Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1-mediated nuclear export.

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor.

Generation and characterisation of an Arabidopsis thaliana f3h/fls1/ans triple mutant that accumulates eriodictyol derivatives.

BACKGROUND: Flavonoids are plant specialised metabolites, which derive from phenylalanine and acetate metabolism. They possess a variety of beneficial characteristics for plants and humans. Several modification steps in the synthesis of tricyclic flavonoids cause for the amazing diversity of flavonoids in plants. The 2-oxoglutarate-dependent dioxygenases (2-ODDs) flavanone 3-hydroxylase (F3H, synonym FHT), flavonol synthase (FLS) and anthocyanidin synthase (ANS, synonym leucoanthocyanidin dioxygenase (LDOX)), catalyse oxidative modifications to the central C ring. They are highly similar and have been shown to catalyse, at least in part, each other's reactions. FLS and ANS have been identified as bifunctional enzymes in many species, including Arabidopsis thaliana, stressing the capability of plants to bypass missing or mutated reaction steps on the way to flavonoid production. However, little is known about such bypass reactions and the flavonoid composition of plants lacking all three central flavonoid 2-ODDs. RESULTS: To address this issue, we generated a f3h/fls1/ans mutant, as well as the corresponding double mutants and investigated the flavonoid composition of this mutant collection. The f3h/fls1/ans mutant was further characterised at the genomic level by analysis of a nanopore DNA sequencing generated genome sequence assembly and at the transcriptomic level by RNA-Seq analysis. The mutant collection established, including the novel double mutants f3h/fls1 and f3h/ans, was used to validate and analyse the multifunctionalities of F3H, FLS1, and ANS in planta. Metabolite analyses revealed the accumulation of eriodictyol and additional glycosylated derivatives in mutants carrying the f3h mutant allele, resulting from the conversion of naringenin to eriodictyol by flavonoid 3'-hydroxylase (F3'H) activity. CONCLUSIONS: We describe the in planta multifunctionality of the three central flavonoid 2-ODDs from A. thaliana and identify a bypass in the f3h/fls1/ans triple mutant that leads to the formation of eriodictyol derivatives. As (homo-)eriodictyols are known as bitter taste maskers, the annotated eriodictyol (derivatives) and in particular the observations made on their in planta production, could provide valuable insights for the creation of novel food supplements.

Comparative CO2 and SiO2 hydratase activity of an enzyme from the siliceous demosponge Suberitesdomuncula.

Silicase, an enzyme that catalyzes the hydrolysis of silicon-oxygen bonds, is a crucial player in breaking down silicates into silicic acid, particularly in organisms like aquatic sponges with siliceous skeletons. Despite its significance, our understanding of silicase remains limited. This study comprehensively examines silicase from the demosponge Suberites domuncula, focusing on its kinetics toward CO2 as a substrate, as well as its silicase and esterase activity. It investigates inhibition and activation profiles with a range of inhibitors and activators belonging to various classes. By comparing its esterase activity to human carbonic anhydrase II, we gain insights into its enzymatic properties. Moreover, we investigate silicase's inhibition and activation profiles, providing valuable information for potential applications. We explore the evolutionary relationship of silicase with related enzymes, revealing potential functional roles in biological systems. Additionally, we propose a biochemical mechanism through three-dimensional modeling, shedding light on its catalytic mechanisms and structural features for both silicase activity and CO2 hydration. We highlight nature's utilization of enzymatic expertise in silica metabolism. This study enhances our understanding of silicase and contributes to broader insights into ecosystem functioning and Earth's geochemical cycles, emphasizing the intricate interplay between biology and the environment.

Sulfonamide-incorporated bis(α-aminophosphonates) as promising carbonic anhydrase inhibitors: Design, synthesis, biological evaluation, and X-ray crystallographic studies.

