In vitro antiproliferative effects of Vatairea macrocarpa (Benth.) Ducke lectin on human tumor cell lines and in vivo evaluation of its toxicity in Drosophila melanogaster.

Tumor cells may develop alterations in glycosylation patterns during the initial phase of carcinogenesis. These alterations may be important therapeutic targets for lectins with antitumor action. This work aimed to evaluate the in vitro cytotoxicity of VML on tumor and non-tumor cells (concentration of 25 µg/mL and then microdiluted) and evaluate its in vivo toxicity at different concentrations (1.8, 3.5 and 7.0 µg/mL), using Drosophila melanogaster. Toxicity in D. melanogaster evaluated mortality rate, as well as oxidative stress markers (TBARS, iron levels, nitric oxide levels, protein and non-protein thiols). The cytotoxicity assay showed that VML had cytotoxic effect on leukemic lines HL-60 (IC50 = 3.5 µg/mL), KG1 (IC50 = 18.6 µg/mL) and K562 (102.0 µg/mL). In the toxicity assay, VML showed no reduction in survival at concentrations of 3.5 and 7.0 µg/mL and did not alter oxidative stress markers at any concentrations tested. Cytotoxicity of VML from HL-60, KG1 and K562 cells could arise from the interaction between the lectin and specific carbohydrates of tumor cells. In contrast, effective concentrations of VML against no-tumor cells human keratinocyte - HaCat and in the D. melanogaster model did not show toxicity, suggesting that VML is a promising molecule in vivo studies involving leukemic cells.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Occurrence of trace elements in print paper products: Non-carcinogenic risk assessment through dermal exposure.

Over time, the risk assessment of dermal exposure to pollutants in print paper products has received considerable attention. Most studies have focused on organic pollutants, especially bisphenol A (BPA). However, little is known about the levels of trace elements in print paper products, despite the knowledge that these elements are components of printing inks and toners. This study was aimed at determining the concentrations of trace elements in 5 types of paper products, namely bulletins, magazines, special events program booklets, handbills, and newspapers. The average daily intake (ADI) of each element was subsequently estimated through dermal exposure to the papers. The detection frequency of the elements of interest was high (nearly 100%) in most paper products, with the exception of chromium, whose detection was low. In contrast, Ag was not detected in any sample. The levels of the elements in the paper products were low and comparable to those found in other personal and consumer products with the potential for skin contact. The range values of estimated ADIs were 1.70-3.90E-08, 2.30-18.2E-10, 2.60-16.4E-09, 3.65-5.75E-08, 1.29-4.38E-08, 6.23-15.6E-10, 1.51-2.80E-10, 1.43-9.16E-09, 0.00-9.47E-09, and 4.68-220E-08 mg/kg bw/day for Mn, Co, Ni, Cu, Zn, As, Cd, Pb, Cr and Fe respectively. These values were well below the dermal standard reference doses (RfD) for each element. The present results indicate that dermal exposure to trace elements from print paper products was low and does not pose significant risks for toxic (non-carcinogenic) effects on humans.

DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain.

Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.

Dioclea violacea lectin increases the effect of neomycin against multidrug-resistant strains and promotes the purification of the antibiotic in immobilized lectin column.

DVL is a Man/Glc-binding lectin from Dioclea violacea seeds that has the ability to interact with the antibiotic gentamicin. The present work aimed to evaluate whether the DVL has the ability to interact with neomycin via CRD and to examine the ability of this lectin to modulate the antibiotic effect of neomycin against multidrug-resistant strains (MDR). The hemagglutinating activity test revealed that neomycin inhibited the hemagglutinating activity of DVL with a minimum inhibitory concentration of 50 mM, indicating that the antibiotic interacts with DVL via the carbohydrate recognition domain (CRD). DVL immobilized on cyanogen bromide-activated Sepharose® 4B bound 41 % of the total neomycin applied to the column, indicating that the DVL-neomycin interaction is efficient for purification processes. Furthermore, the minimum inhibitory concentrations (MIC) obtained for DVL against all strains studied were not clinically relevant. However, when DVL was combined with neomycin, a significant increase in antibiotic activity was observed against S. aureus and P. aeruginosa. These results demonstrate the first report of lectin-neomycin interaction, indicating that immobilized DVL has the potential to isolate neomycin by affinity chromatography. Moreover, DVL increased the antibiotic activity of neomycin against MDR, suggesting that it is a potent adjuvant in the treatment of infectious diseases.

Polycyclic aromatic hydrocarbons (PAHs): Updated aspects of their determination, kinetics in the human body, and toxicity.

