Discovery of non-sulfonamide carbonic anhydrase IX inhibitors through structure-based virtual screening.

Carbonic anhydrase IX (CA IX) is a subtype of the human carbonic anhydrase (hCA) family and exhibits high expression in various solid tumors, rendering it a promising target for tumor therapy. Currently, marketed carbonic anhydrase inhibitors (CAIs) are primarily composed of sulfonamides derivatives, which may have impeded their potential for further expansion. Therefore, we have developed a structure-based virtual screening approach to explore novel CAIs exhibiting distinctive structures and anti-tumor potential in the FDA database. In vitro experiments demonstrated that 3-pyridinemethanol (0.42 µM), procodazole (8.35 µM) and pamidronic acid (8.51 µM) exhibited inhibitory effects on CA IX activity. The binding stability and interaction mode between the CA IX and the hit compounds are further investigated through molecular dynamics simulations and binding free energy calculations. Furthermore, the ADME/Tox prediction results indicated that these compounds exhibited favorable pharmacological properties and minimal toxic side effects. Our study successfully applied computational strategies to discover three non-sulfonamide inhibitors of carbonic anhydrase IX (CA IX) that demonstrate inhibitory activity in vitro. These findings have significant implications for the development of CA IX inhibitors and anti-tumor drugs, contributing to their progress in the field.

Amyloid Beta Oligomers Activate Death Receptors and Mitochondria-Mediated Apoptotic Pathways in Cerebral Vascular Smooth Muscle Cells; Protective Effects of Carbonic Anhydrase Inhibitors.

Amyloid beta (Aß) deposition within the brain vasculature is an early hallmark of Alzheimer's disease (AD), which triggers loss of brain vascular smooth muscle cells (BVSMCs) in cerebral arteries, via poorly understood mechanisms, altering cerebral blood flow, brain waste clearance, and promoting cognitive impairment. We have previously shown that, in brain endothelial cells (ECs), vasculotropic Aß species induce apoptosis through death receptors (DRs) DR4 and DR5 and mitochondria-mediated mechanisms, while FDA-approved carbonic anhydrase inhibitors (CAIs) prevent mitochondria-mediated EC apoptosis in vitro and in vivo. In this study, we analyzed Aß-induced extrinsic and intrinsic (DR- and mitochondria-mediated) apoptotic pathways in BVSMC, aiming to unveil new therapeutic targets to prevent BVSMC stress and death. We show that both apoptotic pathways are activated in BVSMCs by oligomeric Aß42 and Aß40-Q22 (AßQ22) and mitochondrial respiration is severely impaired. Importantly, the CAIs methazolamide (MTZ) and acetazolamide (ATZ) prevent the pro-apoptotic effects in BVSMCs, while reducing caspase 3 activation and Aß deposition in the arterial walls of TgSwDI animals, a murine model of cerebral amyloid angiopathy (CAA). This study reveals new molecular targets and a promising therapeutic strategy against BVSMC dysfunction in AD, CAA, and ARIA (amyloid-related imaging abnormalities) complications of recently FDA-approved anti-Aß antibodies.

PLBD: protein-ligand binding database of thermodynamic and kinetic intrinsic parameters.

We introduce a protein-ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein-ligand crystal structures, enabling structure-thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein-ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein-ligand recognition and could be integrated into small molecule drug design. Database URL https://plbd.org/.

Challenging breast cancer through novel sulfonamide-pyridine hybrids: design, synthesis, carbonic anhydrase IX inhibition and induction of apoptosis.

Background: Among the important key modulators of the tumor microenvironment and hypoxia is a family of enzymes named carbonic anhydrases. Herein, 11 novel sulfonamide-pyridine hybrids (2-12) were designed, synthesized and biologically evaluated for their potential use in targeting breast cancer. Methods & results: The para chloro derivative 7 reported the highest cytotoxic activity against the three breast cancer cell lines used. In addition, compound 7 was found to induce cell cycle arrest and autophagy as well as delaying wound healing. The IC50 of compound 7 against carbonic anhydrase IX was 253 ± 12 nM using dorzolamide HCl as control. Conclusion: This study encourages us to expand the designed library, where more sulfonamide derivatives would be synthesized and studied for their structure-activity relationships.

