Isolation and properties of trypsin inhibitors from the seeds ofLupinus luteus andL. polyphyllus

From dormant seeds of European yellow lupin (Lupinus luteus) and Washington lupin (Lupinus polyphyllus) inhibitors have been isolated that effectively suppress the activity of trypsin and interact nonstochiometrically with chymotrypsin. From their physicochemical properties and aminoacid compositions, the inhibitors from the species of lupin investigated belong to different types.V. F. Kuprevich Institute of Experimental Botany, Belarusian Academy of Sciences, Minsk. A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 879–884, November–December, 1993.     

Untargeted Phytochemical Profile,Antioxidant Capacity and Enzyme Inhibitory Activity of Cultivated and Wild Lupin Seeds from Tunisia

Lupin seeds can represent a valuable source of phenolics and other antioxidant compounds. In this work, a comprehensive analysis of the phytochemical profile was performed on seeds from three Lupinus species, including one cultivar (Lupinus albus) and two wild accessions (Lupinus cossentinii and Lupinus luteus), collected from the northern region of Tunisia. Untargeted metabolomic profiling allowed to identify 249 compounds, with a great abundance of phenolics and alkaloids. In this regard, the species L. cossentinii showed the highest phenolic content, being 6.54 mg/g DW, followed by L. luteus (1.60 mg/g DW) and L. albus (1.14 mg/g DW). The in vitro antioxidant capacity measured by the ABTS assay on seed extracts ranged from 4.67 to 17.58 mg trolox equivalents (TE)/g, recording the highest values for L. albus and the lowest for L. luteus. The DPPH radical scavenging activity ranged from 0.39 to 3.50 mg TE/g. FRAP values varied between 4.11 and 5.75 mg TE/g. CUPRAC values for lupin seeds ranged from 7.20 to 8.95 mg TE/g, recording the highest for L. cossentinii. The results of phosphomolybdenum assay and metal chelation showed similarity between the three species of Lupinus. The acetylcholinesterase (AChE) inhibition activity was detected in each methanolic extract analyzed with similar results. Regarding the butyrylcholinesterase (BChE) enzyme, it was weakly inhibited by the Lupinus extracts; in particular, the highest activity values were recorded for L. albus (1.74 mg GALAE/g). Overall, our results showed that L. cossentinii was the most abundant source of polyphenols, consisting mainly in tyrosol equivalents (5.82 mg/g DW). Finally, significant correlations were outlined between the phenolic compounds and the in vitro biological activity measured, particularly when considering flavones, phenolic acids and lower-molecular-weight phenolics.     

The Effect of Fermentation with Kefir Grains on the Physicochemical and Antioxidant Properties of Beverages from Blue Lupin (Lupinus angustifolius L.) Seeds

Plant derived fermented beverages have recently gained consumers’ interest, particularly due to their intrinsic functional properties and presence of beneficial microorganisms. Three variants containing 5%, 10%, and 15% (w/w) of sweet blue lupin (Lupinus angustifolius L. cv. “Boregine”) seeds were inoculated with kefir grains and incubated at 25 °C for 24 h. After processing, beverages were stored in refrigerated conditions (6 °C) for 21 days. Changes in microbial population, pH, bioactive compounds (polyphenolics, flavonoids, ascorbic acid), reducing sugars, and free amino acids were estimated. Additionally, viscosity, firmness, color, and free radicals scavenging properties were determined. Results showed that lactic acid bacteria as well as yeast were capable of growing well in the lupin matrix without any supplementation. During the process of refrigeration, the viability of the microorganisms was over the recommended minimum level for kefir products. Hydrolysis of polysaccharides as well as increase of free amino acids was observed. As a result of fermentation, the beverages showed excellent DPPH, ABTS^+·, ^·OH, and O₂⁻ radicals scavenging activities with a potential when considering diseases associated with oxidative stress. This beverages could be used as a new, non-dairy vehicle for beneficial microflora consumption, especially by vegans and lactose-intolerant consumers.     

