Residue profiles of peptides with cholesterol esterase and pancreatic lipase inhibitory activities through virtual screening and sequence analysis.

The detailed characterization of the structural features of peptides targeting cholesterol esterase (CEase) or pancreatic lipase (PPL) will benefit the management of hyperlipidemia and obesity. This study employed the Glide SP (standard precision)-peptide method to predict the binding modes of 202 dipeptides and 203 tripeptides to these targets, correlating residue composition and position with binding energy. Strong preferences for Trp, Phe, and Tyr were observed at all positions of potential inhibitory peptides, whereas negatively charged residues Glu and Asp were disfavored. Notably, Arg and aromatic rings significantly influenced the peptide conformation at the active site. Tripeptide IWR demonstrated the high efficacy, with IC50 values of 0.214 mg/mL for CEase and 0.230 mg/mL for PPL. Five novel IWR scaffold-tetrapeptides exhibited promising inhibitory activity. Non-covalent interactions and energy contributions dominated the formation of stable complexes. Our results provide insights for the development of new sequences or peptide-like molecules with enhanced inhibitory activity.

Computational analysis of 3D printing: Selecting the better among newly released materials.

Resin-based three-dimensional (3D) printing finds extensive application in the field of dentistry. Although studies of cytotoxicity, mechanical and physical properties have been conducted for newly released 3D printing resins such as Crowntec (Saremco), Temporary Crown Resin (Formlabs) and Crown & Bridge (Nextdent), the resistance of these materials to esterases in saliva has not been demonstrated at the molecular level. Therefore, in this study, the binding affinities and stability of these new 3D printing resins to the catalytic sites of esterases were investigated using molecular docking and molecular mechanics with Poisson-Bolzmann and surface area solvation (MM/PBSA) methods after active pocket screening. Toxicity predictions of the materials were also performed using ProTox-II and Toxtree servers. The materials were analyzed for mutagenicity, cytotoxicity, and carcinogenicity, and LD50 values were predicted from their molecular structures. The results indicated that out of the three novel 3D printing materials, Nexdent exhibited reduced binding affinity to esterases, indicating enhanced resistance to enzymatic degradation and possessing a superior toxicity profile.

Exploring the inhibitory potential of KPHs-AL-derived GLLF peptide on pancreatic lipase and cholesterol esterase activities.

The effective modulation of pancreatic lipase and cholesterol esterase activities proves critical in maintaining circulatory triglycerides and cholesterol levels within physiological boundaries. In this study, peptides derived from KPHs-AL, produced through the enzymatic hydrolysis of skipjack tuna dark muscle using alkaline protease, have a specific inhibitory effect on pancreatic lipase and cholesterol esterase. It is hypothesized that these peptides target and modulate the activities of enzymes by inducing conformational changes within their binding pockets, potentially impacting the catalytic functions of both pancreatic lipase and cholesterol esterase. Results revealed these peptides including AINDPFIDL, FLGM, GLLF and WGPL, were found to nestle into the binding site groove of pancreatic lipase and cholesterol esterase. Among these, GLLF stood out, demonstrating potent inhibition with IC50 values of 0.1891 mg/mL and 0.2534 mg/mL for pancreatic lipase and cholesterol esterase, respectively. The kinetics studies suggested that GLLF competed effectively with substrates for the enzyme active sites. Spectroscopic analyses, including ultraviolet-visible, fluorescence quenching, and circular dichroism, indicated that GLLF binding induced conformational changes within the enzymes, likely through hydrogen bond formation and hydrophobic interactions, thereby increasing structural flexibility. Molecular docking and molecular dynamics simulations supported these findings, showing GLLF's stable interaction with vital active site residues. These findings position GLLF as a potent inhibitor of key digestive enzymes, offering insights into its role in regulating lipid metabolism and highlighting its potential as functional ingredient.

Identification and molecular binding mechanism of novel pancreatic lipase and cholesterol esterase inhibitory peptides from heat-treated adzuki bean protein hydrolysates.

