Mammalian Cell Toxicity Research Articles

Dimerization and lysine substitution of melittin have differing effects on bacteria.

Melittin is a potent antimicrobial peptide from bee venom that is effective against both Gram-positive and Gram-negative bacteria. However, it is extremely toxic to mammalian cells and, as yet, has no clinical use. Modifications to its amino acid sequence, cyclization, truncation, and dimerization have been attempted in order to reduce its toxicity whilst maintaining its antimicrobial activity. In this study, we targeted the three lysine residues present in melittin and substituted them with lysine homologs containing shorter side chains (ornithine, Orn, diaminobutyric acid, Dab, and diaminopropanoic acid, Dap) and made both parallel and antiparallel melittin dimers to observe how lysine substitution and dimerization affects its activity and toxicity. The antibacterial activity of melittin and its analogs was tested against S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria), and cytotoxicity was tested against the mammalian cell lines HEK293 and H4IIE. Overall, dimerization and lysine substitution exhibited improved antimicrobial activity toward E. coli and limited improvement toward S. aureus. However, mammalian cell toxicity was only marginally reduced compared to native melittin. Interestingly, the parallel dimer was found to be marginally more active than the antiparallel dimer, indicating orientation maybe important for activity, although both dimers were less effective than the native and Lys-analog peptides toward S. aureus. Of the Lys substitutions, Dab and Dap improved melittin's activity toward E. coli. Dimerization and Lys substitution of melittin improved the antimicrobial activity toward Gram-negative bacteria but did not significantly improve its activity toward Gram-positive bacteria. Some analogs also displayed reduced toxicity toward HEK293 and H4IIE cells but overall remained toxic at bactericidal concentrations. Our data indicates that although highly antibacterial, melittin's toxicity is the major drawback in its potential use.

Peptide-mimetic treatment of Pseudomonas aeruginosa in a mouse model of respiratory infection

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2000 deaths in the U.S. annually. While the emergence of resistant bacteria has become ominously common, identification of useful new drug classes has been limited over the past over 40 years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity in mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30–60 min in vitro, and is effective against a range of clinical isolates, including extensively drug resistant strains. In vivo, TM5 significantly reduced bacterial load in the lungs within 24 h compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

Biguanide-Vancomycin Conjugates are Effective Broad-Spectrum Antibiotics against Actively Growing and Biofilm-Associated Gram-Positive and Gram-Negative ESKAPE Pathogens and Mycobacteria.

Strategies to increase the efficacy and/or expand the spectrum of activity of existing antibiotics provide a potentially fast path to clinically address the growing crisis of antibiotic-resistant infections. Here, we report the synthesis, antibacterial efficacy, and mechanistic activity of an unprecedented class of biguanide-antibiotic conjugates. Our lead biguanide-vancomycin conjugate, V-C6-Bg-PhCl (5e), induces highly effective cell killing with up to a 2 orders-of-magnitude improvement over its parent compound, vancomycin (V), against vancomycin-resistant enterococcus. V-C6-Bg-PhCl (5e) also exhibits improved activity against mycobacteria and each of the ESKAPE pathogens, including the Gram-negative organisms. Furthermore, we uncover broad-spectrum killing activity against biofilm-associated Gram-positive and Gram-negative bacteria as well as mycobacteria not observed for clinically used antibiotics such as oritavancin. Mode-of-action studies reveal that vancomycin-like cell wall synthesis inhibition with improved efficacy attributed to enhanced engagement at vancomycin binding sites through biguanide association with relevant cell-surface anions for Gram-positive and Gram-negative bacteria. Due to its potency, remarkably broad activity, and lack of acute mammalian cell toxicity, V-C6-Bg-PhCl (5e) is a promising candidate for treating antibiotic-resistant infections and notoriously difficult-to-treat slowly growing and antibiotic-tolerant bacteria associated with chronic and often incurable infections. More generally, this study offers a new strategy (biguanidinylation) to enhance antibiotic activity and facilitate clinical entry.

