The analysis of methylxanthine fractions obtained from Camellia sinensis cultivated in Turkey and effects on the in vitro inhibition of CYP2D6 enzyme.

Tea is a worldwide consumed herbal beverage and it was aimed in this study to reveal the major fractions of green and black tea in order to enlighten the in vitro inhibition potency on the well-known drug metabolizing enzyme CYP2D6 activity. Methylxanthine fractions were extracted from green and black tea and a yield of 0.265 g (1.06%) for 25 g of dried black tea and 0.302 g (1.2%) for 25 g of green tea was calculated. High-performance liquid chromatography analysis represented that the major components of the methylxanthine fractions were caffeine, theobromine, and theophylline. Methylxanthine content of black tea was 368.25 ± 4.6 µg/ml caffeine, 89.30 ± 2.3 µg/ml theobromine, and 3.40 ± 0.5 µg/ml theophylline, whereas that of green tea was 176.50 ± 3.7 µg/ml caffeine, 53.85 ± 1.4 µg/ml theobromine, and 2.06 ± 0.7 µg/ml theophylline. The results of concentration-dependent inhibition studies were 76% green tea, 75% black tea, and 55% caffeine at concentration of 10 mg/ml. The inhibition rates of green and black tea on CYP2D6 activity were 76% and 75%, respectively, where that of quinidine, the well-known inhibitor of CYP2D6, was 82%. Our results indicate that green and black tea is very likely to modify the CYP2D6 enzyme activity.

A gossypol derivative effectively protects against Zika and dengue virus infection without toxicity.

BACKGROUND: Zika virus (ZIKV) and dengue virus (DENV) cause microcephaly and dengue hemorrhagic fever, respectively, leading to severe problems. No effective antiviral agents are approved against infections of these flaviviruses, calling for the need to develop potent therapeutics. We previously identified gossypol as an effective inhibitor against ZIKV and DENV infections, but this compound is toxic and not suitable for in vivo treatment. RESULTS: In this study, we showed that gossypol derivative ST087010 exhibited potent and broad-spectrum in vitro inhibitory activity against infections of at least ten ZIKV strains isolated from different hosts, time periods, and countries, as well as DENV-1-4 serotypes, and significantly reduced cytotoxicity compared to gossypol. It presented broad-spectrum in vivo protective efficacy, protecting ZIKV-infected Ifnar1-/- mice from lethal challenge, with increased survival and reduced weight loss. Ifnar1-/- mice treated with this gossypol derivative decreased viral titers in various tissues, including the brain and testis, after infection with ZIKV at different human isolates. Moreover, ST087010 potently blocked ZIKV vertical transmission in pregnant Ifnar1-/- mice, preventing ZIKV-caused fetal death, and it was safe for pregnant mice and their pups. It also protected DENV-2-challenged Ifnar1-/- mice against viral replication by reducing the viral titers in the brain, kidney, heart, and sera. CONCLUSIONS: Overall, our data indicate the potential for further development of this gossypol derivative as an effective and safe broad-spectrum therapeutic agent to treat ZIKV and DENV diseases.

The production and biochemical characterization of α-carbonic anhydrase from Lactobacillus rhamnosus GG.

We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, ß, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only ß- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s-1 and kcat/KM of 1.41 × 107 s-1 M-1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms. KEY POINTS: • The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa. • LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1. • Acetazolamide resulted in a marginal inhibitory effect on cell growth.

In vitro inhibition of Mycobacterium tuberculosis ß-carbonic anhydrase 3 with Mono- and dithiocarbamates and evaluation of their toxicity using zebrafish developing embryos.

We investigated a panel of 14 compounds belonging to the monothiocarbamate (MTC) and dithiocarbamate (DTC) series against the ß-carbonic anhydrase 3 (ß-CA3) of Mycobacterium tuberculosis (Mtb). We also evaluated all compounds for toxicity using 1-5-day post fertilisation zebrafish embryos. 11 out of the 14 investigated derivatives showed effective nanomolar or submicromolar in vitro inhibition against the ß-CA3 (KIs 2.4-812.0 nM), and among them four DTCs of the series (8-10 and 12) showed very significant inhibition potencies with KIs between 2.4 and 43 nM. Out of 14 compounds screened for toxicity and safety 9 compounds showed no adverse phenotypic effects on the developing zebrafish larvae at five days of exposure. The results of in vitro inhibition and the toxicological evaluation of our study suggest that 5 compounds are suitable for further in vivo preclinical characterisation in zebrafish model.