A novel series of sulfonamide-incorporated bis(α-aminophosphonates) acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. The synthesized bivalent ligands were tested against five human (h) isoforms, hCA I, hCA II, hCA VII, hCA IX, and hCA XIII. Such derivatives showed high activity and selectivity against the cancer-related, membrane-bound isoform hCA IX, and among them, compound 5h, tetraisopropyl (1,3-phenylenebis{[(4-sulfamoylphenyl)amino]methylene})bis(phosphonate) showed a KI of 15.1 nM, being highly selective against this isoform over all other investigated ones (hCA I/IX = 42; hCA II/IX = 6, hCA VII/IX = 3, hCA XIII/IX = 5). Therefore, compound 5h could be a potential lead for the development of selective anticancer agents. The newly developed sulfonamides were also found effective inhibitors against the cytosolic hCA XIII isoform. Compound 5i displayed the best inhibition against this isoform with a KI of 17.2 nM, equal to that of the well-known inhibitor acetazolamide (AAZ), but significantly more selective over all other tested isoforms (hCA I/XIII = 239; hCA II/XIII = 23, hCA VII/XIII = 2, hCA IX/XIII = 3) compared to AAZ.

Design, synthesis, and biological evaluation of 3-benzenesulfonamide-linked 3-hydrazinoisatin derivatives as carbonic anhydrase inhibitors.

A new series of isatin-linked benzenesulfonamide derivatives (9a-w) were synthesized using the tail approach and assayed for their inhibitory potency against four different human carbonic anhydrase (hCA) isoforms, hCA I, II, IX, and XII. Most of these synthesized compounds exhibited interesting inhibition potency against isoforms hCA I, IX, and XII in the nanomolar range and by taking the standard drug acetazolamide. The most potent compounds in the case of hCA I were 9c (435.8 nM) and 9s (956.4 nM), for hCA IX, 9a (60.5 nM), 9d (95.6 nM), 9g (92.1 nM), and 9k (75.4 nM), and for hCA XII, 9p (84.5 nM). However, these compounds showed more selectivity toward hCA IX over hCA I, II, and XII. Thus, these compounds can be further developed as potential lead molecules for the development of isoform-selective hCA IX inhibitors with further structural modifications.

Identification of 3-(5-cyano-6-oxo-pyridin-2-yl)benzenesulfonamides as novel anticancer agents endowed with EGFR inhibitory activity.

New 5-cyano-6-oxo-pyridine-based sulfonamides (6a-m and 8a-d) were designed and synthesized to potentially inhibit both the epidermal growth factor receptor (EGFR) and carbonic anhydrase (CA), with anticancer properties. First, the in vitro anticancer activity of each target substance was tested using Henrietta Lacks cancer cell line and M.D. anderson metastasis breast cancer cell line cells. Then, the possible CA inhibition against the human CA isoforms I, II, and IX was investigated, together with the EGFR inhibitory activity, with the most powerful derivatives. The neighboring methoxy group may have had a steric effect on the target sulfonamides, which prevented them from effectively inhibiting the CA isoforms while effectively inhibiting the EGFR. The effects of the 5-cyanopyridine derivatives 6e and 6l on cell-cycle disruption and the apoptotic potential were then investigated. To investigate the binding mechanism and stability of the target molecules, thorough molecular modeling assessments, including docking and dynamic simulation, were performed.

Leveraging SARS-CoV-2 Main Protease (Mpro) for COVID-19 Mitigation with Selenium-Based Inhibitors.

The implementation of innovative approaches is crucial in an ongoing endeavor to mitigate the impact of COVID-19 pandemic. The present study examines the strategic application of the SARS-CoV-2 Main Protease (Mpro) as a prospective instrument in the repertoire to combat the virus. The cloning, expression, and purification of Mpro, which plays a critical role in the viral life cycle, through heterologous expression in Escherichia coli in a completely soluble form produced an active enzyme. The hydrolysis of a specific substrate peptide comprising a six-amino-acid sequence (TSAVLQ) linked to a p-nitroaniline (pNA) fragment together with the use of a fluorogenic substrate allowed us to determine effective inhibitors incorporating selenium moieties, such as benzoselenoates and carbamoselenoates. The new inhibitors revealed their potential to proficiently inhibit Mpro with IC50-s in the low micromolar range. Our study contributes to the development of a new class of protease inhibitors targeting Mpro, ultimately strengthening the antiviral arsenal against COVID-19 and possibly, related coronaviruses.

Mono- and three-tailed sugar and iminosugar decorated benzenesulfonamide carbonic anhydrase inhibitors.

