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Campylobacter is a major cause of acute human diarrheal illness. Broiler chickens constitute a primary reservoir for C. jejuni leading to human infection. Consequently, there is a need for developing novel intervention methods. Antimicrobial peptides (AMP) are small proteins which have evolved in most lifeforms to provide defense against microbial infections. To date, over 3000 AMP have been discovered; however, few of them have been analyzed specifically for ability to kill campylobacters. We selected and evaluated a set of 11 unique chemically synthesized AMP for ability to inhibit growth of C. jejuni. Six of the AMP we tested produced zones of inhibition on lawns of C. jejuni. These AMP included: NRC-13, RL-37, Temporin L, Cecropin–Magainin, Dermaseptin, and C12K-2β12. In addition, MIC were determined for Cecropin–Magainin, RL-37 and C12K-2β12 against 15 isolates of Campylobacter representing the three most common pathogenic strains. MIC for campylobacters were approximately 3.1 µg/mL for AMP RL-37 and C12K-2β12. MIC were slightly higher for the Cecropin–Magainin AMP in the range of 12.5 to 100 µg/mL. These AMP are attractive subjects for future study and potential in vivo delivery to poultry to reduce Campylobacter spp. populations.

We investigated a panel of 14 compounds belonging to the monothiocarbamate (MTC) and dithiocarbamate (DTC) series against the β-carbonic anhydrase 3 (β-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 β-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.

9 pages, 4 figures, 2 tables, supplementary data https://doi.org/10.1016/j.etap.2020.103561, 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, This work was financed by Spanish Ministry of Economy, Industry and Competitivity (ref CGL2016-76332-R MINECO/FEDER, UE), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Background The frequent usage of broad-spectrum insecticides like cypermethrin in agriculture activities could lead to the development of resistance in insects like Amsacta albistriga. Objectives The present study was conducted to understand the toxicity of cypermethrin with and without a combination of three enzyme inhibitors (PBO, DEM, and TPP) on A. albistriga using a topical bioassay. Methodology The in vitro and in vivo studies were conducted to understand the effect of three enzyme inhibitors such as piperonyl butoxide (PBO) and diethyl maleate (DEM) and triphenyl phosphate along with six different concentration of cypermethrin on the activities of acetylcholinesterase (AChE), esterase (EST), glutathione S-transferase (GST), and mixed-function oxidase (MFO) of A. albistriga. Results Bioassay shows elevated LC50 for cypermethrin (63.32 ppm) whereas in a combination of PBO cypermethrin LC50 values were reduced into 12.039 ppm followed by TPP combination as 13.234 ppm. In vitro and in vivo inhibition analysis shows AChE inhibition by PBO P < 0.01, esterase inhibition by PBO, and TPP were less; GST inhibition by DEM was observed at P < 0.001. Native PAGE results revealed that the in vitro AChE isoenzyme inhibition could be possible by PBO synergism with cypermethrin in A. albistriga. Conclusions The overall results conclude that the PBO and TPP enzyme inhibitors could be fine synergist molecules when it mixed with cypermethrin insecticide to control and manage the insecticide-resistant Amsacta albistriga in the field.

Summary Soy protein isolates (SPI) films were evaluated as carriers of citral and limonene, and their physical and antifungal properties were evaluated. The presence of antimicrobials in SPI films resulted in changes on colour without affecting the transparency. Films with citral added were more extensible; however, a reduction in tensile strength and elastic modulus was observed in films with limonene added. Aroma compounds addition in films induced a slight increase in water vapour properties in relation to discontinuity of network proteins evidenced by SEM. Besides, FTIR spectra evidenced a partial alteration of SPI secondary structure. Citral was less retained than limonene. The increase in limonene release with high relative humidity was explained by increase in protein chain mobility. SPI films enriched with limonene exhibited strong antifungal activity against the postharvest decay pathogen Penicillium italicum under storage conditions.