Antioxidant activity and phenolic content of herbal infusions from medicinal plants used in Argentina

Currently, teas and herbal infusions represent an important part of the human diet. Most contain phenolic compounds with high antioxidant activity, usually associated with human health protective functions. This attribute defines teas and infusions as nutraceutical foods. In Argentina, several native species are traditionally used for medical purposes. Some of those species are Larrea cuneifolia, Larrea nitida, Grindelia chiloensis, Pteromonnina dictyocarpa, Mandevilla laxa, and Monttea aphylla. The objectives of this study were to analyze the antioxidant power and the scavenging capacity of infusions obtained from those six medicinal plants, to characterize the phenolic profile, and to study in vitro their safety or cytotoxicity. Additionally, the potential use of two infusions as antioxidant additives in a food model was evaluated. The results indicated that the analyzed plant species are rich in phenolic acids (e.g., caffeic, ferulic, and chlorogenic acid), and flavonoids (e.g., quercetin and kaempferol), with high antioxidant power. The infusion blend obtained with G. chiloensis and L. cuneifolia exhibited the highest value of antioxidant capacity measured with the FRAP technique (193.4 μg EAA/mg DW). On the other hand, L. cuneifolia infusion showed the greatest antioxidant capacity determined by FRAP (131.9 ± 5.2 μg EAA/mg DW) and DPPH assays (0.453 mL/mg s). Additionally, L. cuneifolia infusion showed the highest phenolic content (232.8 μg GAE/mg DW) and flavonoid content (153.3 μg QE/mg DW). None of the infusions showed toxicity in mammalian cells, except for G. chiloensis. Furthermore, the L. cuneifolia and L. nitida infusions showed a high inhibitory effect on lipid oxidation in ground beef (55% and 51% at 4 days of storage, respectively). The results suggest that the studied infusions are safe and a rich source of antioxidants, which supports their use in traditional medicine. However, further exhaustive studies of G. chiloensis infusion are needed to ensure its safety, as it has shown cytotoxicity. Besides, it is worthwhile to advance the study of L. cuneifolia and L. nitida as sources of dietary antioxidants, due to their high antioxidant power and ability to protect against lipid peroxidation.Graphical

A series of pyrimidine-based antifungals with anti-mold activity disrupt ER function in Aspergillus fumigatus.

Fungal infections are a major contributor to morbidity and mortality among immunocompromised populations. Moreover, fungal disease caused by molds are difficult to treat and are associated with particularly high mortality. To address the need for new mold-active antifungal drugs, we performed a high-throughput screen with Aspergillus fumigatus, the most common pathogenic mold. We identified a novel, pyrimidine-based chemical scaffold with broad-spectrum antifungal activity including activity against several difficult-to-treat molds. A chemical genetics screen of Saccharomyces cerevisiae suggested that this compound may target the endoplasmic reticulum (ER) and perturb ER function and/or homeostasis. Consistent with this model, this compound induces the unfolded protein response and inhibits secretion of A. fumigatus collagenases. Initial cytotoxicity and pharmacokinetic studies show favorable features including limited mammalian cell toxicity and bioavailability in vivo. Together, these data support the further medicinal chemistry and pre-clinical development of this pyrimidine scaffold toward more effective treatments for life-threatening invasive mold infections.IMPORTANCEInvasive fungal diseases are life-threatening infections caused by fungi in immunocompromised individuals. Currently, there are only three major classes of antifungal drugs available to treat fungal infections; however, these options are becoming even more limited with the global emergence of antifungal drug resistance. To address the need for new antifungal therapies, we performed a screen of chemical compounds and identified a novel molecule with antifungal activity. Initial characterization of this compound shows drug-like features and broad-spectrum activity against medically important fungi. Together, our results support the continued development of this compound as a potential future therapy for these devastating fungal infections.

Negatively charged nanodiscs for the reduction of toxicity and enhanced efficacy of polymyxin B against Acinetobacter baumannii sepsis

The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. Statement of significanceMultidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii, currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.