A collection of novel mono- and three-tailed derivatives based on a sugar (glucose) or an iminosugar (trihydroxy piperidine) featuring a terminal benzenesulfonamide were synthesized to investigate the so-called "sugar" and "azasugar" approach with the aim of exploring the activity and selectivity towards the inhibition of human carbonic anhydrases (hCAs). The synthetic approach relies on a general copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction followed by an amine-isothiocyanate coupling. Biological assays were used to collect subtle information on the role of these single or multiple hydrophilic chains. Among the sugar-based inhibitors, the single-tailed compound 10 was identified as a better inhibitor than the reference compound (AAZ) towards three different hCAs, while, among the three sugar tailed derivatives, potent and selective inhibition was found for compounds 25 and 26. A promising and selective inhibitory activity was discovered for the iminosugar single-tailed compound 31 towards hCA VII (Ki = 9.7 nM).

Substituted furan sulfonamides as carbonic anhydrase inhibitors: Synthesis, biological and in silico studies.

Carbonic Anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide involved in several of biological processes, such as respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. They show wide diversity in tissue distribution and in their subcellular localization. Fifteen novel furyl sulfonamides were designed, synthesized and evaluated against four human isoforms: hCA I, hCA II, hCA IV and hCA IX. Compounds appeared to be very active mostly against hCAI (8) and hCA IV (11) isoforms being more potent than reference drug acetazolamide (AAZ). It should be mentioned that four compounds were more active than AAZ against hCA IX isoform, with compound 13d to be selective against hCA I (SI 70), hCA II (SI 13.5) and hCA IV (SI 20). Furthermore, docking was performed for some of these compounds on all isoforms I order to understand the possible interactions with the active site. The most active compounds showed good bioavailability and drug likeness scores.

Schiff bases as linker in the development of quinoline-sulfonamide hybrids as selective cancer-associated carbonic anhydrase isoforms IX/XII inhibitors: A new regioisomerism tactic.

A novel set of quinoline tailored with the sulfonamide as zinc-binding group (ZBG) has been rationalized and synthesized as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Such hybrids were decorated by a novel elongated imine linker with/without ethylene spacer with variable hydrophobic and lipophilic pockets. Therefore, a regioisomeric tactic has been established, most of which act as efficient inhibitors of the tumor-associated CA isoforms IX and XII. Interestingly, one hybrid 10b displayed an appreciable activity in MCF-7 cell line under normoxic condition (IC50 of 8.42 µM) in comparison to the standard staurosporine (IC50 = 5.34 µM) and excellent activity under hypoxic conditions (IC50 = 1.56 µM) in comparison to staurosporine (IC50 = 4.45 µM). Furthermore, hybrids 8a and 10b encouraged MCF-7 and MDA-MB-231 cell apoptosis alongside promising Bax/Bcl expression ratio change. Docking studies were also, performed and agreed with the biological results. Our SAR study suggested that our regiosiomerization tactic for the quinoline based-sulfonamide molecules led to effective inhibition of tumuor-relevant hCAs IX/XII.

Click chemistry approaches for developing carbonic anhydrase inhibitors and their applications.

Click chemistry reactions constitute an important and relatively new approach in the medicinal chemistry toolbox and offer substantial advantages to medicinal chemists in terms of overcoming the limitations of facile chemical synthesis, increased throughput, yields and improved quality of compound libraries. Over the last two decades, click chemistry has been widely used in different aspects of carbonic anhydrase (CA, EC 4.2.1.1) modulators drug design. This review provides an overview of the general principles and applications of click chemistry in CA-related research, including the design of selective CA inhibitors (CAIs), their applications against several diseases such as innovative anticancer and anti-infective agents, fluorescent and radiopharmaceutical probes as new theranostic agents, as well as their application in protein labelling.

Diversely N-substituted benzenesulfonamides dissimilarly bind to human carbonic anhydrases: crystallographic investigations of N-nitrosulfonamides.

Carbonic anhydrases (CAs) are a zinc metalloenzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton, pivotal for a wide range of biological processes. CAs are involved in numerous pathologies and thus represent valuable drug targets in the treatments of several diseases such as glaucoma, obesity, tumour, neuropathic pain, cerebral ischaemia, or as antiinfectives. In the last two decades, several efforts have been made to achieve selective CA inhibitors (CAIs) employing different drug design approaches. However, N-substitutions on primary sulphonamide groups still remain poorly investigated. Here, we reported for the first time the co-crystallisation of a N-nitro sulphonamide derivative with human (h) CA II pointing out the binding site and mode of inhibition of this class of CAIs. The thorough comprehension of the ligand/target interaction might be valuable for a further CAI optimisation for achieving new potent and selective derivatives.