Intrinsic affinity of protein - ligand binding by differential scanning calorimetry.

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein-ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.

The Protective Effects of Acetazolamide Against Homocysteine-Induced Blood-Brain-Barrier Disruption by Regulating the Activation of the Wnt/ß-Catenin Signaling Pathway.

Acetazolamide (AZA) is a carbonic anhydrase inhibitor (CAI) with neuroprotective effects. Hyperhomocysteinemia is associated with blood-brain-barrier (BBB) disruption in brain disorders. A previous study indicated that AZA might have a new role in brain disorders. However, its function in hyperhomocysteinemia-related BBB disruption has not been reported. Here, we aim to clarify the role of AZA in homocysteine (Hcy)-mediated BBB dysfunction using both in vivo and in vitro assays. We found that AZA improved memory and cognitive function, and reduced brain edema in Hcy-stimulated hyperhomocysteinemia model rats. This protective effect of AZA on hyperhomocysteinemia rats was accompanied by improved BBB permeability and increased expression levels of the tight junction proteins, occludin, and claudin-5. The in vitro assay results show that AZA prevented Hcy-induced cell injury and attenuated the increased permeability in Hcy-treated bEnd.3 brain endothelial cells. The Hcy-induced decrease in occludin and claudin-5, and increase in MMP-2 and MMP-9 expression levels were attenuated by AZA in bEnd.3 cells. Moreover, the Hcy-induced downregulation of the Wnt/ß-catenin signaling pathway in bEnd.3 cells was abolished by AZA. Inhibition of Wnt/ß-catenin by ICG-001 reversed the protective effects of AZA in Hcy-treated bEnd.3 cells. We also prove that this process is mediated by WTAP. These findings suggest that acetazolamide mitigated the Hcy-induced compromised brain vascular endothelial integrity by regulating the activation of the Wnt/ß-catenin signaling pathway.

The gram-negative bacterium Escherichia coli as a model for testing the effect of carbonic anhydrase inhibition on bacterial growth.

Carbonic anhydrases, catalysing the reversible CO2 hydration reaction, contribute in all living organisms to the maintenance of stable metabolic functions depending on intracellular concentrations of carbon dioxide, bicarbonate, and protons. Recent studies have examined how CAs affect bacterial lifecycle, considering these enzymes druggable targets due to interference with their activities by using inhibitors or activators. Here, we propose Escherichia coli cells as a model for testing the effect of acetazolamide (AZA), a potent CA inhibitor, on bacterial survival by evaluating E. coli growth through its glucose consumption. AZA, at concentrations higher than 31.2 µg/mL, was able to impair E. coli growth and glucose uptake. AZA is a good inhibitor of the two recombinant E. coli CAs, the ß-CA CynT2, and the γ-CA EcoCAγ, with KIs of 227 and 248 nM, respectively. This study provides a proof-of-concept, low-cost method for identifying effective CA inhibitors capable of impairing bacterial metabolism.

Biological activity and molecular docking studies of some N-phenylsulfonamides against cholinesterases and carbonic anhydrase isoenzymes.