Antibacterial mechanism of action of crude plantaricin LP21-2 against Escherichia coli and its potential application in yak milk

The mechanism of action of plantaricin against Escherichia coli and its potential application in dairy products remain unclear. In this study, the antibacterial activity and mechanism of action of plantaricin LP21-2 against the E. coli strain ATCC25922 were investigated. The results showed that crude plantaricin LP21-2 inhibited the growth of E. coli_22 as well as inhibited and degraded the biofilm formation in E. coli_22. The mechanism of action of plantaricin LP21-2 against E. coli_22 cells involves the reduction of cell viability and metabolic activity, triggering the leakage of intracellular substances (e.g., DNA, proteins, and ATP) and inducing oxidative stress damage and morphological deformation. Moreover, plantaricin LP21-2 exhibited no hemolytic activity and negligible toxicity in mammalian Caco-2 cells, indicating good biosafety. When yak milk was used as a food model, E. coli_22 viable counts (6.19 ± 0.03 log CFU/ml) were effectively reduced by 0.23, 0.6 and 1.94 log CFU/ml, with the use of crude plantaricin LP21-2 at 1/2, 1 × , and 2 × MIC, respectively, after 12 days of storage at 4 °C. Notably, the addition of crude plantaricin LP21-2 also maintained the nutrient composition, antioxidant parameters, and sensory properties of yak milk. Overall, these findings provide novel insights into plantaricin LP21-2 against E. coli and highlight its promising application in dairy foods.

Development of a Selective and Stable Antimicrobial Peptide.

Antimicrobial peptides (AMPs) are presented as potential scaffolds for antibiotic development due to their desirable qualities including broad-spectrum activity, rapid action, and general lack of susceptibility to current resistance mechanisms. However, they often lose antibacterial activity under physiological conditions and/or display mammalian cell toxicity, which limits their potential use. Identification of AMPs that overcome these barriers will help develop rules for how this antibacterial class can be developed to treat infection. Here we describe the development of our novel synthetic AMP, from discovery through in vivo application. Our evolved AMP, DTr18-dab, has broad-spectrum antibacterial activity and is nonhemolytic. It is active against planktonic bacteria and biofilm, is unaffected by colistin resistance, and importantly is active in both human serum and a Galleria mellonella infection model. Several modifications, including the incorporation of noncanonical amino acids, were used to arrive at this robust sequence. We observed that the impact on antibacterial activity with noncanonical amino acids was dependent on assay conditions and therefore not entirely predictable. Overall, our results demonstrate how a relatively weak lead can be developed into a robust AMP with qualities important for potential therapeutic translation.

In Vitro Bactericidal Activity of Biogenic Copper Oxide Nanoparticles for Neisseria gonorrhoeae with Enhanced Compatibility for Human Cells.

Resistance to antibiotics and antimicrobial compounds is a significant problem for human and animal health globally. The development and introduction of new antimicrobial compounds are urgently needed, and copper oxide nanoparticles (CuO NPs) have found widespread application across various sectors including biomedicine, pharmacy, catalysis, cosmetics, and many others. What makes them particularly attractive is the possibility of their synthesis through biogenic routes. In this study, we synthesized biogenic green tea (GT, Camellia sinensis)-derived CuO NPs (GT CuO NPs) and examined their biophysical properties, in vitro toxicity for mammalian cells in culture, and then tested them against Neisseria gonorrhoeae, an exemplar Gram-negative bacterium from the World Health Organization's Priority Pathogen List. We compared our synthesized GT CuOP NPs with commercial CuO NPs (Com CuO NPs). Com CuO NPs were significantly more cytotoxic to mammalian cells (IC50 of 7.32 μg/mL) than GT CuO NPs (IC50 of 106.1 μg/mL). GT CuO NPs showed no significant increase in bax, bcl2, il6, and il1β mRNA expression from mammalian cells, whereas there were notable rises after treatment with Com CuO NPs. GT-CuO NPs required concentrations of 0.625 and 3.125 μg/mL to kill 50 and 100% of bacteria, respectively, whereas Com-CuO NPs needed concentrations of 15.625 and 30 μg/mL to kill 50 and 100% of bacteria, and the antibiotic ceftriaxone killed 50 and 100% with 3.125 and 30 μg/mL. Gonococci could be killed within 30 min of exposure to GT CuO NPs and the NPs could kill up to 107 within 1 h. In summary, this is the first report to our knowledge that describes the bioactivity of biogenic CuO NPs against N. gonorrhoeae. Our data suggest that biogenic nanoparticle synthesis has significant advantages over traditional chemical routes of synthesis and highlights the potential of GT-CuO NPs in addressing the challenges posed by multidrug-resistant Neisseria gonorrhoeae infections.