Cloning, characterization, and inhibition of the novel ß-carbonic anhydrase from parasitic blood fluke, Schistosoma mansoni.

Schistosoma mansoni is an intestinal parasite with one ß-class carbonic anhydrase, SmaBCA. We report the sequence enhancing, production, catalytic activity, and inhibition results of the recombinant SmaBCA. It showed significant catalytic activity on CO2 hydration in vitro with kcat 1.38 × 105 s-1 and kcat/Km 2.33 × 107 M-1 s-1. Several sulphonamide inhibitors, from which many are clinically used, showed submicromolar or nanomolar inhibitory effects on SmaBCA. The most efficient inhibitor with a KI of 43.8 nM was 4-(2-amino-pyrimidine-4-yl)-benzenesulfonamide. Other effective inhibitors with KIs in the range of 79.4-95.9 nM were benzolamide, brinzolamide, topiramate, dorzolamide, saccharin, epacadostat, celecoxib, and famotidine. The other tested compounds showed at least micromolar range inhibition against SmaBCA. Our results introduce SmaBCA as a novel target for drug development against schistosomiasis, a highly prevalent parasitic disease.

Synthesis and biological evaluation of new 3-substituted coumarin derivatives as selective inhibitors of human carbonic anhydrase IX and XII.

The Carbonic anhydrase isoforms IX and XII play a significant role in regulating the intracellular and extracellular pH in hypoxic tumours abetting the metastasis of solid tumours. Selective and potent inhibitors targeting carbonic anhydrase IX and XII reduce the activity of these isoforms in hypoxic tumours, representing an antitumor and antimetastatic mechanism. Coumarin-based derivatives are selective inhibitors of CA isoforms IX and XII. In this study, we report the design and synthesis of new 3-substituted coumarin derivatives with different functional moieties and their inhibitory activity against various carbonic anhydrase isoforms. We found that the tertiary sulphonamide derivative 6c showed selective inhibition against CA IX with IC50 of 4.1 µM. Similarly, the carbothioamides 7c, 7b and oxime ether derivative 20a exhibited good inhibition against CA IX and CA XII. Additionally, the binding mode was predicted and validated using molecular docking and dynamic simulations.

Selenium-analogs based on natural sources as cancer-associated carbonic anhydrase isoforms IX and XII inhibitors.

In the relentless search for new cancer treatments, organoselenium compounds, and carbonic anhydrase (CA) inhibitors have emerged as promising drug candidates. CA isoforms IX and XII are overexpressed in many types of cancer, and their inhibition is associated with potent antitumor/antimetastatic effects. Selenium-containing compounds, particularly selenols, have been shown to inhibit tumour-associated CA isoforms in the nanomolar range since the properties of the selenium atom favour binding to the active site of the enzyme. In this work, two series of selenoesters (1a-19a and 1b-19b), which gathered NSAIDs, carbo/heterocycles, and fragments from natural products, were evaluated against hCA I, II, IX, and XII. Indomethacin (17b) and flufenamic acid (19b) analogs exhibited selectivity for tumour-associated isoform IX in the low micromolar range. In summary, selenoesters that combine NSAIDs with fragments derived from natural sources have been developed as promising nonclassical inhibitors of the tumour-associated CA isoforms.

Benzenesulfonamide derivatives as Vibrio cholerae carbonic anhydrases inhibitors: a computational-aided insight in the structural rigidity-activity relationships.

Vibrio cholerae causes life-threatening infections in low-income countries due to the rise of antibacterial resistance. Innovative pharmacological targets have been investigated and carbonic anhydrases (CAs, EC: 4.2.1.1) encoded by V. cholerae (VchCAs) emerged as a valuable option. Recently, we developed a large library of para- and meta-benzenesulfonamides characterised by moieties with a different flexibility degree as CAs inhibitors. Stopped flow-based enzymatic assays showed strong inhibition of VchαCA for this library, while lower affinity was detected against the other isoforms. In particular, cyclic urea 9c emerged for a nanomolar inhibition of VchαCA (KI = 4.7 nM) and high selectivity with respect to human isoenzymes (SI≥ 90). Computational studies revealed the influence of moiety flexibility on inhibitory activity and isoform selectivity and allowed accurate SARs. However, although VchCAs are involved in the bacterium virulence and not in its survival, we evaluated the antibacterial activity of such compounds, resulting in no direct activity.