Polycyclic aromatic hydrocarbons (PAHs) are legacy pollutants of considerable public health concern. Polycyclic aromatic hydrocarbons arise from natural and anthropogenic sources and are ubiquitously present in the environment. Several PAHs are highly toxic to humans with associated carcinogenic and mutagenic properties. Further, more severe harmful effects on human- and environmental health have been attributed to the presence of high molecular weight (HMW) PAHs, that is PAHs with molecular mass greater than 300 Da. However, more research has been conducted using low molecular weight (LMW) PAHs). In addition, no HMW PAHs are on the priority pollutants list of the United States Environmental Protection Agency (US EPA), which is limited to only 16 PAHs. However, limited analytical methodologies for separating and determining HMW PAHs and their potential isomers and lack of readily available commercial standards make research with these compounds challenging. Since most of the PAH kinetic data originate from animal studies, our understanding of the effects of PAHs on humans is still minimal. In addition, current knowledge of toxic effects after exposure to PAHs may be underrepresented since most investigations focused on exposure to a single PAH. Currently, information on PAH mixtures is limited. Thus, this review aims to critically assess the current knowledge of PAH chemical properties, their kinetic disposition, and toxicity to humans. Further, future research needs to improve and provide the missing information and minimize PAH exposure to humans.

Crystal structure and specific location of a germin-like protein with proteolytic activity from Thevetia peruviana.

Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical ß-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.

Machaerium acutifolium lectin alters membrane structure and induces ROS production in Candida parapsilosis.

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.

Characterization of Three Osmotin-Like Proteins from Plumeria rubra and Prospection for Adiponectin Peptidomimetics.

BACKGROUND: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet. OBJECTIVE AND METHODS: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2). RESULTS: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide. CONCLUSION: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.

Endocrine disrupting chemicals associated with dry eye syndrome.

BACKGROUND: Dry eye syndrome (DES) is a multifactorial disease that causes changes in the tear film and occurs more frequently in women. Sex hormones (SHs) influence tear production, and SHs imbalance is associated with DES. Endocrine-disrupting chemicals (EDCs) are compounds that can bind to SHs receptors, changing the SHs action in several organs and tissues. METHODS: The levels of 21 EDCs were measured in the urine of DES patients and healthy controls. All individuals were submitted to eye exams for DES and responded to the questionnaire "Ocular Surface Disease Index (OSDI)". DES was considered present when the OSDI score was >20 and one of the DES tests surpassed the established thresholds. RESULTS: Methyl-protocatechuic acid (OHMeP), had higher urine levels in DES individuals than in control individuals (p = 0.0189). On the other hand, triclocarban (TCC) exhibited lower urine levels in DES individuals than in control individuals (p = 0.0081). Statistically significant positive associations were found between Methyl Paraben (MeP), EtP (ethyl paraben) and OHMeP with fluorescein staining test; between TCC and Tear breakup time test and between OHMeP and OSDI score. Significant negative associations were found between EtP and OHMeP and schirmer test; between OHMeP and Tear breakup time test; between TCC and the OSDI score and fluorescein and lissamine staining test.The quadratic discriminant function classified 94.4% of individuals in their groups based on the urine levels of EDCs. CONCLUSION: The following EDCs, MeP, EtP, and OHMeP, were associated with signs and symptoms of DES. TCC had a paradoxical protective effect against DES. These findings suggest that EDCs are associated with DES and the exposure should be included in the investigation of causes and risk factors for DES.

The Galactose-Binding Lectin Isolated from Vatairea macrocarpa Seeds Enhances the Effect of Antibiotics Against Staphylococcus aureus-Resistant Strain.

The use of natural products together with standard antimicrobial drugs has recently received more attention as a strategy to combat infectious diseases caused by multidrug-resistant (MDR) microorganisms. This study aimed to evaluate the capacity of a galactose-binding lectin from Vatairea macrocarpa seeds (VML) to modulate antibiotic activity against standard and MDR Staphylococcus aureus and Escherichia coli bacterial strains. The minimum inhibitory concentration (MIC) obtained for VML against all strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when VML was combined with the antibacterial drugs gentamicin, norfloxacin and penicillin, a significant increase in antibiotic activity was observed against S. aureus, whereas the combination of VML and norfloxacin presented decreased and, hence, antagonistic antibiotic activity against E. coli. By its inhibition of hemagglutinating activity, gentamicin (MIC = 50 mM) revealed its interaction with the carbohydrate-binding site (CBS) of VML. Using molecular docking, it was found that gentamicin interacts with residues that constitute the CBS of VML with a score of - 120.79 MDS. It is this interaction between the antibiotic and the lectin's CBS that may be responsible for the enhanced activity of gentamicin in S. aureus. Thus, our results suggest that the VML can be an effective modulating agent against S. aureus. This is the first study to report the effect of lectins as modulators of bacterial sensitivity, and as such, the outcome of this study could lay the groundwork for future research involving the use of lectins and conventional antibiotics against such infectious diseases such as community-acquired methicillin-resistant S. aureus (MRSA).