In this research, a series of N-phenylsulfonamide derivatives (1-12) were designed, synthesized, and investigated for their inhibitory potencies against carbonic anhydrase isoenzymes I, II, and IX (hCA I, hCA II, and hCA IX) and cholinesterases (ChE), namely, acetylcholinesterase and butyrylcholinesterase. These compounds, whose inhibition potentials were evaluated for the first time, were characterized by spectroscopic techniques (1 H- and 13 C-NMR and FT-IR). CA isoenzyme inhibitors are significant therapeutic targets, especially owing to their preventive/activation potential in the therapy processes of some diseases such as cancer, osteoporosis, and glaucoma. On the other hand, Cholinesterase inhibitors are valuable molecules with biological importance that can be employed in the therapy process of Alzheimer's patients. The results showed that the tested molecules had enzyme inhibition activities ranging from 9.7 to 93.7 nM against these five metabolic enzymes. Among the tested molecules, the methoxy and the hydroxyl group-containing compounds 10, 11, and 12 exhibited more enzyme inhibition activities when compared to standard compounds acetazolamide, sulfapyridine, and sulfadiazine for CA isoenzymes and neostigmine for ChE, respectively. Of these three molecules, compound 12, which had a hydroxyl group in the para position in the aromatic ring, was determined to be the most active molecule against all enzymes. In silico work, molecular docking has also shown similar results and is consistent with the experimental data in the study. As a result, we can say that some of the tested molecules might be used as promising inhibitor candidates for further studies on this topic.

Roles of Carbonic Anhydrases and Carbonic Anhydrase Related Proteins in Zebrafish.

During recent decades, zebrafish (Danio rerio) have become one of the most important model organisms in which to study different physiological and biological phenomena. The research field of carbonic anhydrases (CAs) and carbonic anhydrase related proteins (CARPs) is not an exception to this. The best-known function of CAs is the regulation of acid-base balance. However, studies performed with zebrafish, among others, have revealed important roles for these proteins in many other physiological processes, some of which had not yet been predicted in the light of previous studies and suggestions. Examples include roles in zebrafish pigmentation as well as motor coordination. Disruption of the function of these proteins may generate lethal outcomes. In this review, we summarize the current knowledge of CA-related studies performed in zebrafish from 1993-2021 that was obtained from PubMed search.

Inhibition of Schistosoma mansoni carbonic anhydrase by the antiparasitic drug clorsulon: X-ray crystallographic and in vitro studies.

Clorsulon is an anthelmintic drug that is clinically used against Fasciola hepatica. Due to the presence of two sulfonamide moieties in its core nucleus, which are well recognized as zinc-binding groups, it was proposed that it may be efficacious in the inhibition of parasite carbonic anhydrases (CAs). Proteomic analyses revealed the presence of CA in the tegument of Schistosoma mansoni, and recently the druggability of this target was explored by testing the inhibitory activities of several sulfonamide-based derivatives. According to the principles of drug repurposing, the aim was to demonstrate a putative new mechanism of action of clorsulon and thus widen its antiparasitic spectrum. For this purpose, the inhibitory activity and isoform selectivity of clorsulon was studied using human CA I and S. mansoni CA, revealing different modes of binding of clorsulon that explain its inhibitory potency against the two enzymes. The information obtained in this study could be crucial in the design of more active and selective derivatives.

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.

Synthesis, carbonic anhydrase enzyme inhibition evaluations, and anticancer studies of sulfonamide based thiadiazole derivatives.

The sulfonamide-based thiadiazole derivatives (STDs) with different hydrophobic/hydrophilic substitutions were synthesized to investigate their potentials in carbonic anhydrase inhibition (CAI). The CAI activity of the STDs (4a-4h) and the mechanism of the inhibition kinetics were determined. STD 4f contained both methoxy and Cl groups at benzene ring in STD 4f showed the lowest IC50 value. The molecular docking study confirmed that STDs bind strongly with the active sites of the target protein PDBID 1V9E. With the help of Lineweaver-Burk plots, inhibition kinetics of PDBIR 1V9E protein with STDs were determined. Cytotoxicity was checked against human keratinocyte cell lines and the anticancer properties were determined against MCF-7 cell lines. The electrochemical method was used to investigate the binding study with DNA and CA enzymes. Anticancer studies showed that STDs have weak bonding ability to DNA and strong binding ability with CA. It is concluded that anticancer activity is through CAI rather than by DNA binding.

New Carbonic Anhydrase-II Inhibitors from Marine Macro Brown Alga Dictyopteris hoytii Supported by In Silico Studies.

