Cistus incanus: a natural source of antimicrobial metabolites.

The discovery of natural molecules with antimicrobial properties has become an urgent need for the global treatment of bacterium and virus infections. Cistus incanus, a Mediterranean shrub species, represents a valuable source of phytochemicals with an interesting wide-spectrum antimicrobial potential. In this study, we analysed the spectrum of molecules composing a commercial hydroalcoholic extract of C. incanus finding ellagitannins as the most abundant. The effect of the extract and its main constituents (gallic acid, ellagic acid and punicalin) was assessed as co-treatment during viral (HSV-1, HCoV-229E, SARS-CoV-2) and bacterial infection (Staphylococcus aureus and Escherichia coli) of cells and as pre-treatment before virus infections. The results indicated a remarkable antiviral activity of punicalin against SARS-CoV-2 by pre-treating both the viral and the host cells, and a major sensitivity of S. aureus to the C. incanus extract compared to E. coli. The present study highlights broad antimicrobial potential of C. incanus extract.

Intrinsically disordered Prosystemin discloses biologically active repeat motifs.

The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.

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.

6-Substituted Triazolyl Benzoxaboroles as Selective Carbonic Anhydrase Inhibitors: In Silico Design, Synthesis, and X-ray Crystallography.

Benzoxaborole is currently a scaffold of great relevance in medicinal chemistry. In 2016, it was reported to be a new and valuable chemotype for designing carbonic anhydrase (CA) inhibitors. Herein, using an in silico design, we report the synthesis and characterization of substituted 6-(1H-1,2,3-triazol-1-yl)benzoxaboroles. 6-Azidobenzoxaborole was described for the first time as a molecular platform to prepare libraries of inhibitors by a copper(I)-catalyzed azide-alkyne cycloaddition via a click chemistry strategy. With inhibition constants below 30 nM, some derivatives, such as compound 20, showed efficacy as selective hCA VII and IX inhibitors. The design hypothesis was validated by crystallographic investigation on the hCA II/20 adduct, which provided explanations over the different inhibition behavior observed against the five evaluated hCA isoforms. Overall, this study identified 20 as a new promising lead compound to develop novel anticancer agents targeting the tumor-associated hCA IX but also potent neuropathic pain relievers targeting hCA VII.

A Combined in Silico and Structural Study Opens New Perspectives on Aliphatic Sulfonamides, a Still Poorly Investigated Class of CA Inhibitors.

Aliphatic sulfonamides are an interesting class of carbonic anhydrase inhibitors (CAIs) proven to be effective for several carbonic anhydrase (CA) isoforms involved in pathologic states. Here we report the crystallographic structures of hCA II in complex with two aliphatic sulfonamides incorporating coumarin rings, which showed a good inhibition and selectivity for this isoform. Although these two molecules have a very similar chemical structure, differing only in the substitution of the two aliphatic hydrogen atoms with two fluorine atoms, they adopt a significantly different binding mode within the enzyme active site. Theoretical binding free energy calculations, performed to rationalize these data, showed that a delicate balance of electrostatic and steric effects modulate the protein-ligand interactions. Data presented here can be fruitfully used for the rational design of novel and effective isozyme-specific inhibitor molecules.

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.

Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.

Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.

The Amazing World of IDPs in Human Diseases II.

Intrinsically Disordered Proteins (IDPs) lack stable tertiary and secondary structures and are extensively distributed across eukaryotic cells, playing critical roles in cell signaling and regulation [...].

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.

Tomato Prosystemin Is Much More than a Simple Systemin Precursor.

Systemin (Sys) is an octadecapeptide, which upon wounding, is released from the carboxy terminus of its precursor, Prosystemin (ProSys), to promote plant defenses. Recent findings on the disordered structure of ProSys prompted us to investigate a putative biological role of the whole precursor deprived of the Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant truncated ProSys (ProSys(1-178), the Prosystemin sequence devoid of Sys region). By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests, we demonstrate that truncated ProSys promotes defense barriers in tomato plants through a hormone-independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted in protection against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild-type ProSys is more complex than previously shown, as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of jasmonic acid biosynthesis and the successive activation of a set of defense-related genes, and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity.

Sultam based Carbonic Anhydrase VII inhibitors for the management of neuropathic pain.

We report a series of compounds 1-17 derived from the antiepileptic drug Sulthiame (SLT) from which both the benzenesulfonamide and the sultam moiety were retained. All compounds were tested in vitro for their inhibition activity against the human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, VII, IX and XII isoforms. Among the series, derivatives 1 and 11 showed great enhancement of both inhibition potency and selectivity towards the hCA VII isoform, when compared to the reference SLT drug. The binding mode of 11 within the hCA VII active site was deciphered by means of X-ray crystallography and revealed the sultam moiety being exposed to the rim of the active site. In vivo experiments on a model of neuropathic pain induced by oxaliplatin clearly showed 11 being an effective pain relieving agent and therefore worth of further exploitation towards the validation of the hCA VII as new target for the management of neuropathies.

Zeta-carbonic anhydrases show CS2 hydrolase activity: A new metabolic carbon acquisition pathway in diatoms?