Dioclea violacea lectin modulates the gentamicin activity against multi-resistant strains and induces nefroprotection during antibiotic exposure.

Gentamicin is an aminoglycoside antibiotic used to treat infections of various origins. In the last few decades, the constant use of gentamicin has resulted in increased bacterial resistance and nephrotoxicity in some cases. In this study, we examined the ability of Dioclea violacea lectin (DVL) in modulate the antimicrobial activity of gentamicin and reduce the nephrotoxicity induced by this drug. The minimum inhibitory concentration (MIC) obtained for DVL against all strains studied was not clinically relevant (MIC ≥ 1024 µg/mL). However, when DVL was combined with gentamicin, a significant increase in antibiotic action was observed against Staphylococcus aureus and Escherichia coli. DVL also reduced antibiotic tolerance in S. aureus during 10 days of continuous treatment. In addition, DVL presented a nephroprotective effect, reducing sodium excretion, N-Gal expression and urinary protein, that are important markers of glomerular and tubular injuries. Taken together, studies of inhibition of hemagglutinating activity, fluorescence spectroscopy and molecular docking revealed that gentamicin can interact with DVL via the carbohydrate recognition domain (CRD), suggesting that the results obtained in this study may be directly related to the interaction of DVL-gentamicin and with the ability of the lectin to interact with glycans present in the cells of the peritoneum.

Parkia platycephala lectin enhances the antibiotic activity against multi-resistant bacterial strains and inhibits the development of Haemonchus contortus.

Lectins have been studied in the past few years as an alternative to inhibit the development of pathogenic bacteria and gastrointestinal nematodes of small ruminants. The development of new antibacterial and anthelmintic compounds is necessary owing to the increase in drug resistance among important pathogens. Therefore, this study aimed to evaluate the capacity of a glucose/mannose-binding lectin from Parkia platycephala seeds (PPL) to inhibit the development of Haemonchus contortus and to modulate antibiotic activity against multi-resistant bacterial strains, thereby confirming its efficacy when used in combination with gentamicin. PPL at the concentration of 1.2 mg/mL did not show inhibitory activity on H. contortus in the egg hatch test or the exsheathment assay. However, it did show significant inhibition of H. contortus larval development with an IC50 of 0.31 mg/mL. The minimum inhibitory concentration (MIC) obtained for PPL against all tested bacterial strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when PPL was combined with gentamicin, a significant increase in antibiotic activity was observed against S. aureus and E.coli multi-resistant strains. The inhibition of hemagglutinating activity by gentamicin (MIC = 50 mM) revealed that it may be interacting with the carbohydrate-binding site of PPL. It is this interaction between the antibiotic and lectin carbohydrate-binding site that may be responsible for the enhanced activity of gentamicin against multi-resistant strains. It can be concluded that PPL showed selective anthelmintic effect, inhibiting the development of H. contortus larvae and that it increased the effect of the antibiotic gentamicin against multi-resistant bacterial strains, thus constituting a potential therapeutic resource against resistant bacterial strains and H. contortus.

Mo-CBP3, a 2S albumin from Moringa oleifera, is a complex mixture of isoforms that arise from different post-translational modifications.

Mo-CBP3 is a chitin-binding 2S albumin from Moringa oleifera. This seed storage protein is resistant to thermal denaturation and shows biological activities that might be of practical use, such as antifungal properties against Candida sp., a pathogen that causes candidiasis, and against Fusarium solani, a soil fungus that can cause diseases in plants and humans. Previous work has demonstrated that Mo-CBP3 is a mixture of isoforms encoded by members of a small multigene family. Mature Mo-CBP3 is a small protein (∼14 kDa), constituted by a small chain of approximately 4 kDa and a large chain of 8 kDa, which are held together by disulfide bridges. However, a more comprehensive picture on the spectrum of Mo-CBP3 isoforms which are found in mature seeds, is still lacking. In this work, genomic DNA fragments were obtained from M. oleifera leaves, cloned and completely sequenced, thus revealing new genes encoding Mo-CBP3. Moreover, mass spectrometry analysis showed that the mature protein is a complex mixture of isoforms with a remarkable number of molecular mass variants. Using computational predictions and calculations, most (∼86%) of the experimentally determined masses were assigned to amino acid sequences deduced from DNA fragments. The results suggested that the complex mixture of Mo-CBP3 isoforms originates from proteins encoded by closely related genes, whose products undergo different combinations of distinct post-translational modifications, including cleavage at the N- and C-terminal ends of both subunits, cyclization of N-terminal Gln, as well as Pro hydroxylation, Ser/Thr phosphorylation, and Met oxidation.