Highly selective alkylcoumarates and diterpenoids isolated from leaves of Baccharis quitensis: a search for new hit compounds against Trypanosoma cruzi

In the present work, the hexane extract of aerial parts of Baccharis quitensis Kunth. was subjected to chromatographic fractionation to afford two alkyl phenylpropanoids: n-docosyl (E)-p-coumarate (1) and n-tetracosyl (E)-p-coumarate (2) as well as five diterpenes: ent-kaurenoic acid (3), grandifloric acid (4), 15β-senecioyl-oxy-ent-kaur-16-en-19-oic acid (5), and 15-oxo-ent-kaurenoic acid (6). Using an ex-vivo assay with macrophages infected with Trypanosoma cruzi, compounds 1 and 2 demonstrated high potency against intracellular amastigotes, with EC50 values of 7.5 and 6.9 µM, respectively. Compound 6 revealed a moderate potency against T. cruzi, with an EC50 of 25.6 µM, and compounds 3–5 showed no effectiveness. Additionally, compounds 1–6 compounds presented no toxicity for mammalian cells to the highest tested concentration of 200 µM. Based on potency and the selectivity indexes of 1, 2 and 6, these compounds could be future candidates for optimisation studies for the design of prototypes against Chagas disease.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

Vancomycin-Polyguanidino Dendrimer Conjugates Inhibit Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria and Eradicate Biofilm-Associated S.aureus.

The global challenge of antibiotic resistance necessitates the introduction of more effective antibiotics. Here we report a potentially general design strategy, exemplified with vancomycin, that improves and expands antibiotic performance. Vancomycin is one of the most important antibiotics in use today for the treatment of Gram-positive infections. However, it fails to eradicate difficult-to-treat biofilm populations. Vancomycin is also ineffective in killing Gram-negative bacteria due to its inability to breach the outer membrane. Inspired by our seminal studies on cell penetrating guanidinium-rich transporters (e.g., octaarginine), we recently introduced vancomycin conjugates that effectively eradicate Gram-positive biofilm bacteria, persister cells and vancomycin-resistant enterococci (with V-r8, vancomycin-octaarginine), and Gram-negative pathogens (with V-R, vancomycin-arginine). Having shown previously that the spatial array (linear versus dendrimeric) of multiple guanidinium groups affects cell permeation, we report here for the first time vancomycin conjugates with dendrimerically displayed guanidinium groups that exhibit superior efficacy and breadth, presenting the best activity of V-r8 and V-R in single broad-spectrum compounds active against ESKAPE pathogens. Mode-of-action studies reveal cell-surface activity and enhanced vancomycin-like killing. The vancomycin-polyguanidino dendrimer conjugates exhibit no acute mammalian cell toxicity or hemolytic activity. Our study introduces a new class of broad-spectrum vancomycin derivatives and a general strategy to improve or expand antibiotic performance through combined mode-of-action and function-oriented design studies.

Cleistocalyx nervosum var. paniala berry extract and cyanidin-3-glucoside inhibit hepatotoxicity and apoptosis.