4 pages, 2 figures, 1 table, Tetrabromobisphenol A (TBBPA), a brominated flame retardant used in synthetic polymers and electronics, is present in the aquatic environment and recent evidence suggests it can be potentially biomagnified in the marine ecosystem. However, the toxicity of TBBPA in the marine biota has not been investigated in detail. In this study we aimed to understand the role of carboxylesterases (CEs) in xenobiotic metabolism under the exposure of marine organisms to a chemical of environmental concern, TBBPA. Specifically, we tested for in vitro inhibition of CE activity in a range of marine organisms covering different ecological niches, from species from low (mussels and copepods), medium (sardines and anchovies) and high trophic levels (tuna). The results revealed that the highest inhibition of CE activity to 100 μM TBBPA was recorded in mussels (66.5% inhibition) and tunids (36.3-76.4%), whereas copepods and small pelagic fish showed comparatively lower effects (respectively, 30% and 36.5-55.6%). Our results suggest that CE-mediated detoxification and physiological processes could be compromised in TBBPA-exposed organisms and could ultimately affect humans as many of them are market species, The funding was from AIMCOST (CGL2016-76332-R MINECO/FEDER, UE) and PELWEB (CTM2017-88939-R) projects of the Spanish Ministry of Economy and Competitiveness. JN was funded by the Spanish National Program Ramón y Cajal (RYC-2015-17809), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

The inhibition activities and mechanisms of four anthocyanins including cyanidin-3-glucoside, cyanidin-3,5-glucoside, cyanidin-3-rutinoside, and peonidin-3-glucoside, against porcine pancreatic α-amylase were investigated through in vitro and in silico studies. The in vitro inhibition study demonstrated that the four anthocyanins competitively inhibited porcine pancreatic α-amylase, which was later verified by the in silico molecular docking study that showed all the anthocyanins bound exclusively to the active site of porcine pancreatic α-amylase. Cyanidin-3-glucoside was found to have the highest inhibition activity with the K i value of 0.014 mM, followed by cyanidin-3-rutinoside, cyanidin-3,5-glucoside, and peonidin-3-glucoside with the K i value of 0.019, 0.020, and 0.045 mM, respectively. Results obtained from the in silico study also showed that the four anthocyanins were surrounded by the side chains of the active site of porcine pancreatic α-amylase, among which the side chain of GLU233 was supposed to play a key role in imparting the inhibition activity of anthocyanins.

The genome of Mycobacterium tuberculosis (Mtb) encodes three β-carbonic anhydrases (CAs, EC 4.2.1.1) that are crucial for the life cycle of the bacterium. The Mtb β-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 β-CAs showed that β-CA inhibitors effectively inhibited the growth of pathogenic bacteria. The in vitro and in vivo studies clearly demonstrated that β-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 β-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.

The development of novel inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) represents a viable approach to alleviate Alzheimer's disease. Thirty-six halogenated 2-hydroxy-N-phenylbenzamides (salicylanilides) with various substitution patterns and their esters with phosphorus-based acids were synthesized in yields of 72% to 92% and characterized. They were evaluated for in vitro inhibition of AChE from electric eel and BuChE from equine serum using modified Ellman's spectrophotometric method. The benzamides exhibited a moderate inhibition of AChE with IC50 values in a narrow concentration range from 33.1 to 85.8 mu M. IC50 values for BuChE were higher (53.5-228.4 mu M). The majority of derivatives inhibit AChE more efficiently than BuChE and are comparable or superior to rivastigmine-an established cholinesterases inhibitor used in the treatment of Alzheimer's disease. Phosphorus-based esters especially improved the activity against BuChE with 5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite 5c superiority (IC50 = 2.4 mu M). This derivative was also the most selective inhibitor of BuChE. It caused a mixed inhibition of both cholinesterases and acted as a pseudo-irreversible inhibitor. Several structure-activity relationships were identified, e.g., favouring esters and benzamides obtained from 5-halogenosalicylic acids and polyhalogenated anilines. Both 2-hydroxy-N-phenylbenzamides and esters share convenient physicochemical properties for blood-brain-barrier penetration and thus central nervous system delivery. ých inhibitorů acetylcholinesterázy (AChE) a butyrylcholinesterázy (BuChE) představuje životaschopný přístup ke zmírnění Alzheimerovy choroby. Třicet šest halogenovaných 2-hydroxy-N-fenylbenzamidů (salicylanilidů) s různými substitučními vzory a jejich estery s kyselinami na bázi fosforu bylo syntetizováno ve výtěžcích 72% až 92% a charakterizováno. Byly hodnoceny in vitro inhibice AChE z elektrického úhoře a BuChE z koňského séra pomocí modifikované Ellmanovy spektrofotometrické metody. Benzamidy vykazovaly mírnou inhibici AChE s hodnotami IC50 v úzkém koncentračním rozmezí od 33,1 do 85,8 mu M. Hodnoty IC50 pro BuChE byly vyšší (53,5 až 228,4 uM). Většina derivátů inhibuje AChE účinněji než BuChE a je srovnatelná nebo lepší než rivastigmin-zavedený inhibitor cholinesteráz používaný při léčbě Alzheimerovy choroby. Estery na bázi fosforu zvláště zlepšily aktivitu proti BuChE s 5-chlor-2 - {[4- (trifluormethyl) fenyl] karbamoyl} fenyl diethylfosfitem 5c (IC50 = 2,4 um M). Tento derivát byl také nejelektivnějším inhibitorem BuChE. Způsobil smíšenou inhibici obou cholinesteráz a působil jako pseudo-ireverzibilní inhibitor. Bylo identifikováno několik vztahů mezi strukturou a aktivitou, např. Zvýhodněné estery a benzamidy získané z 5-halogenenosalicylových kyselin a polyhalogenovaných anilinů. Jak 2-hydroxy-N-fenylbenzamidy, tak estery sdílejí vhodné fyzikálně-chemické vlastnosti pro pronikání hematoencefalickou bariérou, a tím dodávku centrálního nervového systému.