First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase.

The worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase.

Mycobacterium tuberculosis ß-Carbonic Anhydrases: Novel Targets for Developing Antituberculosis Drugs.

The genome of Mycobacterium tuberculosis (Mtb) encodes three ß-carbonic anhydrases (CAs, EC 4.2.1.1) that are crucial for the life cycle of the bacterium. The Mtb ß-CAs have been cloned and characterized, and the catalytic activities of the enzymes have been studied. The crystal structures of two of the enzymes have been resolved. In vitro inhibition studies have been conducted using different classes of carbonic anhydrase inhibitors (CAIs). In vivo inhibition studies of pathogenic bacteria containing ß-CAs showed that ß-CA inhibitors effectively inhibited the growth of pathogenic bacteria. The in vitro and in vivo studies clearly demonstrated that ß-CAs of not only mycobacterial species, but also other pathogenic bacteria, can be targeted for developing novel antimycobacterial agents for treating tuberculosis and other microbial infections that are resistant to existing drugs. In this review, we present the molecular and structural data on three ß-CAs of Mtb that will give us better insights into the roles of these enzymes in pathogenic bacterial species. We also present data from both in vitro inhibition studies using different classes of chemical compounds and in vivo inhibition studies focusing on M. marinum, a model organism and close relative of Mtb.

Investigation of salicylanilide and 4-chlorophenol-based N-monosubstituted carbamates as potential inhibitors of acetyl- and butyrylcholinesterase.

Based on the presence of carbamate moiety, twenty salicylanilide N-monosubstituted carbamates concomitantly with their parent salicylanilides and five newly prepared 4-chlorophenyl carbamates obtained from isocyanates were investigated using Ellman's method for their in vitro inhibitory activity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum. The carbamates and salicylanilides exhibited mostly a moderate inhibition of both cholinesterase enzymes with IC50 values ranging from 5 to 235 µM. IC50 values for AChE were in a narrower concentration range when compared to BChE, but many of the compounds produced a balanced inhibition of both cholinesterases. The derivatives were comparable or superior to rivastigmine for AChE inhibition, but only a few of carbamates also for BChE. Several structure-activity relationships were identified, e.g., N-phenethylcarbamates produce clearly favourable BChE inhibition. The compounds also share convenient physicochemical properties for CNS penetration.

Carbonic Anhydrase Inhibitors as Novel Drugs against Mycobacterial ß-Carbonic Anhydrases: An Update on In Vitro and In Vivo Studies.

Mycobacteria cause a variety of diseases, such as tuberculosis, leprosy, and opportunistic diseases in immunocompromised people. The treatment of these diseases is problematic, necessitating the development of novel treatment strategies. Recently, ß-carbonic anhydrases (ß-CAs) have emerged as potential drug targets in mycobacteria. The genomes of mycobacteria encode for three ß-CAs that have been cloned and characterized from Mycobacterium tuberculosis (Mtb) and the crystal structures of two of the enzymes have been determined. Different classes of inhibitor molecules against Mtb ß-CAs have subsequently been designed and have been shown to inhibit these mycobacterial enzymes in vitro. The inhibition of these centrally important mycobacterial enzymes leads to reduced growth of mycobacteria, lower virulence, and impaired biofilm formation. Thus, the inhibition of ß-CAs could be a novel approach for developing drugs against the severe diseases caused by pathogenic mycobacteria. In the present article, we review the data related to in vitro and in vivo inhibition studies in the field.

In vitro inhibition effect of some coumarin compounds on purified human serum paraoxonase 1 (PON1).

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro effect of some hydroxy and dihydroxy ionic coumarin derivatives (1-20) on purified PON1 activity was investigated. Among these compounds, derivatives 11-20 are water soluble. In investigated compounds, compounds 6 and 13 were found the most active (IC50 = 35 and 34 µM) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

In vitro inhibition effects of some coumarin derivatives on human erythrocytes glucose-6-phosphate dehydrogenase activities.