Excessive oxidative toxicity in liver cells is a significant risk factor that can cause cellular injury, leading to the development of chronic liver disease (CLD). Natural anthocyanins have been shown to prevent the harmful effects of oxidative toxicity in mammalian cells. Ripe Cleistocalyx nervosum var. paniala berry fruits are rich in anthocyanins, which have been reported to possess many health benefits. Therefore, this study examined the protective effect of ethanolic fruit extract of C. nervosum var. paniala (CNPE) against hydrogen peroxide (H2O2)-induced oxidative damage and cell death in human hepatoma HepG2 cells. Results showed that CNPE had strong antioxidant capabilities and high amounts of total phenolics and anthocyanins. HPLC analysis showed that CNPE consists of cyanidin-3-glucoside (C3G). Our investigations found that HepG2 cells pretreated with CNPE or anthocyanin C3G inhibited H2O2-induced cellular damage and apoptosis by increasing the viability of cells, the expression of antiapoptotic Bcl-2 protein, and the activities of cellular antioxidant enzymes, namely SOD, CAT, and GPx. Moreover, both CNPE and C3G significantly suppressed expression of apoptotic proteins (Bax and cytochrome c) and the activities of cleaved caspase-9 and caspase-3 caused by H2O2. Our results indicate that CNPE and C3G can suppress H2O2-induced hepatotoxicity and cell death through stimulation of endogenous antioxidant enzyme activities and inhibition of apoptosis pathway in HepG2 cells. These findings might support development of CNPE as an alternative natural product for preventing CLD.

Effect of Star Topology Versus Linear Polymers on Antifungal Activity and Mammalian Cell Toxicity.

The global increase in invasive fungal infections and the emergence of drug-resistant strains demand the urgent development of novel antifungal drugs. In this context, synthetic polymers with diverse compositions, mimicking natural antimicrobial peptides, have shown promising potential for combating fungal infections. This study investigates how altering polymer end-groups and topology from linear to branched star-like structures affects their efficacy against Candida spp., including clinical isolates. Additionally, the polymers' biocompatibility is accessed with murine embryonic fibroblasts and red blood cells invitro. Notably, a low-molecular weight star polymer outperforms both its linear polymeric counterparts and amphotericin B (AmpB)in terms of an improved therapeutic index and reduced haemolytic activity, despite a higher minimum inhibitory concentration against Candida albicans (C. albicans) SC5314 (16-32µgmL-1 vs 1µgmL-1 for AmpB). These findings demonstrate the potential of synthetic polymers with diverse topologies as promising candidates for antifungal applications.

Establishing Quantifiable Guidelines for Antimicrobial α/β-Peptide Design: A Partial Least-Squares Approach to Improve Antimicrobial Activity and Reduce Mammalian Cell Toxicity.

Antimicrobial peptides (AMPs) are promising candidates to combat pathogens that are resistant to conventional antimicrobial drugs because they operate through mechanisms that involve membrane disruption. However, the use of AMPs in clinical settings has been limited, at least in part, by their susceptibility to proteolytic degradation and their lack of selectivity toward pathogenic microbes vs mammalian cells. We recently reported on the design of α- and β-peptide oligomers structurally templated upon the naturally occurring α-helical AMP aurein 1.2. These α/β-peptide oligomers are more proteolytically stable than aurein 1.2 and have several other attributes that render them attractive as alternatives to conventional AMPs. This study describes the influence of peptide physicochemical properties on the broad-spectrum activity of aurein 1.2-based α/β-peptide mimics against nine bacterial, fungal, and mammalian cell lines. We used a partial least-squares regression (PLSR)-supervised machine learning model to quantify and visualize relationships between experimentally determined physicochemical properties (e.g., hydrophobicity, charge, and helicity) and experimentally measured cell-type-specific activities of 21 peptides in a 149-member α/β-peptide library. Using this approach, we identified several peptides that were predicted to exhibit enhanced broad-spectrum selectivity, a measure that evaluates antimicrobial activity relative to mammalian cell toxicity compared to aurein 1.2. Experimental validation demonstrated high model predictive performance, and characterization of compounds with the highest broad-spectrum selectivity revealed peptide hydrophobicity, helicity, and helical rigidity to be strong predictors of broad-spectrum selectivity. The most selective peptide identified from the model prediction has more than a 13-fold improvement in broad-spectrum selectivity than that of aurein 1.2, demonstrating the ability of using PLSR models to identify quantitative structure-function relationships for nonstandard amino acid-containing peptides. Overall, this work establishes quantifiable guidelines for the rational design of helical antimicrobial α/β-peptides and identifies promising new α/β-peptides with significantly reduced mammalian toxicities and improved antifungal and antibacterial activities relative to aurein 1.2.