Heat-treated adzuki bean protein hydrolysates exhibit lipid-reducing properties; however, few studies have reported pancreatic lipase (PL) and cholesterol esterase (CE) inhibitory effects and elucidated the underlying mechanisms. In this study, we accomplished the identification of antiobesity peptides through peptide sequencing, virtual screening, and in vitro experiments. Furthermore, the mechanisms were investigated via molecular docking. The findings reveal that the action of pepsin and pancreatin resulted in the transformation of intact adzuki bean protein into smaller peptide fragments. The < 3 kDa fraction exhibited a high proportion of hydrophobic amino acids and displayed superior inhibitory properties for both PL and CE. Five novel antiobesity peptides (LLGGLDSSLLPH, FDTGSSFYNKPAG, IWVGGSGMDM, YLQGFGKNIL, and IFNNDPNNHP) were identified as PL and CE inhibitors. Particularly, IFNNDPNNHP exhibited the most robust biological activity. These peptides exerted their inhibitory action on PL and CE by occupying catalytic or substrate-binding sites through hydrogen bonds, hydrophobic interactions, salt bridges, and π-π stacking.

Novel Plant-Protein (Quinoa) Derived Bioactive Peptides with Potential Anti-Hypercholesterolemic Activities: Identification, Characterization and Molecular Docking of Bioactive Peptides.

Hypercholesterolemia remains a serious global public health concern. Previously, synthetic anti-hypercholesterolemic drugs were used for ameliorating this condition; however, long-term usage presented several side-effects. In this regard, natural products as an adjunct therapy has emerged in recent times. This study aimed to produce novel bioactive peptides with anti-hypercholesterolemic activity (cholesterol esterase (CEase) and pancreatic lipase (PL)) from quinoa protein hydrolysates (QPHs) using three enzymatic hydrolysis methods (chymotrypsin, protease and bromelain) at 2-h hydrolysis intervals (2, 4, and 6 h). Chymotrypsin-generated hydrolysates showed higher CEase (IC50: 0.51 mg/mL at 2 h) and PL (IC50: 0.78 mg/mL at 6 h) inhibitory potential in comparison to other derived hydrolysates and intact quinoa proteins. Peptide profiling by LC-MS QTOF and in silico interaction with target enzymes showed that only four derived bioactive peptides from QPHs could bind in the active site of CEase, whereas twelve peptides could bind in the active site of PL. Peptides QHPHGLGALCAAPPST, HVQGHPALPGVPAHW, and ASNLDNPSPEGTVM were identified to be potential CEase inhibitors, and FSAGGLP, QHPHGLGALCAAPPST, KIVLDSDDPLFGGF, MFVPVPH, and HVQGHPALPGVPAHW were identified as potential PL inhibitors on the basis of the maximum number of reactive residues in these bioactive peptides. In conclusion, QPHs can be considered as an alternative therapy for the treatment of hypercholesterolemia.

Effect of Abutilon indicum (L) Extract on Adipogenesis, Lipolysis and Cholesterol Esterase in 3T3-L1 Adipocyte Cell Lines.

Abutilon indicum (L) is an Indian traditional plant used for the treatment of diabetes and heart diseases. The present study is to evaluate the functional of A. indicum leaf extract as insulin like character to inhibit lipolysis and stimulates Adipogenesis activity. The ability of the A. indicum leaf extract in anti-obesity effect of Adipogenesis, lipolysis and cholesterol esterase functions can be predicted by using 3T3-L1 adipocyte cell lines. Substances were isolated from A. indicum leaves and the double filtered crude sample were used for Adipogenesis, lipolysis and cholesterol esterase activity using 3T3-L1 adipocytes at different concentrations. We used differential media-I, differential media-II and maintenance media (MM1) at concentrations of 20, 40, 60, 80, 100, 200 and 400 µg/mL respectively. In addition to the extract, there is a significance increase in glycerol release (p < 0.001) compared with crude and reference compounds. Cholesterol esterase activity predicts the IC50 = 27.11 µg/mL of orlistat positive control compare with IC50 = 8.158 µg/mL of crude extract. Based on the observation, A. indicum leaf extract can promotes lipolysis and differentiated adipocytes. It is potentially used as adjuvant in the treatment of Type 2 diabetes.