In continuation of phytochemical investigations of the methanolic extract of Dictyopteris hoytii, we have obtained twelve compounds (1-12) through column chromatography. Herein, three compounds, namely, dimethyl 2-bromoterepthalate (3), dimethyl 2,6-dibromoterepthalate (4), and (E)-3-(4-(dimethoxymethyl)phenyl) acrylic acid (5) are isolated for the first time as a natural product, while the rest of the compounds (1, 2, 6-12) are known and isolated for the first time from this source. The structures of the isolated compounds were elucidated by advanced spectroscopic 1D and 2D NMR techniques including 1H, 13C, DEPT, HSQC, HMBC, COSY, NEOSY, and HR-MS and comparison with the reported literature. Furthermore, eight compounds (13-20) previously isolated by our group from the same source along with the currently isolated compounds (1-12) were screened against the CA-II enzyme. All compounds, except 6, 8, 14, and 17, were evaluated for in vitro bovine carbonic anhydrase-II (CA-II) inhibitory activity. Eventually, eleven compounds (1, 4, 5, 7, 9, 10, 12, 13, 15, 18, and 19) exhibited significant inhibitory activity against CA-II with IC50 values ranging from 13.4 to 71.6 µM. Additionally, the active molecules were subjected to molecular docking studies to predict the binding behavior of those compounds. It was observed that the compounds exhibit the inhibitory potential by specifically interacting with the ZN ion present in the active site of CA-II. In addition to ZN ion, two residues (His94 and Thr199) play an important role in binding with the compounds that possess a carboxylate group in their structure.

Carbonic Anhydrase IV Selective Inhibitors Counteract the Development of Colitis-Associated Visceral Pain in Rats.

Persistent pain affecting patients with inflammatory bowel diseases (IBDs) is still very difficult to treat. Carbonic anhydrase (CA) represents an intriguing pharmacological target considering the anti-hyperalgesic efficacy displayed by CA inhibitors in both inflammatory and neuropathic pain models. The aim of this work was to evaluate the effect of inhibiting CA IV, particularly when expressed in the gut, on visceral pain associated with colitis induced by 2,4-di-nitrobenzene sulfonic acid (DNBS) in rats. Visceral sensitivity was assessed by measuring animals' abdominal responses to colorectal distension. Repeated treatment with the selective CA IV inhibitors AB-118 and NIK-67 effectively counteracted the development of visceral pain induced by DNBS. In addition to pain relief, AB-118 showed a protective effect against colon damage. By contrast, the anti-hyperalgesic activity of NIK-67 was independent of colon healing, suggesting a direct protective effect of NIK-67 on visceral sensitivity. The enzymatic activity and the expression of CA IV resulted significantly increased after DNBS injection. NIK-67 normalised CA IV activity in DNBS animals, while AB-118 was partially effective. None of these compounds influenced CA IV expression through the colon. Although further investigations are needed to study the underlying mechanisms, CA IV inhibitors are promising candidates in the search for therapies to relieve visceral pain in IBDs.

Determination of intracellular protein-ligand binding affinity by competition binding in-cell NMR.

Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.

Privileged scaffolds in medicinal chemistry: Studies on pyrazolo[1,5-a]pyrimidines on sulfonamide containing Carbonic Anhydrase inhibitors.

We report for the first time a small series of compounds endowed in vitro with inhibitory properties for the human (h) expressed Carbonic Anhydrase (CAs, E.C. 4.2.1.1) enzymes of physiological interest (i.e. I, II, VA, IX and XII) and bearing the pyrazolo[1,5-a]pyrimidine (PP) scaffold at the tail section. Among the series reported, 1a-3a, 7a, 8a, 1b and 2b resulted effective ligands and with good selectivities for the hCAs II, IX or XII. In consideration of the nearly matching KI values of 7a for both the hCA II and IX (i.e. 26.4 and 23.0 nM respectively) we explored its binding mode within the CA IX mimic isoform by means of X-ray crystal experiments on the corresponding adduct.