Thermal studies on protein isolates of white lupin seeds (<Emphasis Type="Italic">Lupinus albus</Emphasis>)

This study used TG, DSC, and SDS-PAGE techniques to study protein isolates (PIs) in the powder form obtained from lupin seeds flour Lupinus albus. Different methods of preparing PIs were tested, resulting in final products that were different only in relation to the yield and protein content. The results of the protein analysis by SDS-PAGE showed that the same protein fractions were present in the lupin seeds and in the obtained PIs. This result shows that the process of extraction was not damaging to the composition of the original protein. On the other hand, the results of the thermal analysis (DSC and TG–DTG curves) obtained for the different PIs, led to the detection of changes in the protein conformation through the ΔH values, which in general decreased with increasing values of pH and ionic strength in the experimental conditions of extraction.     

Uptake of weathered p,p′-DDE by plant species effective at accumulating soil elements

Ten plant species previously shown to accumulate inorganic elements effectively from natural solids were grown under field conditions in p,p′-dichlorodiphenyldichloroethane (p,p′-DDE) contaminated soil. The plant species, which included rye, mustard, canola, vetch, pigeonpea, clover, peanut, and 3 cultivars of white lupin, represented both monocots and dicots, as well as two major families within the dicots: the Brassicaceae and the Fabaceae. The plants varied widely in their ability to phytoextract and translocate weathered p,p′-DDE. The percentage of contaminant phytoextracted ranged from 0.06% (white lupin) to 0.22% (clover, vetch), and the translocation factors (TF; contaminant concentration ratio of stems to roots) ranged from 0.04 (clover, white lupin) to 0.37 (canola). An inverse relationship exists between the amount of contaminant in the roots as measured by the root BCF (bioconcentration factor; dry weight contaminant concentration ratio of root to soil) and the TF. Duplicate mounds of each species were periodically amended with nitrogen (N), phosphorus (P), nitrogen and phosphorus together (N/P); a minus phosphorus treatment involved the addition of AlSO₄ to the soil prior to planting. The effect of nutrient regime on plant biomass, p,p′-DDE uptake and translocation, and inorganic element content varied greatly among the 10 plant species. For some species (rye, vetch, pigeonpea, clover, white lupin), reductions or non-significant changes in p,p′-DDE uptake were observed under the nutrient treatments and were not correlated with plant biomass effects. For mustard, canola, and peanut, the percentage of p,p′-DDE phytoextracted in the various treatments was more than doubled and was directly correlated with a two-fold increase in total plant biomass. Although it is generally assumed that fertilizer amendments will enhance the phytoremediation of organic and inorganic pollutants, the data here suggest that such effects are highly species specific and in some cases may actually decrease remediation potential.     

Preliminary approaches for the development of a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method for the detection and label-free semi-quantitation of the main storage proteins of Lupinus albus in foods

Since recent literature has indicated that white lupin (Lupinus albus) may be a useful source of hypocholesterolemic proteins to be used in functional food formulation, our final goal is the development of a fast and automated high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the detection and the label-free semi-quantitation of the main lupin globulins in lupin foods and food ingredients. We present here some preliminary results in this direction. As a first step a total protein extract (TPE-WF) from lupin flakes was pre-fractionated by anion-exchange chromatography and each fraction was digested with trypsin and analyzed by HPLC/ESI-MS/MS. Subsequently, the tryptic digest of TPE-WF was directly analyzed by HPLC/ESI-MS/MS without any pre-fractionation. Eventually, in order to test the applicability of the method to real samples, a lupin beverage and two lupin protein isolates were analyzed. Both Mascot and Spectrum Mill MS Proteomics Workbench software were used to identify the protein composition in these samples and Spectrum Mill was used also to test the possibility of developing a label-free semi-quantitation method based on peptide hits. Encouraging results were obtained especially in the detection of the hypocholesterolemic component beta-conglutin.     