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.

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, β-carbonic anhydrases (β-CAs) have emerged as potential drug targets in mycobacteria. The genomes of mycobacteria encode for three β-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 β-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 β-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.

Gençer, Nahit (Balikesir Author), 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 mu M) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives., Balikesir University Research project 2014/54

Statine-based inhibitors of Plasmepsin II (PLMII) coupled with Primaquine have been designed using the `double-drug' approach. The IC50 values for PLMII inhibition ranged from 0.59 to 400 nM and the best IC50 value for inhibition of Plasmodium falciparum growth in vitro was 0.4 μM, which represent a remarkable improvement compared to other statine-based PLMII inhibitors.

Çakır, Ümit (Balıkesir author), 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., Scientific Research Council of Balikesir University

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

WOS: 000311310400015, PubMed: 22971832, 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 inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76 U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K (i) of 0.0080 +/- 0.256 and 0.0003 +/- 0.018 mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K (i) of 0.0010 +/- 0.173 mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC50 of 0.012, 0.022 and 0.003 mM values, respectively. IC50 value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178 mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

Non-carboxylic acid containing bioisosteres of (5-arylidene-2,4-dioxothiazolidin-3-yl)acetic acids, which are active as aldose reductase (ALR2) inhibitors, were designed by replacing the carboxylic group with the trifluoromethyl ketone moiety. The in vitro evaluation of the ALR2 inhibitory effects of these trifluoromethyl substituted derivatives led to the identification of two inhibitors effective at low micromolar doses. It was further confirmed that a carboxylic chain on N-3 of the thiazolidinedione scaffold is a determining requisite to obtain the highest efficacy levels; however, it is not essential for the interaction with the target enzyme and it can be replaced by different polar groups, thus obtaining less ionised or unionised inhibitors.

Made available in DSpace on 2022-04-28T19:54:01Z (GMT). No. of bitstreams: 0 Previous issue date: 1994-01-01 In vitro inhibition of the of spores germination of Alternaria solani by iprodione, chlorothalonil, and anilazine at different dosages was studied. The highest concentration of active ingredient studied for each fungicide was equivalent to that recommended for the control of the early blight, under field conditions: 0.75; 1.80 and 1.44 g, respectively, of iprodione, chlorothalonil and anilazine per litre of water. A series of two-fold dilutions of each original concentration was studied in additional nine experiments. Each of the three fungicides showed total in vitro spore inhibition at the highest rate, at six hours of incubation. At nine hours, only analazine maintained its full inhibition activity. The inhibition activity of iprodione decreased suddenly after 1/2 dilution, so that at the 1/8 dilution a total loss of inhibitory activity was observed. Chlorothalonil showed a progressive and slighter decrease of its activity as the dilution rate increased. Analizine showed a high inhibitory activity at higher dilutions, without any loss up to 1/128 dilution. Even at 1/512 dilution, its activity was so high that only 20% of spore germination was observed at six or nine hours of incubation. Area de Olericultura e Plantas Aar. Departamento de Horticultura UNESP Campus, CEP 14870-000 Jaboticabal Area de Olericultura e Plantas Aar. Departamento de Horticultura UNESP Campus, CEP 14870-000 Jaboticabal