Purification and biophysical characterization of a mannose/N-acetyl-d-glucosamine-specific lectin from Machaerium acutifolium and its effect on inhibition of orofacial pain via TRPV1 receptor.

A new mannose/N-acetyl-dglucosamine-specific lectin, named MaL, was purified from seeds of Machaerium acutifolium by precipitation with ammonium sulfate, followed by affinity and ion-exchange chromatography. MaL haemagglutinates either native rabbit erythrocytes or those treated with proteolytic enzymes. MaL is highly stable by the ability to maintain its haemagglutinating activity after exposure to temperatures up to 50 °C. The lectin haemagglutinating activity was optimum between pH 6.0 and 7.0 and inhibited after incubation with d-mannose and N-acetyl-d-glucosamine and α-methyl-d-mannopyranoside. MaL is a glycoprotein with relative molecular mass of 29 kDa (α-chain), 13 kDa (ß-chain) and 8 kDa (γ-chain) with secondary structure composed of 3% α-helix, 44% ß-sheet, 21% ß-turn, and 32% coil. The orofacial antinociceptive activity of the lectin was also evaluated. MaL (0.03 mg mL-1) reduced orofacial nociception induced by capsaicin, an effect that occurred via carbohydrate recognition domain interaction, suggesting an interaction of MaL with the transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor. Our results confirm the potential pharmacological relevance of MaL as an inhibitor of acute orofacial mediated by TRPV1.

Structural and enzymatic characterization of Peruvianin­I, the first germin-like protein with proteolytic activity.

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin­I an interesting model for further understanding of the action of laticifer fluids in plant defense.

Structural analysis and anthelmintic activity of Canavalia brasiliensis lectin reveal molecular correlation between the carbohydrate recognition domain and glycans of Haemonchus contortus.

Haemonchus contortus is one of the most economically important parasites infecting small ruminants worldwide. This nematode has shown a great ability to develop resistance to anthelmintic drugs, calling for the development of alternative control approaches. Because lectins recognize and bind to specific carbohydrates and glycan structures present in parasites, they can be considered as an alternative to develop new antiparasitic drugs. Accordingly, this work aimed to investigate the anthelmintic effect of Canavalia brasiliensis (ConBr) lectin against H. contortus and to evaluate a possible interaction of ConBr with glycans of this parasite by molecular docking. ConBr showed significant inhibition of H. contortus larval development with an IC50 of 0.26 mg mL-1. Molecular docking assays revealed that glycans containing the core trimannoside [Man(α1-3)Man(α1-6)Man] of H. contortus interact in the carbohydrate recognition domain of ConBr with an interaction value of MDS = -248.77. Our findings suggest that the inhibition of H. contortus larval development is directly related to the recognition of the core trimannoside present in the glycans of these parasites. This work is the first to report on the structure-function relationships of the anthelmintic activity of plant lectins.

Pre-clinical evaluation of quinoxaline-derived chalcones in tuberculosis.

New effective compounds for tuberculosis treatment are needed. This study evaluated the effects of a series of quinoxaline-derived chalcones against laboratorial strains and clinical isolates of M. tuberculosis. Six molecules, namely N5, N9, N10, N15, N16, and N23 inhibited the growth of the M. tuberculosis H37Rv laboratorial strain. The three compounds (N9, N15 and N23) with the lowest MIC values were further tested against clinical isolates and laboratory strains with mutations in katG or inhA genes. From these data, N9 was selected as the lead compound for further investigation. Importantly, this chalcone displayed a synergistic effect when combined with moxifloxacin. Noteworthy, the anti-tubercular effects of N9 did not rely on inhibition of mycolic acids synthesis, circumventing important mechanisms of resistance. Interactions with cytochrome P450 isoforms and toxic effects were assessed in silico and in vitro. The chalcone N9 was not predicted to elicit any mutagenic, genotoxic, irritant, or reproductive effects, according to in silico analysis. Additionally, N9 did not cause mutagenicity or genotoxicity, as revealed by Salmonella/microsome and alkaline comet assays, respectively. Moreover, N9 did not inhibit the cytochrome P450 isoforms CYP3A4/5, CYP2C9, and CYP2C19. N9 can be considered a potential lead molecule for development of a new anti-tubercular therapeutic agent.

Cloning of cDNA sequences encoding cowpea (Vigna unguiculata) vicilins: Computational simulations suggest a binding mode of cowpea vicilins to chitin oligomers.

Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding ß-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of ß-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to ß-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of ß-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut.