Diacetylcurcumin: a novel strategy against Enterococcus faecalis biofilm in root canal disinfection.

Aim: We evaluated Diacetylcurcumin (DAC), a derivative of curcumin, for its antibacterial and antibiofilm properties against Enterococcus faecalis. Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined, along with antibiofilm potential and toxicity in Galleria mellonella. Additionally, in silico computational analysis was performed to understand its mechanisms of action. Results & conclusion: DAC demonstrated significant antibacterial effects, with MIC and MBC values of 15.6 and 31.25 µg/ml, respectively, and reduced biofilm formation. A synergistic effect, reducing biofilm by 77%, was observed when combined with calcium hydroxide. G. mellonella toxicity tests confirmed DAC's safety at tested concentrations, suggesting its potential for use in root canal disinfection products.

Diacetylcurcumin (DAC) comes from turmeric, a natural spice often used in food. DAC may have the ability to fight germs, including the bacteria Enterococcus faecalis. We tested DAC's ability to kill E. faecalis and stopping the formation of films of the bacteria. We found that a small amount of DAC did kill E. faecalis. When used with calcium hydroxide, DAC works even better to reduce the formation of bacterial films by 77%. DAC is safe to be used on teeth, so may be a useful ingredient for preserving mouth health.

Antifungal activity of 3,3'-dimethoxycurcumin (DMC) against dermatophytes and Candida species.

Dermatomycosis is an infection with global impacts caused especially by dermatophytes and Candida species. Current antifungal therapies involve drugs that face fungal resistance barriers. This clinical context emphasizes the need to discover new antifungal agents. Herein, the antifungal potential of 10 curcumin analogs was evaluated against four Candida and four dermatophyte species. The most active compound, 3,3'-dimethoxycurcumin, exhibited minimum inhibitory concentration values ranging from 1.9‒62.5 to 15.6‒62.5 µg ml-1 against dermatophytes and Candida species, respectively. According to the checkerboard method, the association between DMC and terbinafine demonstrated a synergistic effect against Trichophyton mentagrophytes and Epidermophyton floccosum. Ergosterol binding test indicated DMC forms a complex with ergosterol of Candida albicans, C. krusei, and C. tropicalis. However, results from the sorbitol protection assay indicated that DMC had no effect on the cell walls of Candida species. The in vivo toxicity, using Galleria mellonella larvae, indicated no toxic effect of DMC. Altogether, curcumin analog DMC was a promising antifungal agent with a promising ability to act against Candida and dermatophyte species.

Combinatorial effect of fluconazole, itraconazole, and terbinafine with different culture extracts of Candida parapsilosis and Trichophyton spp. against Trichophyton rubrum.

Onychomycosis is a nail infection caused by dermatophytes, non-dermatophyte fungi, and yeasts, especially Candida species. The present study evaluated the combinatorial effect of different cultured extracts of Candida parapsilosis and Trichophyton mentagrophytes and Trichophyton rubrum with fluconazole, itraconazole, and terbinafine against clinical isolates of Trichophyton rubrum. In addition, investigation of the action of the extracts on the wall or membrane was performed. Pure and mixed cultures of Candida parapsilosis and dermatophytes were filtered through a 0.2-µm membrane and submitted to liquid-liquid extraction using ethyl acetate. After a checkerboard, trial with drugs was performed to evaluate the synergistic interaction with the extract. The results obtained for the minimum inhibitory concentration (MIC) of extracts against the T. rubrum strain in isolation were 500-8000 µg/mL. The MIC range for fluconazole, itraconazole, and terbinafine were 2-32 µg/mL, 0.25-0.5 µg/mL, 0.03-64 µg/mL, respectively. However, when the extract was combined with drugs, the MIC values decreased: extracts 1.9-1000 µg/mL, fluconazole 0.25-4, itraconazole 0.03-0.06 µg/mL, and terbinafine 0.001-0.02 µg/mL. The MIC values of the extracts in the Roswell Park Memorial Institute 1640 medium (RPMI) supplemented with sorbitol did not change, suggesting any action on the cell wall. However, in the presence of RPMI supplemented with ergosterol, MIC values of the extracts increased by up to 2×, indicating action on the fungal cell membrane. A synergistic action was observed between products and drugs, detecting a decrease in MIC values. There is potential and a new therapeutic perspective for fungal control.