A comparative analysis of anti-lipidemic potential of soybean (Glycine max) protein hydrolysates obtained from different ripening stages: Identification, and molecular interaction mechanisms of novel bioactive peptides.

This study aims to investigate the potentials of mature (MSPHs) and young (YSPHs) soybean enzymatic protein hydrolysates for the inhibition of pancreatic lipase (PL) and cholesterol esterase (C-Ease) enzymes. Higher proteins degradation levels were recorded with Bromelain compared to Flavourzyme and Alcalase, and upon hydrolysis, improved PL and C-Ease inhibition performances were displayed compared to unhydrolyzed proteins. Afterwards, six PHs with potent anti-lipidemic activities were selected for sequencing using LC-MS QTOF and molecular binding studies. Peptides FPFPRPPHQ, QCCAFEM, FAPEFLK from MSPHs and SFFFPFELPRE, FMYL, PFLL, FPLL, LPHF from YSPHs were predicted to possess potent inhibitory activities against PL. Furthermore, FPFPRPPHQ, FMYL, MMLM from MSPHs, and SFFFPFELPRE from YSPHs were predicted to be potent inhibitors of C-Ease. FPFPRPPHQ and SFFFPFELPRE derived from MSPHs and YSPHs, respectively, demonstrated effective inhibition potentialities against both PL and C-Ease. Therefore, mature and young soybean-derived protein hydrolysates could be recognized as a potential ingredient in the management of hypercholesterolemia.

Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis.

Burkholderia stabilis strain FERMP-21014 secretes cholesterol esterase (BsChe), which is used in clinical settings to determine serum cholesterol levels. Previously, we constructed an expression plasmid with an endogenous constitutive promoter to enable the production of recombinant BsChe. In this study, we obtained one mutant strain with 13.1-fold higher BsChe activity than the wild type, using N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen. DNA-sequencing analysis revealed that the strain had lost chromosome 3 (∆Chr3), suggesting that the genes hindering BsChe production may be encoded on Chr3. We also identified common mutations in the functionally unknown BSFP_068720/30 genes in the top 10 active strains generated during transposon mutagenesis. As BSFP_068720/30/40 comprised an operon on Chr3, we created the BSFP_068720/30/40 disruption mutant and confirmed that each disruption mutant containing the expression plasmid exhibited ~ 16.1-fold higher BsChe activity than the wild type. Quantitative PCR showed that each disruption mutant and ΔChr3 had a ~ 9.4-fold higher plasmid copy number than the wild type. Structural prediction models indicate that BSFP_068730/40 is structurally homologous to the structural maintenance of chromosomes (SMC) protein MukBE, which is responsible for chromosome segregation during cell division. Conversely, BSFP_068720/30/40 disruption did not lead to a Chr3 drop-out. These results imply that BSFP_068720/30/40 is not a SMC protein but is involved in destabilizing foreign plasmids to prevent the influx of genetic information from the environment. In conclusion, the disruption of BSFP_068720/30/40 improved plasmid stability and copy number, resulting in exceptionally high BsChe production. KEY POINTS: • Disruption of BSFP_068720/30/40 enabled mass production of Burkholderia Che/Lip. • BSFP_068730/40 is an SMC protein homolog not involved in chromosome retention. • BSFP_068720/30/40 is likely responsible for the exclusion of exogenous plasmids.

Bioaccessibility of Plant Sterols in Wholemeal Rye Bread Using the INFOGEST Protocol: Influence of Oral Phase and Enzymes of Lipid Metabolism.