Analysis of oxidised and reduced phytochelatins in pea and lupin plants using HPLC/MSn

Plants exposed to heavy metals activate a detoxification system capable of chelating and transporting these harmful ions to vacuoles. Phytochelatins–low molecular weight oligopeptides containing thiols such as glutathione and cystein–have been reported to play a very important role in this respect. High performance liquid chromatography coupled to the electospray ion trap mass spectrometer (HPLC-ESI-IT-MS) was used for identification of phytochelatins induced by Cd²⁺ and Pb²⁺ in roots, stems and leaves of pea (Pisum sativum L.) and yellow lupin (Lupinus luteus L.). This approach enabled unambiguous identification of phytochelatins in plant tissues and detection of phytochelatins and homophytochelatins in reduced as well as in oxidised form. Significant differences were detected in phytochelatin relative amounts and profiles in different parts of plants treated with heavy metals. Roots of both plant species contained mainly reduced phytochelatins, reduced and oxidised forms of these peptides were observed in stems in similar amounts, whereas only the oxidised phytochelatins were present in leaves.     

Studies on Bis(5′-Adenosyl)-P1, P4-Tetraphosphate,Appppa, and Some Synthetic Isosteric and Isopolar Phosphonate Analogues Using the Specific Appppaases from Artemia and Lupin

Abstract

We have recently reported the synthesis and separation of three diastereoisomers of diadenosine 5′, 5″-P¹,P⁴-(P¹, P⁴-dithiO-P², P³-methylene)-tetraphosphate, (Ap₅pCH₂pp₅A), and demonstrated their resistance to hydrolysis by and competitive inhibition of the asymmetrica1 NppppNase from Artemia (1), while Guranowski et al.(2) have studied the ability of four phosphonate analogues of AppppA to act as substrates and inhibitors of the lupin and E. coli NppppNases, lupin phosphodiesterase I, and the yeast AppppA phosphorylase.     

Effect of three 2-allyl-p-mentha-6,8-dien-2-ols on inhibition of mild steel corrosion in 1 M HCl

2-Allyl-p-mentha-6,8-dien-2-ols P1−P3 synthesized from carvone P are tested as corrosion inhibitors of steel in 1 M HCl using weight loss measurements, potentiodynamic polarisation and impedance spectroscopy (EIS) methods. The addition of 2-allyl-p-mentha-6,8-dien-2-ols reduced the corrosion rate. Potentiodynamic polarisation studies clearly reveal that the presence of inhibitors does not change the mechanism of hydrogen evolution and that they act essentially as cathodic inhibitors. 2-Allyl-p-mentha-6,8-dien-2-ols tested adsorb on the steel surface according to Langmuir isotherm. From the adsorption isotherm some thermodynamic data for the adsorption process are calculated and discussed. EIS measurements show the increase of the charge-transfer resistance with the inhibitor concentration. The highest inhibition efficiency (92%) is obtained for P1 at 3 g/L. The corrosion rate decreases with the rise of temperature. The corresponding activation energies are determined.     

Artificial intelligence-aided discovery of prolyl hydroxylase 2 inhibitors to stabilize hypoxia inducible factor-1α and promote angiogenesis

From ZINC database with a total of 1.8 million small molecules, four compounds are identified as prolyl hydroxylase 2 inhibitors through a virtual screening workflow that sequentially incorporates machine learning, molecular docking, and molecular dynamics. Among them, compound 103, (E)-5-(5-((2-(1H-tetrazol-5-yl)hydrazineylidene)methyl)furan-2-yl)isoindoline-1,3-dione, promotes the migration and capillary tube formation capacity of human umbilical vein endothelial cells through enhancing the stability of hypoxia inducible factor-1α and increasing the level of vascular endothelial growth factor.     