Antibacterial properties and carbonic anhydrase inhibition profiles of azido sulfonyl carbamate derivatives.

Aim: The aim of this study was to identify inhibition of carbonic anhydrase I and II (CA I and II) isozymes by azido sulfonyl carbamates through both in vitro and in silico approaches and also to determine the drug-likeness properties and antibacterial activities of azido sulfonyl carbamates. Methods & Results:In vitro inhibition and molecular docking studies of azido sulfonyl carbamate derivatives (1-4) on isozymes were performed. Except for derivative 4, all derivatives inhibited human CA I and II. Almost all compounds had antibacterial effects. The docking results showed that compound 3 had the best results, with binding energy of -8.20 kcal/mol for human CA I and -8.24 kcal/mol for human CA II. Conclusion: Molecule 4 inhibited only CA I. Its usage as a potential chemotherapeutic agent specific to the CA I isozyme may be considered.

Design, synthesis, characterization, in vitro and in silico evaluation of novel imidazo[2,1-b][1,3,4]thiadiazoles as highly potent acetylcholinesterase and non-classical carbonic anhydrase inhibitors.

Imidazole and thiadiazole derivatives display an extensive application in pharmaceutical chemistry, and they have been investigated as bioactive molecules for medicinal chemistry purposes. Classical carbonic anhydrase (CA) inhibitors are based on sulfonamide groups, but inhibiting all CA isoforms nonspecifically, thereby causing undesired side effects, is the main drawback of these types of inhibitors. Here we reported an investigation of novel 2,6-disubstituted imidazo[2,1-b][1,3,4]thiadiazole derivatives (9a-k, 10a, and 11a) and 2,5,6-trisubstituted imidazo[2,1-b][1,3,4]thiadiazole derivatives (12a-20a) that do not possess the zinc-binding sulfonamide group for the inhibition of human carbonic anhydrase (hCA, EC 4.2.1.1) I and II isoforms and also of acetylcholinesterase (AChE, EC 3.1.1.7). Imidazo[2,1-b][1,3,4]thiadiazoles demonstrated low nanomolar inhibitory activity against hCA I, hCA II, and AChE (KIs are in the range of 23.44-105.50 nM, 10.32-104.70 nM, and 20.52-54.06 nM, respectively). Besides, compound 9b inhibit hCA I up to 18-fold compared to acetazolamide, while compound 10a has a 5-fold selectivity towards hCA II. The synthesized compounds were also evaluated for their cytotoxic effects on the L929 mouse fibroblast cell line. Molecular docking simulations were performed to elucidate these inhibitors' potential binding modes against hCA I and II isoforms and AChE. The novel compounds reported here can represent interesting lead compounds, and the results presented here might provide further structural guidance to discover and design more potent hCA and AChE inhibitors.

Modulating the Efficacy of Carbonic Anhydrase Inhibitors through Fluorine Substitution.

The insertion of fluorine atoms and/or fluoroalkyl groups can lead to many beneficial effects in biologically active molecules, such as enhanced metabolic stability, bioavailability, lipophilicity, and membrane permeability, as well as a strengthening of protein-ligand binding interactions. However, this "magic effect" of fluorine atom(s) insertion can often be meaningless. Taking advantage of the wide range of data coming from the quest for carbonic anhydrase (CA) fluorinated inhibitors, this Minireview attempts to give "general guidelines" on how to wisely insert fluorine atom(s) within an inhibitor moiety to precisely enhance or disrupt ligand-protein interactions, depending on the target location of the fluorine substitution in the ligand. Multiple approaches such as ITC, kinetic and inhibition studies, X-ray crystallography, and NMR spectroscopy are useful in dissecting single binding contributions to the overall observed effect. The exploitation of innovative directions made in the field of protein and ligand-based fluorine NMR screening is also discussed to avoid misconduct and finely tune the exploitation of selective fluorine atom insertion in the future.

Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII.

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.