Nematostatic activity of isoprenylated guanidine alkaloids from Pterogyne nitens and their interaction with acetylcholinesterase.

Although new nematicides have appeared, the demand for new products less toxic and more efficient for the control of plant-parasitic nematodes are still high. Consequently, studies on natural secondary metabolites from plants, to develop new nematicides, have increased. In this work, nineteen extracts from eleven Brazilian plant species were screened for activity against Meloidogyne incognita. Among them, the extracts of Piterogyne nitens showed a potent nematostatic activity. The alkaloid fraction obtained from the ethanol extract of leaves of P. nitens was more active than the coming extract. Due to the promising activity from the alkaloid fraction, three isoprenylated guanidine alkaloids isolated from this fraction, galegine (1), pterogynidine (2), and pterogynine (3) were tested, showing similar activity to the alkaloid fraction, which was comparable to that of the positive control Temik at 250 µg/mL. At lower concentrations (125-50 µg/mL), compound 2 showed to be the most active one. As several nematicides act through inhibition of acetylcholinesterase (AChE), the guanidine alkaloids were also employed in two in vitro AChE assays. In both cases, compound 2 was more active than compounds 1 and 3. Its activity was considered moderated compared to the control (physostigmine). Compound 2 was selected for an in silico study with the electric eel (Electrophorus electricus) AChE, showing to bind mostly to the same site of physostigmine in the AChEs, pointing out that this could be the mechanism of action for this compound. These results suggested that the guanidine alkaloids 1,2 and 3 from P. nitens are promising for the development of new products to control M. incognita, especially guanidine 2, and encourage new investigations to confirm the mechanism of action, as well as to determine the structure-activity relationship of the guanidine alkaloids.

Chalcone T4 Inhibits RANKL-Induced Osteoclastogenesis and Stimulates Osteogenesis In Vitro.

Chalcones are phenolic compounds produced during the biosynthesis of flavonoids that have numerous biological activities, including anti-inflammatory, antioxidant and anticancer. In this in vitro study, we investigate a newly synthesized chalcone (Chalcone T4) in the context of bone turnover, specifically on the modulation of osteoclast differentiation and activity and osteoblast differentiation. Murine macrophages (RAW 264.7) and pre-osteoblasts (MC3T3-E1) were used as models of osteoclasts and osteoblasts, respectively. Differentiation and activity osteoclasts were induced by RANKL in the presence and absence of non-cytotoxic concentrations of Chalcone T4, added in different periods during osteoclastogenesis. Osteoclast differentiation and activity were assessed by actin ring formation and resorption pit assay, respectively. Expression of osteoclast-specific markers (Nfatc1, Oscar, Acp5, Mmp-9 and Ctsk) was determined by RT-qPCR, and the activation status of relevant intracellular signaling pathways (MAPK, AKT and NF-kB) by Western blot. Osteoblast differentiation and activity was induced by osteogenic culture medium in the presence and absence of the same concentrations of Chalcone T4. Outcomes assessed were the formation of mineralization nodules via alizarin red staining and the expression of osteoblast-related genes (Alp e Runx2) by RT-qPCR. Chalcone T4 reduced RANKL-induced osteoclast differentiation and activity, suppressed Oscar, Acp5 and Mmp-9 expression, and decreased ERK and AKT activation in a dose-dependent manner. Nfact1 expression and NF-kB phosphorylation were not modulated by the compound. Mineralized matrix formation and the expression of Alp and Runx2 by MC3T3-E1 cells were markedly stimulated by Chalcone T4. Collectively, these results demonstrate that Chalcone T4 inhibits in osteoclast differentiation and activity and stimulates osteogenesis, which indicates a promising therapeutic potential in osteolytic diseases.