Bioaccessibility of plant sterols (PS) in an enriched wholemeal rye bread was evaluated, for the first time, using the INFOGEST protocol without gastric lipase (GL) and cholesterol esterase (CE), with GL or GL + CE. Moreover, human chewing and an in vitro oral phase (simulated salivary fluid and α-amylase) were evaluated for this purpose. The addition of GL decreased the bioaccessibility of total PS (from 23.8 to 18.5%), whereas the use of GL + CE does not significantly affect PS bioaccessibility. The in vitro oral phase resulted in an ineffective homogenization of the fresh vs partially dried and milled bread, reducing the bioaccessibility of total (from 20.2 to 12.8%) and individual PS. The INFOGEST digestion including the use of GL and CE, as well as an oral phase with human chewing, is proposed for the assessment of PS bioaccessibility in a solid matrix such as wholemeal rye bread since it more closely approximates the in vivo situation.

Structural characterization and hypolipidemic activity of Gracilaria lemaneiformis polysaccharide and its degradation products.

This research aimed to analyze structural characterization and hypolipidemic activity in vitro of G. lemaneiformis polysaccharide (GLP) and its degradation products. The results presented that the content of galacturonic acid declined and glucuronic acid level enhanced, average particle size decreased from 99.9 µm to 25.7 µm, and color brightness of polysaccharide strengthened after degraded by H2O2-Vc. There was no significant change in thermal stability of polysaccharide before and after degradation. It was observed in AFM analysis, polysaccharide changed to smaller, delicacy and dispersion after degradation. As seen in FT-IR, H2O2-Vc degradation never change the structure of polysaccharide. Polysaccharide and its degradation products showed a significant inhibition effect on pancreatic lipase and cholesterol esterase in a dose-dependent manner, which presented the mixed type of competitive and non-competitive for pancreatic lipase, and non-competitive for cholesterol esterase, respectively. The fluorescence quenching type was static on pancreatic lipase and dynamic on cholesterol esterase.

Investigation of Antidiabetic Effect of Pistacia atlantica Leaves by Activity-Guided Fractionation and Phytochemical Content Analysis by LC-QTOF-MS.

In this study, the antidiabetic, antiobesity, antioxidant, and antihyperlipidemic effects potential of Pistacia atlantica Desf. leaves were evaluated by in vitro methods. The effects of the leaves of the plant on pancreatic lipase, pancreatic cholesterol esterase, and PTP1B enzymes were investigated for the first time and it was observed that leaf methanol extract (IC50: 123.67 ± 0.40 µg/ml) and n-hexane sub-extract (IC50: 61.03 ± 0.11 µg/ml) had much stronger effects on pancreatic cholesterol esterase enzyme than simvastatin (IC50: 142.30 ± 5.67 µg/ml). The methanolic extract of P. atlantica leaves exerted strong inhibitory effect on the enzymes (α-amylase and α-glucosidase) effective on carbohydrate digestion. It was thought that the methanol extract could provide significant benefits against oxidative stress in diabetes mellitus since it showed antioxidant activities (DPPH radical scavenging activity and reducing power) as strong as reference compounds (ascorbic acid and quercetin). Qualitative and quantitative analyzes of rutin (0.328 ± 0.000 g/100 g dry extract), methyl gallate (5.245 ± 0.014 g/100 g dry extract), quercetin-3-O-glucoside (0.231 ± 0.000 g/100 g dry extract), and gallic acid (0.528 ± 0.127 g/100 g dry extract) in methanol extract were performed by RP-HPLC. The phytochemical content of the active sub-fraction obtained from the leaf methanol extract by activity-guided fractionation and column chromatography studies was characterized by LC-QTOF-MS. The presence of trigalloylglucose, digalloylglucose, and methyl gallate in the G6 coded sub-fraction obtained by chromatographic techniques from the ethyl acetate sub-extract, which has the highest inhibitory effect on α-amylase and α-glucosidase enzymes, was determined by LC-QTOF-MS. In addition to the G5 coded subfraction, a strong α-glucosidase enzyme inhibitory activity was also observed in the G6 coded sub-fraction, and methyl gallate, methyl digallate, 2″-O-galloyl-quercetin-3-O-hexoside, and myricetin-3-O-hexoside were identified in this sub-fraction. This study displayed that the methanol extract of P. atlantica leaves could be a potential source for bioactive compounds with antidiabetic effects by showing inhibitory effects on enzymes involved in carbohydrate digestion.