Inhibitory effects of some synthesized dihydroxycoumarin compounds on purified G6PD were investigated. For this purpose, initially human erythrocyte G6PD was purified 7069-fold in a yield of 33.6% by using ammonium sulfate precipitation and affinity chromatography which includes 2',5'-ADP Sepharose 4B. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enzyme activity was determined spectrophotometrically according to Beutler method at 340 nm. 6,7-Dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) were used as dihydroxycoumarin compounds. This study has demonstrated that G6PD activity is very highly sensitive to study coumarin derivatives.

Evaluation of (S)- and (R)-misonidazole as GPX inhibitors: synthesis, characterization including circular dichroism and in vitro testing on bovine GPx-1.

Racemic misonidazole, a radiosensitizer formally used in radiation therapy of cancer and to date still applied, was once reported to exhibit strong inhibitory effects on mouse glutathione peroxidases (GPX). This appeared to qualify misonidazole as a lead structure for the development of novel GPX inhibitors to cause oxidative stress in chemotherapy-resistant tumors. A unique feature of misonidazole as an inhibitor of GPX is the absence of a thiol functionality. Therefore, it was expected to selectively target inhibition devoid of promiscuous interactions with cations and sulfhydryl groups. We synthesized the isomers of misonidazole and analyzed the ability of chiroptical high-performance liquid chromatography (HPLC) to identify the particular enantiomers. Due to the chiral pool synthesis, the assignment of the correct configuration could be verified. Finally, we evaluated both isomers for their inhibitory activities on bovine erythrocyte GPx-1, which is 87% homologous to the human enzyme. Despite the previously reported inhibition of racemic misonidazole on the less homologous mouse GPx-1, we did not find any significant inhibitory activity on the bovine enzyme for either isomer. Though misonidazole appears unlikely to be an inhibitor of human GPx-1 activity, we still spotlight misonidazole as a promising fragment-like lead structure in general.

An Improved Test Method for Assaying the Inhibition of Bioflavonoids on Xanthine Oxidase Activity in†vitro.

The difference on inhibitory effects of bioflavonoids inhibiting XOD activity assayed by varying test methods cause of us to be further in consideration. The reported test method creating a micro-environment surrounding XOD in the absence of ⋅O2 -, which is seemly different from the assay in vivo. So, the vitro test method for assaying XOD activity is necessary to be improved for selection of potential inhibitors in the presence of ⋅O2 -. The inhibitory results demonstrated that bioflavonoids of MY, DMY, QUE and LUT are capable to be on effective IC50 values, but others are not. As well, their resulting inhibitions determined by the improved test method are much less than that reported in the literature, indicating that their chemical affinities with XOD become weaker. Moreover, DMY assayed on the inhibitions of XOD in the improved test method performs to be a better inhibitor, as compared to the assay of the reported test methods. Abasing on the transformation of DMY into MY in the presence of ⋅O2 -, the good inhibition of DMY on XOD activity can be explained by the synergistic effect of MY.

Exploring the Therapeutic Potential of Coumarin-thiazolotriazole Pharmacophores for SARS-CoV-2 Spike Protein through In-vitro and In-silico Evaluation.

INTRODUCTION: The pandemic caused by SARS-CoV-2 significantly impacted human life around the globe. Numerous unexpected modifications of the SARS-CoV-2 genome have resulted in the emergence of new types and have caused great concern globally. METHOD: Inhibitory effects of bioactive phytochemicals derived from natural and synthetic sources are promising for pathogenic viruses. in vitro and in silico techniques were used in the current study to identify novel inhibitors of coumarin clubbed thiazolo[3,2-b][1,2,4]triazoles against the SARS-CoV-2 spike protein. RESULT: Interestingly, all the tested molecules demonstrated substantial inhibition of spike protein with 91.81-57.90% inhibition. The spike protein was remarkably inhibited by compounds 6k (91.83%), 6j (89.75%), 6m (87.69%),6i (86.60%), 6l (85.40%), 6h (84.70%), 6l (84.70%), 6g (83.40%), 6b (82.60%), 6f (81.90%), while compounds 6d 6a, 6c, and 6e exhibited significant activity against spike protein with 79.60%, 77.10%, 75.30%, and 57.90% inhibition, respectively. The binding mechanism of these novel inhibitors with spike protein was deduced in silico, which reflects that the active molecules firmly bind with the receptor binding domain (RBD) of spike protein, thereby inhibiting its function. CONCLUSION: The combined in vitro and in silico investigations unfold the therapeutic potential of coumarin-thiazolotriazole scaffolds in the treatment of SARS-CoV-2 infection.