Binding Free Energy (BFE) Calculations and Quantitative Structure–Activity Relationship (QSAR) Analysis of Schistosoma mansoni Histone Deacetylase 8 (smHDAC8) Inhibitors

Histone-modifying proteins have been identified as promising targets to treat several diseases including cancer and parasitic ailments. In silico methods have been incorporated within a variety of drug discovery programs to facilitate the identification and development of novel lead compounds. In this study, we explore the binding modes of a series of benzhydroxamates derivatives developed as histone deacetylase inhibitors of Schistosoma mansoni histone deacetylase (smHDAC) using molecular docking and binding free energy (BFE) calculations. The developed docking protocol was able to correctly reproduce the experimentally established binding modes of resolved smHDAC8–inhibitor complexes. However, as has been reported in former studies, the obtained docking scores weakly correlate with the experimentally determined activity of the studied inhibitors. Thus, the obtained docking poses were refined and rescored using the Amber software. From the computed protein–inhibitor BFE, different quantitative structure–activity relationship (QSAR) models could be developed and validated using several cross-validation techniques. Some of the generated QSAR models with good correlation could explain up to ~73% variance in activity within the studied training set molecules. The best performing models were subsequently tested on an external test set of newly designed and synthesized analogs. In vitro testing showed a good correlation between the predicted and experimentally observed IC₅₀ values. Thus, the generated models can be considered as interesting tools for the identification of novel smHDAC8 inhibitors.     

Current literature highlights - August 2002.

Cdk4 Inhibitors: Cyclins and cyclin-dependent kinases (Cdks) play important roles in regulation of the cell cycle. In particular, D-type cyclins, which are activated by rearrangement or amplification in several tumours, associate Cdk4/6. Cyclin D-Cdk4/6 complexes phosphorylate the retinoblastoma protein (pRB) and regulate the cell cycle during G₁/S transition. Loss of function or deletion of p16^ink4a (endogenous Cdk4/6 specific inhibitor protein) frequently occurs in clinical cancer cells. As a next generation of Cdk inhibitors, selective inhibitors of only one target Cdk are expected to cause cell cycle arrest specifically. Suppression of tumour growth by G₁ arrest is thought to reduce the stress for normal cells more than in other phases, because normal cells are usually resting in the G₀-G₁ phase. Thus, the design of Cdk4 selective inhibitors that cause cell cycle arrest in the G₁ phase has been attempted [2] (Structure-based generation of a new class of potent Cdk4 inhibitors: New de novo design strategy and library design, Honma, T. et. al., J. Med. Chem., 44, (2001), 4615-4627). To obtain highly selective and potent Cdk4 inhibitors a structure-based design was performed which consisted of lead generation of a new class of Cdk4 inhibitors based on a Cdk4 homology model, and enhancement of Cdk4 selectivity of lead compounds over Cdk1/2 and other kinases based on the binding modes and structural differences between Cdk4 and other kinases. This methodology was applied to search the Available Chemicals Directory and 382 commercial compounds were selected for screening in cyclin D-Cdk4 assays at concentrations up to 1mM. From this set, 18 compounds were found which possessed an IC₅₀ value of under 500 mM. From these hits, a class of diarylureas were identified with the potential for parallel synthesis follow up to validate the potential of the scaffold and to obtain preliminary SAR. 410 Urea compounds were then designed and synthesised as singles in solution, the design based on the diarylurea hits, and they were screened in a Cdk4 inhibition assay. One of the most potent compounds isolated was (i) which possessed an IC₅₀ value of 34 nM. This work has utilised a structure-based lead generation approach consisting of homology modelling of the target protein, construction of a library of compounds, followed by modification of hits obtained based on predicted binding mode. This strategy has provided potent compounds from a new class of diarylurea Cdk4 inhibitors and may lay the foundation for further work to improve potency in this series.     