Sterol bioaccessibility in a plant sterol-enriched beverage using the INFOGEST digestion method: Influence of gastric lipase, bile salts and cholesterol esterase.

This study evaluates the influence of increasing bile salts and the addition of key enzymes of the lipidic metabolism in the INFOGEST digestion method on sterol bioaccessibility from a plant sterol (PS)-enriched beverage. The assayed modifications were increasing concentration of bovine bile salts (10 vs. 17.5 mM), and addition of gastric lipase (GL) (60U/mL), cholesterol esterase (CE) (0.075 or 2U/mL) or both. Compared to the original method (10 mM bile salts without enzymes), the assayed conditions significantly reduced bioaccessibility of individual (from 11.3 to 19.7 to 5.1-16.6%) and total PS (13.7 to 6.9-8.0%), and cholesterol (52.8 to 20.9-26.1%), except only when CE is added not allowing cholesterol quantification. The bioaccessibility achieved when lipolytic enzymes were tested was similar for all sterols. For a more physiological approach to in vivo conditions, incorporation of bile salts (10 mM), GL (60U/mL) and CE (0.075U/mL) to the INFOGEST method is proposed, although it increases the cost compared to the established method.

Identification and synthesis of selective cholesterol esterase inhibitor using dynamic combinatorial chemistry.

In this study, the concept of dynamic combinatorial chemistry (DCC) was applied to explore novel cholesterol esterase (CEase) inhibitors. In the presence of enzyme, two substrates (A1H3 and A2H3) were amplified from the dynamic combinatorial library (DCL), which was generated through reversible acylhydrazone formation reaction. In the in vitro biological evaluation, compound A1H3 exhibited not only potent (IC50 in nanomolar range) but also selective inhibition (>120 folds of selectivity for CEase over AChE). Furthermore, the binding pattern and possible binding mechanism were investigated in the kinetic experiment and molecular docking study, respectively.

Identification and characterization of cholesterol esterase and lipase inhibitory peptides from amaranth protein hydrolysates.

Human diet is undergoing a shift towards plant-based diet as a sustainable source of protein compared to animal-derived protein. In this study, cholesterol esterase (CEase) and pancreatic lipase (PL) inhibitory activities of amaranth protein hydrolysates (APHs) were studied. Bromelain, chymotrypsin, and actinase E were used for generating APHs at 2, 4 & 6 h of hydrolysis. Higher PL inhibiting potential were observed in bromelain-derived APHs (IC50 = 0.38-0.66 mg/mL) in comparison to intact amaranth proteins (IC50 = 3.93 mg/mL). Bromelain-4 h hydrolysates (AB4) demonstrated significant inhibitory potential for both CEase (IC50 = 0.47 mg/mL) and PL (IC50 = 0.48 mg/mL) activity. Peptide identification in AB-4 hydrolysate revealed that among 17 bioactive peptides, three peptides (FPFPPTLGY, FGAPR, and FPFVPAPT) were predicted as potential PL inhibitors and only one peptide (FPFVPAPT) was predicted as CEase inhibitor based on the number of substrate binding sites on active site of the enzymes. This is the first study providing insights into amaranth protein derived bioactive peptide possessing CEase and LIP inhibitory potential.

Comparison of the Japan Society of Clinical Chemistry reference method and CDC method for HDL and LDL cholesterol measurements using fresh sera.

