Commikuanoids A-C: New cycloartane triterpenoids with exploration of carbonic anhydrase-II inhibition from the resins of Commiphora kua by in vitro and in silico molecular docking.

Three new cycloartane triterpenoids, commikuanoids A-C (1-3), together with four known compounds 4-7, were isolated from the resin of Commiphora kua. Their structures were confirmed by advanced NMR techniques such as 1D (1H and 13C) and 2D (HMBC, HSQC, COSY, NOESY and NOE) and high-resolution mass spectrometry (HRMS). Five compounds (1-5) were screened for in vitro carbonic anhydrase II (CA II) inhibitory activity. All the tested compounds demonstrated significant activity against CA II with IC50 values ranging from 4.9-19.6 µM. Moreover, the binding pattern of each compound in the binding site of CA-II was predicted through in silico molecular docking approach. It was observed that compounds 2, 4, and 5 binds with the Zn ion present in the active site of CA II, while compounds 1 and 3 mediated hydrogen bonding with Thr199 of CA-II, and all the compounds showed good binding score (> - 5 kcal/mol).

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.

Intracellular Binding/Unbinding Kinetics of Approved Drugs to Carbonic Anhydrase II Observed by in-Cell NMR.

Candidate drugs rationally designed in vitro often fail due to low efficacy in vivo caused by low tissue availability or because of unwanted side effects. To overcome the limitations of in vitro rational drug design, the binding of candidate drugs to their target needs to be evaluated in the cellular context. Here, we applied in-cell NMR to investigate the binding of a set of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. Some compounds were originally developed toward other targets and were later found to inhibit CAs. We observed strikingly different dose- and time-dependent binding, wherein some drugs exhibited a more complex behavior than others. Specifically, some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, therefore preventing the quantitative formation of a stable protein-ligand complex. Such observations could be correlated to the known off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design.

Visualizing drug binding interactions using microcrystal electron diffraction.

Visualizing ligand binding interactions is important for structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially reduced lattice defects make small macromolecular crystals attractive targets for studying drug binding using microcrystal electron diffraction (MicroED). However, so far no drug binding interactions could unambiguously be resolved by electron diffraction alone. Here, we use MicroED to study the binding of a sulfonamide inhibitor to human carbonic anhydrase isoform II (HCA II). We show that MicroED data can efficiently be collected on a conventional transmission electron microscope from thin hydrated microcrystals soaked with the clinical drug acetazolamide (AZM). The data are of high enough quality to unequivocally fit and resolve the bound inhibitor. We anticipate MicroED can play an important role in facilitating in-house fragment screening for drug discovery, complementing existing methods in structural biology such as X-ray and neutron diffraction.

A Proof-of-Concept Fragment Screening of a Hit-Validated 96-Compounds Library against Human Carbonic Anhydrase II.

Fragment screening is a powerful tool to identify and characterize binding pockets in proteins. We herein present the results of a proof-of-concept screening campaign of a versatile 96-entry fragment library from our laboratory against the drug target and model protein human carbonic anhydrase II. The screening revealed a novel chemotype for carbonic anhydrase inhibition, as well as less common non-covalent interaction types and unexpected covalent linkages. Lastly, different runs of the PanDDA tool reveal a practical hint for its application.

Two flavonoid-based compounds from Murraya paniculata as novel human carbonic anhydrase isozyme II inhibitors detected by a resazurin yeast-based assay.

Human carbonic anhydrase isozyme II has been used as protein target for disorder treatment including glaucoma. Current clinically used sulfonamide-based CA inhibitors can induce side effects, and so alternatives are required. This study aimed to investigate a natural CA inhibitor from Murraya paniculata. The previously developed yeast-based assay was used to screen 14 compounds isolated from M. paniculata and identified by NMR analysis for anti-human CA isozyme II (hCAII) activity. Cytotoxicity of the compounds was also tested using the same yeast-based assay but in a different cultivation condition. Two flavonoid candidate compounds, 5, 6, 7, 8, 3', 4', 5'-heptamethoxyflavone (4) and 3 ,5, 7, 8, 3', 4', 5'-heptamethoxyflavone (9), showed potent inhibitory activity against hCAII with a minimal effective concentration of 10.8 and 21.5 µM, respectively, while they both exhibited no cytotoxic effect even at the highest concentration tested (170 µM). The results from an in vitro esterase assay of the two candidates confirmed their hCAII inhibitory activity with IC50 values of 24.0 and 34.3 µM, respectively. To investigate the potential inhibition mechanism of compound 4, in silico molecular docking was performed using the FlexX and Swissdock software. This revealed that compound 4 coordinated with the Zn2+ ion in the hCAII active site through its methoxy oxygen at a distance of 1.60 Å (FlexX) or 2.29 Å (Swissdock). The interaction energy of compound 4 with hCAII was -13.36 kcal/mol. Thus, compound 4 is a potent novel flavonoid-based hCAII inhibitor and may be useful for further anti-CAII design and development.