Synthesis by Microwave Irradiation, Molecular Structural Analysis and Trypanocidal Activity of Novel Pyrazole-tetrahydropyrimidine Derivatives.

A series of new eight 2-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1,4,5,6-tetrahydropyrimidines 1(a-h) were synthesized by microwave irradiation technique. In vitro phenotypic screening was performed to evaluate the effect of these compounds on intracellular amastigotes forms of Trypanosoma cruzi</i>, the etiological agent of Chagas disease. Compounds 1(a-h) were synthesized from pyrazole-carbonitriles 2(a-h) employing microwave irradiation (50W) for 10-20 minutes. Physicochemical properties were calculated using OSIRIS DataWarrior. The toxic effect on mammalian cells (Vero Cells) and the trypanocidal activity against Trypanosoma cruzi</i> (Dm28c-Luc) were also evaluated. Compounds 1(a-h) were obtained in 24-94% yields. They were completely characterized by Fourier Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR) and High-Resolution Mass Spectrometry (HRMS) analyses. The derivatives showed low trypanocidal activity, with IC50 ranging from 47.16 to > 100 μM, with lower activity than benznidazole (1.93 μM) used as reference drug. The attractive features of this synthetic methodology are mild conditions, short reaction time, and low power. All derivatives showed low toxicity in mammalian cells, good oral bioavailability, and did not violate Lipinski´s rule of 5.

Cathepsin D overexpression in the nervous system rescues lethality and Aβ42 accumulation of cathepsin D systemic knockout invivo.

The lysosome is responsible for protein and organelle degradation and homeostasis and the cathepsins play a key role in maintaining protein quality control. Cathepsin D (CTSD), is one such lysosomal protease, which when deficient in humans lead to neurolipofuscinosis (NCL) and is important in removing toxic protein aggregates. Prior studies demonstrated that CTSD germ-line knockout-CtsdKO (CDKO) resulted in accumulation of protein aggregates, decreased proteasomal activities, and postnatal lethality on Day 26±1. Overexpression of wildtype CTSD, but not cathepsin B, L or mutant CTSD, decreased α-synuclein toxicity in worms and mammalian cells. In this study we generated a mouse line expressing human CTSD with a floxed STOP cassette between the ubiquitous CAG promoter and the cDNA. After crossing with Nestin-cre, the STOP cassette is deleted in NESTIN+cells to allow CTSD overexpression-CTSDtg (CDtg). The CDtg mice exhibited normal behavior and similar sensitivity to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced neurodegeneration. By breeding CDtg mice with CDKO mice, we found that over-expression of CTSD extended the lifespan of the CDKO mice, partially rescued proteasomal deficits and the accumulation of Aβ42 in the CDKO. This new transgenic mouse provides supports for the key role of CTSD in protecting against proteotoxicity and offers a new model to study the role of CTSD enhancement invivo.

A novel palladium complex with a coumarin-thiosemicarbazone hybrid ligand inhibits Trypanosoma cruzi release from host cells and lowers the parasitemia in vivo.