OBJECTIVES: In 2009, the Japan Society of Clinical Chemistry (JSCC) recommended a reference method for the measurement of serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels. This automated method uses cholesterol esterase-cholesterol dehydrogenase to measure cholesterol levels in fractions obtained after ultracentrifugation and dextran sulfate/magnesium chloride precipitation. In the present study, using fresh samples, we compared the LDL-C and HDL-C levels measured using this method with those measured using the traditional Centers for Disease Control and Prevention (CDC)-beta-quantification (BQ) method. DESIGN: and methods: Using both the JSCC and CDC-BQ methods, LDL-C/HDL-C levels were measured in 47 non-diseased and 126 diseased subjects, whose triglyceride levels were lower than 11.29 â€‹mmol/L (1000 â€‹mg/dL). RESULTS: For LDL-C, the equation of the line representing the correlation between the two methods was y â€‹= â€‹0.991x + 0.009 â€‹mmol/L; r â€‹= â€‹0.999; and Sy/x â€‹= â€‹0.025 â€‹mmol/L, where x is the mean LDL-C level measured using the CDC-BQ method. Similarly, for HDL-C, the equation of the line representing the correlation between the two methods was y â€‹= â€‹0.988x + 0.041 â€‹mmol/L, r â€‹= â€‹0.999, and Sy/x â€‹= â€‹0.019 â€‹mmol/L, where x is the mean HDL-C level measured using the CDC-BQ method. CONCLUSIONS: The JSCC method agreed with the CDC-BQ method in cases of both non-diseased and diseased subjects, including those with dyslipidemia.

New insights into the cholesterol esterase- and lipase-inhibiting potential of bioactive peptides from camel whey hydrolysates: Identification, characterization, and molecular interaction.

Novel antihypercholesterolemic bioactive peptides (BAP) from peptic camel whey protein hydrolysates (CWPH) were generated at different time, temperature, and enzyme concentration (%). Hydrolysates showed higher pancreatic lipase- (PL; except 3 CWPH) and cholesterol esterase (CE)-inhibiting potential, as depicted by lower half-maximal inhibitory concentration values (IC50 values) compared with nonhydrolyzed camel whey proteins (CWP). Peptide sequencing and in silico data depicted that most BAP from CWPH could bind active site of PL, whereas as only 3 peptides could bind the active site of CE. Based on higher number of reactive residues in the BAP and greater number of substrate binding sites, FCCLGPVPP was identified as a potential CE-inhibitory peptide, and PAGNFLPPVAAAPVM, MLPLMLPFTMGY, and LRFPL were identified as PL inhibitors. Molecular docking of selected peptides showed hydrophilic and hydrophobic interactions between peptides and target enzymes. Thus, peptides derived from CWPH warrant further investigation as potential candidates for adjunct therapy for hypercholesterolemia.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Addition of either gastric lipase or cholesterol esterase to improve both ß-cryptoxanthin ester hydrolysis and micellarization during in vitro digestion of fruit pulps.

Using the INFOGEST in vitro digestion protocol adapted to carotenoids, the impact of additional rabbit gastric lipase (RGL) on the hydrolysis extent of ß-cryptoxanthin esters was evaluated for the first time, and compared with the addition of porcine cholesterol esterase (CEL). Both the modifications increased the hydrolysis of (all-E)-ß-cryptoxanthin esters from mandarin and peach pulps, although the outcomes were different. Addition of RGL consistently increased the average hydrolysis extent from 55.2% to 59.5% in mandarin pulp and from 22.7% to 48.8% in peach pulp (p < 0.05). The addition of CEL produced lower hydrolysis extents, i.e., 58.5% in mandarin (not statistically significant) and 28.4% in peach (p < 0.05), compared to those obtained with RGL. The hydrolysis extent positively correlated with the carotenoid ester concentration in both matrices. Bioaccessibility values were higher in mandarin pulp (range 32-34%) compared to those in peach pulp (range 16-21%), and were associated with the hydrolysis extent of the carotenoid esters during digestion. Addition of RGL and CEL produced no significant (p < 0.05) effect on the overall carotenoid bioaccessibility values of mandarin, while positively affected those in peach. Altogether these results corroborate that the hydrolysis extent of xanthophyll esters limits bioaccessibility. Additionally, hydrophobicity of the carotenoid inversely correlates with micellarization, as free (all-E)-xanthophylls micellarized in a higher extent compared to (all-E)-ß-carotene and xanthophyll esters. The new information of our results is that the addition of rabbit gastric lipase substantially contributes to the hydrolysis of ß-cryptoxanthin esters from fruit pulps, and consequently, to increase carotenoid bioaccessibility, being even more effective than CEL.