Further validation of strecker-type α-aminonitriles as a new class of potent human carbonic anhydrase II inhibitors: hit expansion within the public domain using differential scanning fluorimetry leads to chemotype refinement.

Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase (bCA) led to further validation of this new class of inhibitors and identification a new, refined chemotype represented by inhibitors with 10-improved potency. [Formula: see text].

Screening of benzenesulfonamide in combination with chemically diverse fragments against carbonic anhydrase by differential scanning fluorimetry.

The differential scanning fluorimetry (DSF) screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (bCA) delivered >100 hits that either caused, on their own, a significant thermal shift (ΔTm, °C) in the protein melting temperature or significantly influenced the thermal shift observed for BSA alone. Three hits based on 1,2,3-triazole moiety represent the periphery of the recently reported potent inhibitors of hCA II, IX and XII which were efficacious in vivo. Such a re-discovery of suitable BSA periphery essentially validates the new fragment-based approach to the discovery of future CAIs. Structures of other validated fragment hits are reported.

Syntesis of thio- and seleno-acetamides bearing benzenesulfonamide as potent inhibitors of human carbonic anhydrase II and XII.

A novel series of thio- and seleno-acetamides bearing benzenesulfonamide were synthetized and tested as human carbonic anhydrase inhibitors. These compounds were tested for the inhibition of four human (h) isoforms, hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX/XII). Several derivatives showed potent inhibition activity in low nanomolar range such as 3a, 4a, 7a and 8a. Furthermore, based on the tail approach we explain the interesting and selective inhibition profile of compound such as 5a and 9a, which were more selective for hCA I, 9b which was selective for hCA II, 3f selective for hCA IX and finally, 3e and 4b selective for hCA XII, over the other three isoforms. They are interesting leads for the development of more effective and isoform-selective inhibitors.

Discovery of new ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as carbonic anhydrase I, II, IX and XII inhibitors.

A series of twenty novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties substituted on one side with aromatic amines and on the other side with dimethylamine, morpholine and piperidine is reported. The compounds were synthesized from the 4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido)benzensulfonamide (1) by using stepwise nucleophilic substitution of the chlorine atoms of cyanuric chloride. The intermediates 2(a-e) and final compounds 3(a-o) were tested for their efficiency as carbonic anhydrase (CA) inhibitors against four selected physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, namely, the cytosolic ones hCA I and II, and the transmembrane, tumor associated ones hCA IX, and XII. The compounds 2a, 2e and 3m showed the highest activity for hCA IX with Kis in the range of 11.8-14.6 nM. Most of the compounds showed high hCA IX selectivity over the abundant off-target isoforms hCA I and II. Since hCA IX is a validated drug target for anticancer/antimetastatic agents, these isoform-selective and potent inhibitors may be considered of interest for further medicinal/pharmacologic studies.

Synthesis, characterization, crystal structure of novel bis-thiomethylcyclohexanone derivatives and their inhibitory properties against some metabolic enzymes.

In this study, a series of novel bis-thiomethylcyclohexanone compounds (3a-3j) were synthesized by the addition of thio-Michael to the bis-chalcones under mild reaction conditions. The bis-thiomethylcyclohexanone derivatives (bis-sulfides) were characterized by 1H NMR, 13C NMR, FTIR and elemental analysis techniques. Furthermore, the molecular and crystal structures of 3h, 3i and 3j compounds were determined by single crystal X-ray diffraction studies. In this study, X-ray crystallography provided an alternative and often-complementary means for elucidating functional groups at the enzyme inhibitory site. Acetylcholinesterase (AChE) is a member of the hydrolase protein super family and has a significant role in acetylcholine-mediated neurotransmission. Here, we report the synthesis and determining of novel bis-thiomethylcyclohexanone compounds based hybrid scaffold of AChE inhibitors. The newly synthesized bis-thiomethylcyclohexanone compounds showed Ki values of in range of 39.14-183.23 nM against human carbonic anhydrase I isoenzyme (hCA I), 46.03-194.02 nM against human carbonic anhydrase II isoenzyme (hCA II), 4.55-32.64 nM against AChE and 12.77-37.38 nM against butyrylcholinesterase (BChE). As a result, novel bis-thiomethylcyclohexanone compounds can have promising anti Alzheimer drug potential and record novel hCA I, and hCA II enzymes inhibitor.

Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues.

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small-molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1 H NMR techniques. 2-Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small-molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand-binding site and could be abrogated by quenching with a molar excess of hydroxylamine.

Discovery of ß-Adrenergic Receptors Blocker-Carbonic Anhydrase Inhibitor Hybrids for Multitargeted Antiglaucoma Therapy.

The combination of a ß-adrenergic receptors (AR) blocker and a carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in eye drops formulations is one of the most clinically used treatment for glaucoma. A novel approach consisting of single-molecule, multitargeted compounds for the treatment of glaucoma is proposed here by designing compounds which concomitantly interact with the ß-adrenergic and CA targets. Most derivatives of the two series of benzenesulfonamides incorporating 2-hydroxypropylamine moieties reported here exhibited striking efficacy against the target hCA II and XII, whereas a subset of compounds also showed significant modulation of ß1- and ß2-ARs. X-ray crystallography studies provided rationale for the observed hCA inhibition. The best dual-agents decreased IOP more effectively than clinically used dorzolamide, timolol, and the combination of them in an animal model of glaucoma. The reported evidence supports the proof-of-concept of ß-ARs blocker-CAI hybrids for antiglaucoma therapy with an innovative mechanism of action.

Development of drug discovery screening system by molecular interaction kinetics-mass spectrometry.

RATIONALE: Drug discovery studies invariably require qualitative and quantitative analyses of target compounds at every stage of drug discovery. We have developed a system combining molecular interaction analysis and mass spectrometry (LC-MS) using the principle of nanopore optical interferometry (nPOI) called molecular interaction kinetics-mass spectrometry (MIK-MS). Since nPOI has high binding capacity, the bond-dissociated compound can be directly detected using LC-MS. In this study, we use carbonic anhydrase II (CAII) as a ligand and apply six small compounds as analytes and report the affinity analysis using MIK-MS. METHODS: CAII was immobilized onto a COOH sensor chip using standard amine coupling. A reference surface was prepared by activating and subsequently blocking the surface under identical conditions. An amount of 50 µL of mix solution was injected over the reference channel and sample channel for CAII immobilization. The solutions eluting from the sensor chip were collected from the waste-line of the SKi Pro system every 30 s. Reconstructed elution samples were then injected into the LC-MS/MS system. RESULTS: A mixture containing furosemide, acetazolamide, 4-sulfamoylbenzoic acid, 5-(dimethylamino)-1-naphthalene sulfonamide (DNSA), sulfanilamide and sulpiride (15 µM each) was injected into the CAII-immobilized sensor chip, and the fractions eluted from the SKi Pro system were collected and subjected to selected reaction monitoring LC-MS characterization. Specific results were obtained for acetazolamide, DNSA, furosemide and sulpiride. The results suggest that the association-dissociation curve of a mixed sample can be obtained by one-time MIK-MS analysis. CONCLUSIONS: Six small-molecule binders of CAII were analyzed quantitatively using nPOI and MIK-MS, and the results were compared to published surface plasmon resonance (SPR) results. The nPOI and SPR results show good agreement, confirming the reliability of the analysis. Time-dependent binding results may be obtained by our MS sensorgram approach. Drugs that meet medical needs in a short period are required; this nPOI-LC-MS system is considered an important tool for rapid drug discovery.

Ellagitannin and flavonoid constituents from Agrimonia pilosa Ledeb. with their protein tyrosine phosphatase and acetylcholinesterase inhibitory activities.

A new ellagitannin, agritannin (1), a new flavone glycoside, agriflavone (2), and another flavone glycoside with spectroscopic data reported for the first time, kaempferol-3-O-[(S)-3-hydroxy-3-methylglutaryl (1→6)]-ß-d-glucoside (3), along with 16 known compounds were isolated from the aerial parts of Agrimonia pilosa Ledeb. These compounds were evaluated for PTP1B inhibitory activity. Among them, compounds 9 and 18 displayed potential inhibitory activity against PTP1B with IC50 values of 7.14±1.75 and 7.73±0.24µM, respectively. In addition, compound 1 showed significant inhibitory effect with an IC50 value of 17.03±0.09µM. Furthermore, these compounds were tested in AChE inhibitory assays. Most of them were found to have moderate inhibitory effects, with IC50 values ranging from 60.20±1.09 to 92.85±1.12µM. Except compounds 3, 8, and 18 were inactive.

3D QSAR studies, pharmacophore modeling, and virtual screening of diarylpyrazole-benzenesulfonamide derivatives as a template to obtain new inhibitors, using human carbonic anhydrase II as a model protein.

A 3D-QSAR modeling was performed on a series of diarylpyrazole-benzenesulfonamide derivatives acting as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The compounds were collected from two datasets with the same scaffold, and utilized as a template for a new pharmacophore model to screen the ZINC database of commercially available derivatives. The datasets were divided into training, test, and validation sets. As the first step, comparative molecular field analysis (CoMFA), CoMFA region focusing and comparative molecular similarity indices analysis (CoMSIA) in parallel with docking studies were applied to a set of 41 human (h) CA II inhibitors. The validity and the prediction capacity of the resulting models were evaluated by leave-one-out (LOO) cross-validation approach. The reliability of the model for the prediction of possibly new CA inhibitors was also tested.

Natural product inhibitors of carbonic anhydrase I and II isoenzymes: osajin and pomiferin.

The aim of this study is to purify carbonic anhydrase I and II isoenzymes from human erythrocyte, isolate two natural products osajin (OSJ) and pomiferin (PMF) from Maclura pomifera fruits, and evaluate the in vitro effect of these natural metabolites on these isoenzymes. These natural products may be used as starting points for drug discovery (like drugs used in several therapeutic applications, including antiglaucoma activity). For the purification procedure, the Sepharose-4B-l-tyrosine-sulphonamide affinity chromatography was used. Column chromatography and thin layer chromatography methods were used for isolation of OSJ and PMF from M. pomifera fruits and their chemical structures were elucidated by IR, 1D, and 2D NMR methods. We compared inhibitory effects of these natural products with inhibitory effects of phenolic compounds and found that these products demonstrated average inhibition effects. We thought that this study will give inspiration to scientists interested in this issue.

Bile acids and their oxo derivatives: Potential inhibitors of carbonic anhydrase I and II, androgen receptor antagonists and CYP3A4 substrates.

Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico-chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal-phase thin-layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure-activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α-hydroxy-12-oxo-5ß-cholanoic and 3α-hydroxy-7-oxo-5ß-cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α-hydroxy-12-oxo-5ß-cholanoic, 3α-hydroxy-7-oxo-5ß-cholanoic, 3,7,12-trioxo-5ß-cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry.

Investigation of the inhibitory properties of some phenolic standards and bee products against human carbonic anhydrase I and II.

Polyphenols are important secondary products of plants with the potential to inhibit carbonic anhydrases. The aim of this study was to investigate the inhibition effects of various phenolic standards, honey, propolis, and pollen species on human carbonic anhydrase I and II. The inhibition values (IC50) of the phenolics (gallic acid, protocatechuic acid, quercetin, catechin, tannic acid, and chrysin) ranged from 0.009 to 0.32 µg/mL, tannic acid emerging as the best inhibitor. The inhibition values of three different types of honey, heather, rhododendron, and chestnut ranged between 2.32 and 25.10 µg/mL, the chestnut honeys exhibiting the best inhibition. The ethanolic extracts of pollen and propolis exhibited good inhibitory properties, with IC50 values between 0.486 and 3.320 µg/mL. In order to evaluate the phenolic composition of bee products, phenolic profiles and total phenolic contents (TFC) were also measured. The inhibition ranking among the natural products studied was phenolic standards > propolis > pollen > honeys, and inhibition was related to TFC.

Microwave-assisted extraction, HPLC analysis, and inhibitory effects on carbonic anhydrase I, II, VA, and VII isoforms of 14 blueberry Italian cultivars.

The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.