In this work, two analogous coumarin-thio and semicarbazone hybrid compounds were prepared and evaluated as a potential antichagasic agents. Furthermore, palladium and platinum complexes with the thiosemicarbazone derivative as ligand (L1) were obtained in order to establish the effect of metal complexation on the antiparasitic activity. All compounds were fully characterized both in solution and in solid state including the resolution of the crystal structure of the palladium complex by X-ray diffraction methods. Unexpectedly, all experimental and theoretical characterizations in the solid state, demonstrated that the obtained palladium and platinum complexes are structurally different: [PdCl(L1)] and [PtCl2(HL1)]. All the studied compounds lower the proliferation of the amastigote form of Trypanosoma cruzi while some of them also have an effect on the trypomastigote stage. Additionally, the compounds inhibit T. cruzi release from host cells in variable extents. The Pd compound presented a remarkable profile in all the in vitro experiments, and it showed no toxicity for mammalian cells in the assayed concentrations. In this sense, in vivo experiments were performed for this compound using an acute model of Chagas disease. Results showed that the complex significantly lowered the parasite count in the mice blood with no significant toxicity.

Amyloid Fibrils of Pisum sativum L. Vicilin Inhibit Pathological Aggregation of Mammalian Proteins.

Although incurable pathologies associated with the formation of highly ordered fibrillar protein aggregates called amyloids have been known for about two centuries, functional roles of amyloids have been studied for only two decades. Recently, we identified functional amyloids in plants. These amyloids formed using garden pea Pisum sativum L. storage globulin and vicilin, accumulated during the seed maturation and resisted treatment with gastric enzymes and canning. Thus, vicilin amyloids ingested with food could interact with mammalian proteins. In this work, we analyzed the effects of vicilin amyloids on the fibril formation of proteins that form pathological amyloids. We found that vicilin amyloids inhibit the fibrillogenesis of these proteins. In particular, vicilin amyloids decrease the number and length of lysozyme amyloid fibrils; the length and width of β-2-microglobulin fibrils; the number, length and the degree of clustering of β-amyloid fibrils; and, finally, they change the structure and decrease the length of insulin fibrils. Such drastic influences of vicilin amyloids on the pathological amyloids' formation cause the alteration of their toxicity for mammalian cells, which decreases for all tested amyloids with the exception of insulin. Taken together, our study, for the first time, demonstrates the anti-amyloid effect of vicilin fibrils and suggests the mechanisms underlying this phenomenon.

The Use of a Barley-Based Well to Define Cationic Betaglucan to Study Mammalian Cell Toxicity Associated with Interactions with Biological Structures.

Among potential macromolecule-based pharmaceuticals, polycations seem particularly interesting due to their proven antimicrobial properties and use as vectors in gene therapy. This makes an understanding of the mechanisms of these molecules' interaction with living structures important, so the goal of this paper was to propose and carry out experiments that will allow us to characterize these phenomena. Of particular importance is the question of toxicity of such structures to mammalian cells and, in the work presented here, two lines, normal fibroblasts 3T3-L1 and A549 lung cancer, were used to determine this. In this work, three well-defined cationic derivatives of barley-derived betaglucans obtained in a reaction with glycidyltrimethylammonium chloride (BBGGTMAC) with different degrees of cationization (50, 70, and 100% per one glucose unit) and electrostatic charge were studied. The studies address interactions of these polymers with proteins (bovine serum proteins and BSA), nucleic acids (DNA), glycosaminoglycans (heparin), and biological membranes. The results described in this study make it possible to indicate that toxicity is most strongly influenced by interactions with biological membranes and is closely related to the electrostatic charge of the macromolecule. The presentation of this observation was the goal of this publication. This paper also shows, using fluorescently labeled variants of polymers, the penetration and impact on cell structure (only for the polymer with the highest substitution binding to cell membranes is observed) by using confocal and SEM (for the polymer with the highest degree of substitution, and the appearance of additional structures on the surface of the cell membrane is observed). The labeled polymers are also tools used together with dynamic light scattering and calorimetric titration to study their interaction with other biopolymers. As for the interactions with biological membranes, lipid Langmuir monolayers as model membrane systems were used.