Functionalized magnetic nanomaterials for electrochemical biosensing of cholesterol and cholesteryl palmitate.

Synthesis and functionalization of magnetite nanoparticles (Fe3O4) was achieved with the view to covalently bind both cholesterol oxidase and cholesterol esterase biorecognition agents for the development of free and total cholesterol biosensors. Prior to enzyme attachment, Fe3O4 was functionalized with 3-aminopropyltriethoxysilane (APTES) and polyamidoamine (PAMAM) dendrimer. Characterization of the material was performed by FT-IR and UV spectroscopy, SEM/EDX surface analysis and electrochemical investigations. The response to cholesterol and its palmitate ester was examined using cyclic voltammetry. Optimum analytical performance for the free cholesterol biosensor was obtained using APTES-functionalized magnetite with a sensitivity of 101.9 µA mM-1 cm-2, linear range 0.1-1 mM and LOD of 80 µM when operated at 37 °C. In the case of the total cholesterol biosensor, the best analytical performance was obtained using PAMAM dendrimer-modified magnetite with sensitivity of 73.88 µA mM-1 cm-2 and linear range 0.1-1.5 mM, with LOD of 90 µM. A stability study indicated that the free cholesterol biosensors retained average activity of 98% after 25 days while the total cholesterol biosensors retained 85% activity upon storage over the same period. Graphical abstract Schematic representation of cholesterol esterase and oxidase loaded magnetic nanoparticles (Fe3O4@APTES or Fe3O4@APTES-PAMAM) generating hydrogen peroxide from cholesterol palmitate.

ω-Quinazolinonylalkyl aryl ureas as reversible inhibitors of monoacylglycerol lipase.

The serine hydrolase monoacylglycerol lipase (MAGL) is involved in a plethora of pathological conditions, in particular pain and inflammation, various types of cancer, metabolic, neurological and cardiovascular disorders, and is therefore a promising target for drug development. Although a large number of irreversible-acting MAGL inhibitors have been discovered over the past years, there are only few compounds known so far which inhibit the enzyme in a reversible manner. Therefore, much effort is put into the development of novel chemical entities showing reversible inhibitory behavior, which is thought to cause less undesired side effects. To explore a wide range of chemical structures as MAGL binders, we have applied a virtual screening approach by docking small molecules into the crystal structure of human MAGL (hMAGL) and envisaged a library of 45 selected compounds which were then synthesized. Biochemical investigations included the determination of the inhibitory potency on hMAGL and two related hydrolases, i.e. human fatty acid amide hydrolase (hFAAH) and murine cholesterol esterase (mCEase). The most promising candidates from theses analyses, i.e. three ω-quinazolinonylalkyl aryl ureas bearing alkyl spacers of three to five methylene groups, exhibited IC50 values of 20-41 µM and reversible, detergent-insensitive behavior towards hMAGL. Among these compounds, the inhibitor 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)butyl)urea (96) was selected for further kinetic characterization, yielding a dissociation constant Ki = 15.4 µM and a mixed-type inhibition with a pronounced competitive component (α = 8.94). This mode of inhibition was further supported by a docking experiment, which suggested that the inhibitor occupies the substrate binding pocket of hMAGL.