In silico exploration of anti-Alzheimer's compounds present in methanolic extract of Neolamarckia cadamba bark using GC–MS/MS

Alzheimer’s disease (AD) can be treated by the inhibition of Beta Amyloid protein (Aβ) and inhibition of Acetylcholinesterase (ACHE). Anti-Alzheimer’s potential phytoconstituents from Neolamarckia cadamba methanolic bark extracts were identified through GC–MS/MS analysis and in silico molecular docking analysis. Powdered bark sample was subjected to extract by soxhlet extractor with n-hexane, chloroform and methanol solvents respectively. The methanolic extract was taken for GC–MS/MS analysis, the observed chromatogram was revealed the presence of 61 constituents in the methanolic extract, 59 new phytoconstituents were identified which were not reported earlier as constituents any part of N. cadamba. GC–MS/MS detected phytoconstituents were analysed through the docking analysis by iGEMDOCK software against Aβ (PDB ID: 2LMN) and ACHE (PDB ID: 3LII) and compared with standard known inhibitors of galantamine and curcumin. Docking analysis binding energy was determined and verified by Discovery studio visulaizer. Both inhibition assay top 5 best dock energy compounds were analysed through the in silico modeling through admetSAR web portal for parameters of intestinal absorption, blood brain barrier permeation, carcinogencity, and acute oral toxicity were determined. From that heptadecanoic acid, 16-methyl-, methyl ester; beta-sitosterol acetate and octadecanoic acid, 2-hydroxy-, methyl ester inhibitors were identified. Further the top lead successful compound of each target molecular interactions were detected by LigPlot analysis. From this research these three compounds are best to treat AD than standard. Isolation of individual compounds would, however, help to find new compounds for other diseases and lead molecules for AD were identified.     

Characterization of plants and seaweeds based corrosion inhibitors against microbially influenced corrosion in a cooling tower water environment

This study compares the inhibitory activities of methanolic extraction of various plants including Artemisia pallens (MEAP), mangrove leaves like Rhizophora mangle (MERM), Avicennia marina (MEAM) and seaweeds such as Pandia povanica (MEPP), Sargassum tenerrimum (MEST) on the corrosion of mild steel (MS) coupons that were incubated on Pseudomonas stutzeri (P. stutzeri) SKR4 strain isolated from the cooling tower water (CTW). The activities of inhibitors are found using GCMS analysis and interactions between microbes and inhibitors were examined in the test for antibacterial activity, minimal inhibition concentration, biofilm formation assay. These all show an excellent inhibitory effect against P. stutzeri. The weight loss, impedance spectroscopy, and Tafel polarization tests used to validate the corrosion investigations show that the inhibitors MEAP-75, MERM-71, MEAM-69, MEPP-66 and MEST-63 % are effective at inhibiting corrosion at 25 ppm. According to Potentiodynamic polarization plots, these five inhibitors act as mixed-type inhibitors. The surface investigation of MS metals by FTIR, SEM, XRD to examine the biofilm surface and it revealed deep pitting corrosion in the bacterial system. In the conclusion, eco-friendly green inhibitors have controlled the biocorrosion in cooling tower water and are recommended for usage in industries as an alternative to environmentally hazardous inhibitors.     

Looking glass mechanism-based inhibition of peptidylglycine α-amidating monooxygenase

Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many peptide hormones, entails sequential enzymatic action by peptidylglycine α-monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). We have previously demonstrated that PAM and PGL exhibit strict tandem reaction stereospecificities, with PAM producing exclusively α-hydroxyglycine moieties of absolute configuration (S), and PGL being reactive only toward (S)-α-hydroxyglycines, and we have also shown that PAM exhibits strict P₂-subsite stereospecificity toward both peptide substrates and peptidyl competitive inhibitors. Herein, it is reported that the inhibitory stereochemistry of olefinic mechanism-based amidation inhibitors differs from the strict subsite stereospecificity exhibited by PAM toward substrates and reversible competitive inhibitors. Kinetic analyses of mechanism-based irreversible inhibition of PAM by the (S)- and (R)-enantiomers of 5-acetamido-4-oxo-6-phenyl-2-hexenoic acid were carried out using the rigorous progress curve method. The two enantiomers were found to exhibit very similar values of K_I and k_inact and in both cases kinetic analysis confirmed that irreversible inhibition occurs strictly at the substrate binding site with no ESI complex being formed during the catalytic processing of these irreversible inhibitors. Molecular docking studies were carried out to help rationalize the sharp contrast in the stereospecificity of PAM toward irreversible inhibitors versus substrates and competitive inhibitors. The results revealed that, in contrast to substrates, both docked enantiomers of the olefinic irreversible inhibitors are well-positioned to undergo catalytic processing at the Cu center that gives rise to irreversible inhibition. Taken together, these results provide one of the first clear examples where the stereospecificity of a particular enzyme toward mechanism-based irreversible inhibitors differs from that for substrates and competitive inhibitors.     

Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure–activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.     

Isolation, chemical composition, and structural features of polysaccharides fromLupinus species

Polysaccharides ofLupinus angustifolius (Bryansk 123 and Krystall varieties) andLupinus luteus (SG-91 variety) are extracted and hydrolyzed. Their chemical composition and structural features are studied. Optimal conditions for extraction, hydrolysis, and purification of the lupine polysaccharides are found. Structural features of the isolated pectins are determined based on¹³C NMR and IR spectroscopy.Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 32–34, January–February, 2000.     

Efficient synthesis and biological activity of enantiomeric pairs of thiolactomycin and its 3-demethyl derivative

The title total synthesis was achieved by employing deconjugative asymmetric α-sulfenylation of the chiral 3-(α,β,γ,δ-unsaturated acyl)oxazolidin-2-one with a 3,3-dimethoxypropyl methanethiosulfonate as a key step. From the biological activity assay carried out using the title compounds, it appeared evident that in vitro antibacterial and mammalian type I FAS inhibitory activity can be cleanly separated by changing not only the substituent at the C₃-position but also the absolute configuration at the C₅-position, and that unnatural (S)-(−)-3-demethylthiolactomycin and its congeners might be usable as selective mammalian type I FAS inhibitors.     

Convenient preparation of pinometostat and related 5′-deoxy-5′-amino adenosine derivatives as well as their activity against DOT1L

From adenosine and 2′-C-Me adenosine, a 3-step route towards nucleoside DOT1L inhibitors, including pinometostat, EPZ5677, and FED1, was established. With useful structural-activity relationship information, the newly prepared 2′-C-Me adenosine derivatives contribute to the limited repertoire of ribose-modified nucleoside DOT1L inhibitors. In general, this new synthetic method will facilitate not only the study of nucleoside DOT1L inhibitors, but also the synthetic and medicinal chemistry research of 5′-deoxy-5′-amino adenosine derivatives.     

Evaluation of crude methanolic mangrove leaves extract for antibiofilm efficacy against biofilm-forming bacteria on a cooling tower wastewater system

The present study demonstrated microbial corrosion protection of MS 1010 on cooling tower water using plant-based inhibitors derived from methanolic extraction of dry mangrove leaves (R.mangle and A.marina), and its assessment of antibacterial activity against corrosive bacteria (B.megaterium) was investigated. FT-IR and GC–MS analyzed the inhibitors component and corrosion behavior of MS 1010 on cooling water, with and without inhibitors were analyzed by EIS and Tafel studies. GC–MS spectra confirmed the presence of Myo-Inositol, 4-C-methyl and chromene as major constituents presented on the R.mangle whereas Lupeol, trifluoroacetate and beta-amyrin compounds were found on the A.marina. In the cooling water, these two inhibitors demonstrated outstanding antibacterial activity and controlled biofilm growth. As plant-based inhibitors were used in cooling water systems, EIS data showed a significant increase in R_ct value when compared to the control system. Tafel plot indicates inhibitors have mixed inhibitory effects and for the systems with and without inhibitors, the i_corr value was 1.5649A and 2.0875A, respectively. At the optimal dose of 25 ppm, the inhibitory efficiency of MERM and MEAM was 81% and 80%, respectively. The overall discussion reveals that inhibitor substances can be absorbed on the metal surface and then act as a dual role in inhibiting corrosive bacterial growth and barrier to the corrosion process in the cooling water system.