2H-chromene and 7H-furo-chromene derivatives selectively inhibit tumour associated human carbonic anhydrase IX and XII isoforms.

Tumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors.

Discovery of sulfonamide-tethered isatin derivatives as novel anticancer agents and VEGFR-2 inhibitors.

In this work, new isatin-based sulphonamides (6a-i, 11a-c, 12a-c) were designed and synthesised as potential dual VEGFR-2 and carbonic anhydrase inhibitors with anticancer activities. Firstly, all target isatins were examined for in vitro antitumor action on NCI-USA panel (58 tumour cell lines). Then, the most potent derivatives were examined for the potential CA inhibitory action towards the physiologically relevant hCA isoforms I, II, and tumour-linked hCA IX isoform, in addition, the VEGFR-2 inhibitory activity was evaluated. The target sulphonamides failed to inhibit the CA isoforms that could be attributable to the steric effect of the neighbouring methoxy group, whereas they displayed potent VEGFR-2 inhibitory effect. Following that, isatins 11b and 12b were tested for their influence on the cell cycle disturbance, and towards the apoptotic potential. Finally, detailed molecular modelling analyses, including docking and molecular dynamics, were carried out to assess the binding mode and stability of target isatins.

Structure-guided identification of a selective sulfonamide-based inhibitor targeting the human carbonic anhydrase VA isoform.

In recent years, multistep hybrid computational protocols have attracted attention for their application in the drug discovery of enzyme inhibitors. So far, there are large collections of human carbonic anhydrase (hCA) inhibitors, but only a few of them selectively inhibit the mitochondrial isoforms hCA VA and VB as potential therapeutics in obesity treatment. Most sulfonamide-based inhibitors show poor selectivity for inhibiting isoforms of therapeutic interest over ubiquitous hCA I and hCA II. Herein, we propose a combination of ligand- and structure-based approaches to generate pharmacophore models for hCA VA inhibitors. Then, we performed a virtual screening (VS) campaign on a database of commercially available sulfonamides. Finally, the in silico screening followed by docking studies suggested several "hit compounds" that demonstrated to inhibit hCA VA at a low nanomolar concentration in a stopped-flow CO2 hydrase assay. Notably, the best candidate, 2-(3,4-dihydro-2H-quinolin-1-yl)-N-(4-sulfamoylphenyl)acetamide (code name VAME-28) proved to be a potent hCA VA inhibitor (Ki value of 54.8 nM) and a more selective agent over hCA II when compared to the reference compound topiramate.

Benzenesulfonamides with trisubstituted triazole motif as selective carbonic anhydrase I, II, IV, and IX inhibitors.

Twenty novel 1,2,3-triazole benzenesulfonamides featuring nitrile 8a-g, carbothioamide 9a-f, and N'-hydroxycarboximidamide 10a-g functionalities were designed and synthesized to improve potency and selectivity as carbonic anhydrase inhibitors (CAIs). The synthesized 1,2,3-triazole compounds were tested in vitro as CAIs against four physiologically and pharmacologically relevant isoforms of human carbonic anhydrase (hCA I, II, IV, and IX). Compounds 8a-g, 9a-f, and 10a-g displayed variable inhibition constants ranging from 8.1 nM to 3.22 µM for hCA I, 4.7 nM to 0.50 µM for hCA II, 15.0 nM to 3.7 µM for hCA IV, and 29.6 nM to 0.27 µM for hCA IX. As per the inhibition data profile, compounds 9a-e exhibited strong efficacy for hCA IV, whereas the inhibition was found to be somewhat diminished in the case of hCA IX by nearly all the compounds. A computational protocol based on docking and MM-GBSA was conducted to reveal the plausible interactions of the targeted sulfonamides within the hCA II and IX binding sites. The outcomes of appending various functionalities at the C-4 position of the 1,2,3-triazole motif over the inhibition potential and selectivity of the designed sulfonamides were examined with a potential for the discovery of new isoform selective CAIs. The CAI and SAR data established the significance of the synthesized 1,2,3-triazoles as building blocks for developing CAI drugs.