In silico and in vitro study of Mycobacterium tuberculosis H37Rv uncharacterized protein (RipD): an insight on tuberculosis therapeutics.

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is responsible for the highest global health problem, with the deaths of millions of people. With prevalence of multiple drug resistance (MDR) strains and extended therapeutic times, it is important to discover small molecule inhibitors against novel hypothetical proteins of the pathogen. In this study, a virtual screening protocol was carried out against MtbH37Rv hypothetical protein RipD (Rv1566c) for the identification of potential small molecule inhibitors. The 3D model of the protein structure binding site was used for virtual screening (VS) of inhibitors from the Pathogen Box, followed by its validation through a molecular docking study. The stability of the protein-ligand complex was assessed using a 150 ns molecular dynamics simulation. MM-PBSA and MM-GBSA are the two approaches that were used to perform the trajectory analysis and determine the binding free energies, respectively. The ligand binding was observed to be stable across the entire time frame with an approximate binding free energy of -22.9916 kcal/mol. The drug-likeness of the inhibitors along with a potential anti-tuberculosis compound was validated by ADMET prediction software. Furthermore, a CFU inhibition assay was used to validate the best hit compound's in vitro inhibitory efficacy against a non-pathogenic Mycobacterium smegmatis MC2155 under low nutrient culture conditions. The study reported that the compound proposed in our study (Pathogen Box ID: MMV687700) will be useful for the identification of potential inhibitors against Mtb in future.

Diffusible Compounds Produced by Hanseniaspora osmophila and Gluconobacter cerinus Help to Control the Causal Agents of Gray Rot and Summer Bunch Rot of Table Grapes.

Gray and summer bunch rot are important diseases of table grapes due to the high economic and environmental cost of their control with synthetic fungicides. The ability to produce antifungal compounds against the causal agents Botrytis, Aspergillus, Penicillium, and Rhizopus of two microorganisms isolated from table grapes and identified as Hanseniaspora osmophila and Gluconobacter cerinus was evaluated. In dual cultures, both biocontrol agents (together and separately) inhibited in vitro mycelial growth of these pathogens. To identify the compounds responsible for the inhibitory effect, extractions were carried out with organic solvents from biocontrol agents separately. Through dual cultures with pathogens and pure extracts, only the hexane extract from H. osmophila showed an inhibitory effect against Botrytis cinerea. To further identify these compounds, the direct bioautography technique was used. This technique made it possible to determine the band displaying antifungal activity at Rf = 0.05-0.2. The compounds present in this band were identified by GC-MS and compared to the NIST library. The most abundant compounds, not previously reported, corresponded to alkanes, ketones, alcohols, and terpenoids. H. osmophila and G. cerinus have the potential to control the causal agents of gray and summer bunch rot of table grapes.

Novel preparation of substituted oxazolines condensed to d-ring of estrane skeleton and characterization of their antiproliferative properties.

A simple and efficient synthesis of novel estrone 16α,17α-oxazoline derivatives substituted at the D ring (compounds 6a-g) is described. The reduction of 16α-azido-3-methoxyestra-1,3,5-trien-17-one (1) in methanol in the presence of CeCl3 under the condition of the Luche reaction produced two epimeric azido alcohol (16α-azido-17α-hydroxy and 16α-azido-17ß-hydroxy) derivatives of estra-1,3,5(10)-triene-3-methyl ether (compounds 2 and 3) in a yield of 90% and 7.6%. The reaction of the sterically unhindered 16α-azido-17α-hydroxy-estra-1,3,5(10)-triene-3-methyl ether (2) with a range of benzaldehydes under the condition of the Schmidt rearrangement yielded d-ring substituted estrone 16α,17α-oxazoline derivatives 6a-g. The in vitro antiproliferative activities of compounds 1, 2, 3, 6a-g were also determined by means of MTT assays on a panel of human cancer cell lines HeLa, SiHa, C-33 A, A2780, MCF-7, MDA-MB-231 and T47D.

The use of an in vitro approach to assess marine invertebrate carboxylesterase responses to chemicals of environmental concern.