CDCA1 is a very peculiar member of the Carbonic Anhydrase (CA) family. It has been the first enzyme to show an efficient utilization of Cd(II) ions in Nature and a unique adaptation capability to live on the surface ocean. Indeed, in this environment, which is extremely depleted in essential metal ions, CDCA1 can utilize Zn(II) or Cd(II) as catalytic metal to support the metabolic needs of fast growing diatoms. In this paper we demonstrate a further catalytic versatility of this enzyme by using a combination of X-ray crystallography, molecular dynamics simulations and enzymatic experiments. First we identified the CO2 binding site and the way in which this substrate travels from the environment to the enzyme active site. Then, starting from the observation of a structural similarity with the substrate entry route of CS2 hydrolase from Acidanius A1-3, we hypothesized and demonstrated that also CS2 is a substrate for CDCA1. This finding is new and unexpected since until now only few CS2 hydrolases have been characterized, and none of them is reported to have any CO2 hydratase action. The physiological implications of this supplementary catalytic activity still remain to be unveiled. We suggest here that it could represent another ability of diatoms expressing CDCA1 to adapt to the external environment. Indeed, the ability of this enzyme to convert CS2 could represent an alternative source of carbon acquisition for diatoms, in addition to CO2.

The Amazing World of IDPs in Human Diseases.

It has been clearly established that some proteins or protein regions are devoid of any stable secondary and/or tertiary structure under physiological conditions, but still possess fundamental biological functions [...].

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.

Carbonic Anhydrase Inhibitors Targeting Metabolism and Tumor Microenvironment.

The tumor microenvironment is crucial for the growth of cancer cells, triggering particular biochemical and physiological changes, which frequently influence the outcome of anticancer therapies. The biochemical rationale behind many of these phenomena resides in the activation of transcription factors such as hypoxia-inducible factor 1 and 2 (HIF-1/2). In turn, the HIF pathway activates a number of genes including those involved in glucose metabolism, angiogenesis, and pH regulation. Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA IX and XII, actively participate in these processes and were validated as antitumor/antimetastatic drug targets. Here, we review the field of CA inhibitors (CAIs), which selectively inhibit the cancer-associated CA isoforms. Particular focus was on the identification of lead compounds and various inhibitor classes, and the measurement of CA inhibitory on-/off-target effects. In addition, the preclinical data that resulted in the identification of SLC-0111, a sulfonamide in Phase Ib/II clinical trials for the treatment of hypoxic, advanced solid tumors, are detailed.

2-Mercaptobenzoxazoles: a class of carbonic anhydrase inhibitors with a novel binding mode to the enzyme active site.

2-Mercaptobenzoxazole is a widely used organic scaffold in medicinal chemistry. By means of kinetic and structural studies, we demonstrate that this molecule can effectively be used to inhibit hCAs showing a peculiar binding mode. The results reported here can pave the way for the development of selective CA inhibitors.

Phenyl(thio)phosphon(amid)ate Benzenesulfonamides as Potent and Selective Inhibitors of Human Carbonic Anhydrases II and VII Counteract Allodynia in a Mouse Model of Oxaliplatin-Induced Neuropathy.

Human carbonic anhydrase (CA; EC 4.2.1.1) isoforms II and VII are implicated in neuronal excitation, seizures, and neuropathic pain (NP). Their selective inhibition over off-target CAs is expected to produce an anti-NP action devoid of side effects due to promiscuous CA modulation. Here, a drug design strategy based on the observation of (dis)similarities between the target CA active sites was planned with benzenesulfonamide derivatives and, for the first time, a phosphorus-based linker. Potent and selective CA II/VII inhibitors were identified among the synthesized phenyl(thio)phosphon(amid)ates 3-22. X-ray crystallography depicted the binding mode of phosphonic acid 3 to both CAs II and VII. The most promising derivatives, after evaluation of their stability in acidic media, were tested in a mouse model of oxaliplatin-induced neuropathy. The most potent compound racemic mixture was subjected to HPLC enantioseparation, and the identification of the eutomer, the (S)-enantiomer, allowed to halve the dose totally relieving allodynia in mice.

Biochemical and Structural Insights into Carbonic Anhydrase XII/Fab6A10 Complex.

6A10 is a CA XII inhibitory monoclonal antibody, which was demonstrated to reduce the growth of cancer cells in vitro and in a xenograft model of lung cancer. It was also shown to enhance chemosensitivity of multiresistant cancer cell lines and to significantly reduce the number of lung metastases in combination with doxorubicin in mice carrying human triple-negative breast cancer xenografts. Starting from these data, we report here on the development of the 6A10 antigen-binding fragment (Fab), termed Fab6A10, and its functional, biochemical, and structural characterization. In vitro binding and inhibition assays demonstrated that Fab6A10 selectively binds and inhibits CA XII, whereas immunohistochemistry experiments highlighted its capability to stain malignant glioma cells in contrast to the surrounding brain tissue. Finally, the crystallographic structure of CA XII/Fab6A10 complex provided insights into the inhibition mechanism of Fab6A10, showing that upon binding, it obstructs the substrate access to the enzyme active site and interacts with CA XII His64 freezing it in its out conformation. Altogether, these data indicate Fab6A10 as a new promising therapeutic tool against cancer.

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.