhibitors, whereas sulfamide, sulfate, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. Finding effective TvaCA1 inhibitors may be useful for developing new antiprotozoan drugs.

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.

Looking toward the Rim of the Active Site Cavity of Druggable Human Carbonic Anhydrase Isoforms.

We report the synthesis and biochemical evaluation of a series of substituted 4-(4-aroylpiperazine-1-carbonyl)benzenesulfonamides (5a-s) developed as inhibitors of druggable carbonic anhydrase (CA) isoforms, as tools for the identification of new therapeutics. X-ray crystallography confirmed that this class of benzenesulfonamides binds CAs through the canonical anchoring of the benzenesulfonamide moiety to the metal ion and a tail-mediated recognition of the middle/top area of the active site cavity. Compound 5e (R = 2-Cl) demonstrated relevant selectivity toward brain-expressed hCA VII. The best balancing in binding affinity and selectivity toward tumor-expressed hCA IX/hCA XII over ubiquitous hCA I/hCA II was found for inhibitor 5o (R = 3-NO2). Notably 5b (R = 2-F) proved to be the most efficacious inhibitor of hCA XII for which computational studies elucidated the CA recognition process.

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.

Exploring structural properties of potent human carbonic anhydrase inhibitors bearing a 4-(cycloalkylamino-1-carbonyl)benzenesulfonamide moiety.

Guided by the crystal structure of 4-(3,4-dihydroquinolin-1(2H)-ylcarbonyl)benzenesulfonamide 3 in complex with hCA II (PDB code 4Z0Q), a novel series of cycloalkylamino-1-carbonylbenzenesulfonamides was designed and synthesized. Thus, we replaced the quinoline ring with an azepine/piperidine/piperazine nucleus and introduced further modifications on cycloalkylamine nucleus by means the installation of hydrophobic/hydrophilic functionalities able to establish additional contacts in the middle area of the enzyme cavity. Among the synthesized compounds, the derivatives 7a, 7b, 8b exhibited a remarkable inhibition for hCA II and the brain-expressed hCA VII in subnanomolar range. The binding of these molecules to the target enzymes was characterized by means of a crystallographic analysis, providing a clear snapshot of the most important interactions established by this class of inhibitors into the hCA II and hCA VII catalytic site. Notably, our results showed that the benzylpiperazine tail of compound 8b is oriented both in hCA II and in hCA VII toward a poorly explored region of the active site. These features should be further investigated for the design of new isoform selective CA inhibitors.

Protective Role of Carbonic Anhydrases III and VII in Cellular Defense Mechanisms upon Redox Unbalance.

Under oxidative stress conditions, several constitutive cellular defense systems are activated, which involve both enzymatic systems and molecules with antioxidant properties such as glutathione and vitamins. In addition, proteins containing reactive sulfhydryl groups may eventually undergo reversible redox modifications whose products act as protective shields able to avoid further permanent molecular oxidative damage either in stressful conditions or under pathological circumstances. After the recovery of normal redox conditions, the reduced state of protein sulfhydryl groups is restored. In this context, carbonic anhydrases (CAs) III and VII, which are human metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate and proton, have been identified to play an antioxidant role in cells where oxidative damage occurs. Both proteins are mainly localized in tissues characterized by a high rate of oxygen consumption, and contain on their molecular surface two reactive cysteine residues eventually undergoing S-glutathionylation. Here, we will provide an overview on the molecular and functional features of these proteins highlighting their implications into molecular processes occurring during oxidative stress conditions.

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.

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.

Disclosing the Interaction of Carbonic Anhydrase IX with Cullin-Associated NEDD8-Dissociated Protein 1 by Molecular Modeling and Integrated Binding Measurements.

Human Carbonic Anhydrase (hCA) IX is a membrane-associated member of the CA enzyme family, involved in solid tumor acidification. This enzyme is a marker of tumor hypoxia and a prognostic factor for several human cancers. In a recent paper, we showed that CA IX interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes. A functional role for this interaction was also identified, since lower CA IX levels were observed in cells with decreased CAND1 expression via shRNA-mediated interference. In this paper, we describe the identification of the structural determinants responsible for the CA IX/CAND1 interaction by means of a multidisciplinary approach, consisting of binding assay measurements, molecular docking, and site-directed mutagenesis. These data open a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CAND1/CA IX interaction provides the molecular basis to design molecules able to destabilize it. Due to the proposed function of CAND1 in stabilizing CA IX, these molecules could represent an efficient tool to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and the metastatic spread of tumors.

Probing Molecular Interactions between Human Carbonic Anhydrases (hCAs) and a Novel Class of Benzenesulfonamides.

On the basis of X-ray crystallographic studies of the complex of hCA II with 4-(3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide (3) (PDB code 4Z1J ), a novel series of 4-(1-aryl-3,4-dihydro-1H-isoquinolin-2-carbonyl)benzenesulfonamides (23-33) was designed. Specifically, our idea was to improve the selectivity toward druggable isoforms through the introduction of additional hydrophobic/hydrophilic functionalities. Among the synthesized and tested compounds, the (R,S)-4-(6,7-dihydroxy-1-phenyl-3,4-tetrahydroisoquinoline-1H-2-carbonyl)benzenesulfonamide (30) exhibited a remarkable inhibition for the brain-expressed hCA VII (Ki = 0.20 nM) and selectivity over wider distributed hCA I and hCA II isoforms. By enantioselective HPLC, we solved the racemic mixture and ascertained that the two enantiomers (30a and 30b) are equiactive inhibitors for hCA VII. Crystallographic and docking studies revealed the main interactions of these inhibitors into the carbonic anhydrase (CA) catalytic site, thus highlighting the relevant role of nonpolar contacts for this class of hCA inhibitors.

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.