Carboxylesterases (CEs) are key enzymes which catalyse the hydrolysis reactions of multiple xenobiotics and endogenous ester moieties. Given their growing interest in the context of marine pollution and biomonitoring, this study focused on the in vitro sensitivity of marine invertebrate CEs to some pesticides, pharmaceuticals, personal care products and plastic additives to assess their potential interaction on this enzymatic system and its suitability as biomarkers. Three bivalves, one gastropod and two crustaceans were used and CEs were quantified following current protocols set for mammalian models. Four substrates were screened for CEs determination and to test their adequacy in the hepatic fraction measures of the selected invertebrates. Two commercial recombinant human isoforms (hCE1 and hCE2) were also included for methodological validation. Among the invertebrates, mussels were revealed as the most sensitive to xenobiotic exposures while gastropods were the least as well as with particular substrate-specific preferences. Among chemicals of environmental concern, the plastic additive tetrabromobisphenol A displayed the highest CE-inhibitory capacity in all species. Since plastic additives easily breakdown from the polymer and may accumulate and metabolise in marine biota, their interaction with the CE key metabolic/detoxification processes may have consequences in invertebrate's physiology, affect bioaccumulation and therefore trophic web transfer and, ultimately, human health as shellfish consumers.

Development of quinoline-based hybrid as inhibitor of methionine aminopeptidase 1 from Leishmania donovani.

Methionine aminopeptidase 1 (MetAP1) is a target for drug discovery against many adversaries and a potential antileishmanial target for its role in N-terminal methionine processing. As an effort towards new inhibitor discovery against methionine aminopeptidase 1 from Leishmania donovani (LdMetAP1), we have synthesized a series of quinoline-based hybrids, that is (Z)-5-((Z)-benzylidine)-2-(quinolin-3-ylimino)thiazolidin-4-ones (QYT-4a-i) whose in vitro screening led to the discovery of a novel inhibitor molecule (QYT-4h) against LdMetAP1. The compound QYT-4h showed nearly 20-fold less potency for human MetAP1 and had drug-like features. Time-course kinetic assays suggested QYT-4h acting through a competitive mode by binding to the metal-activated catalytic site. Notably, QYT-4h was most potent against the physiologically relevant Mn(II) and Fe(II) supplemented forms of LdMetAP1 and less potent against Co(II) supplemented form. Surface plasmon resonance and fluorescence spectroscopy demonstrated high affinity of QYT-4h for LdMetAP1. Through molecular modelling and docking studies, we found QYT-4h binding at the LdMetAP1 catalytic pocket occupying both the catalytic and substrate binding sites mostly with hydrogen bonding and hydrophobic interactions which provide structural basis for its promising potency. These results demonstrate the feasibility of employing small-molecule inhibitors for selective targeting of LdMetAP1 which may find use to effectively eliminate leishmaniasis.

COVID-19: Analysis of Drug Repositioning Practice.

An outbreak of a new coronavirus disease (COVID-19) in China in December 2019 became the epicenter for the spread of a global pandemic. The SARS-CoV-2 coronavirus causes a cascade of respiratory diseases similar to severe acute respiratory syndrome (SARS). Currently, there is no effective, specific, and safe treatment for COVID-19 to suppress the virus in the human body. The present study searched for pharmacological substances with antiviral activity for possible drug repositioning based on experimental and theoretical information in a series of publications on in vitro assays of agents against SARS-CoV-2. An analysis identified 46 well-known pharmaceutical substances that could be used for drug repositioning to create a therapy for COVID-19.

In vitro Inhibition of Clinical Isolates of Otitis Media Pathogens by the Probiotic Streptococcus salivarius BLIS K12.

Otitis media is a common childhood infection, frequently requiring antibiotics. With high rates of antibiotic prescribing and increasing antibiotic resistance, new strategies in otitis media prevention and treatment are needed. The aim of this study was to assess the in vitro inhibitory activity Streptococcus salivarius BLIS K12 against otitis media pathogens. Efficacy of the bacteriocin activity of S. salivarius BLIS K12 against the otitis media isolates was assessed using the deferred antagonism test. Overall, 48% of pathogenic isolates exhibited some growth inhibition by S. salivarius BLIS K12. S. salivarius BLIS K12 can inhibit the in vitro growth of the most common pathogens.