Evaluation of efficacy of new chalcone-based endodontic irrigant against dual biofilm Enterococcus faecalis and Candida albicans: a study in vitro.

The aim of this research was to develop a chalcone-based endodontic irrigant for cleaning and disinfecting the root canal. Minimal inhibitory concentration (MIC) experiments in C. albicans and E. faecalis strains with different aminochalcones (AM) were carried out, and the compound that presented the best activity against both pathogens was chosen. The formulation of an endodontic irrigant was elaborated, tested in mono and dual specie biofilms. Disks were sterilized and then incubated with E. faecalis, C. albicans and E. faecalis and C. albicans mixed for 72 h for biofilm maturation. After contamination, samples were divided in 4 experimental groups and 2 positive control group as follows: Group1: Irrigant; Group2: Irrigant + AM-38; Group3: Chlorhexidine 2% (positive control) and, Group 4: 1.0% sodium hypochlorite (positive control). The samples were analyzed by CFU/ml count. The sample was taken to sonicador to remove the cells and then plated. The toxicity was determined in vitro with human gingival fibroblast cells (HGF) and in vivo using the Galleria mellonella model. Formulation showed antimicrobial activity, with MIC on C. albicans 15.6 and E. faecalis 7.8 µg/ml. Treatment with formulation in concentration 156 µg/ml significantly reduced mono or dual species biofilm formation and viability (p < 0.05). The results were significant against C. albicans and E. faecalis and did not show toxicity in cells and G. mellonella. In general, the formulation showed effective antibiofilm activity, significantly reducing microorganisms, opening paths in search of new endodontic irrigants.

Natural Compounds as Non-Nucleoside Inhibitors of Zika Virus Polymerase through Integration of In Silico and In Vitro Approaches.

Although the past epidemic of Zika virus (ZIKV) resulted in severe neurological consequences for infected infants and adults, there are still no approved drugs to treat ZIKV infection. In this study, we applied computational approaches to screen an in-house database of 77 natural and semi-synthetic compounds against ZIKV NS5 RNA-dependent RNA-polymerase (NS5 RdRp), an essential protein for viral RNA elongation during the replication process. For this purpose, we integrated computational approaches such as binding-site conservation, chemical space analysis and molecular docking. As a result, we prioritized nine virtual hits for experimental evaluation. Enzymatic assays confirmed that pedalitin and quercetin inhibited ZIKV NS5 RdRp with IC50 values of 4.1 and 0.5 µM, respectively. Moreover, pedalitin also displayed antiviral activity on ZIKV infection with an EC50 of 19.28 µM cell-based assays, with low toxicity in Vero cells (CC50 = 83.66 µM) and selectivity index of 4.34. These results demonstrate the potential of the natural compounds pedalitin and quercetin as candidates for structural optimization studies towards the discovery of new anti-ZIKV drug candidates.

Biological Membranes as Targets for Natural and Synthetic Compounds.

Biological membranes are responsible for all types of regulation and compound transfer, as well as information flow between and within eukaryotic and prokaryotic cells [...].

A new reduced chalcone-derivative affects the membrane permeability and electric potential of multidrug-resistant Enterococcusfaecalis.

The emergence and spread of multidrug-resistant (MDR) enterococci and other Gram-positive bacteria represents a severe problem due to the lack of effective therapeutic alternatives. Natural products have long been an important source of new antibacterial scaffolds and can play a key role in the current antibiotic crisis. Enterococci are predominantly non-pathogenic gastrointestinal commensal bacteria, but among them, Enterococcus faecalis and Enterococcus faecium represent the species that account for most clinically relevant infections. The emergence of MDR enterococci has reduced the available antibiotic treatment options and highlights the need to develop new antimicrobial compounds. In the search for new hit compounds against MDR Enterococcus spp., natural-derived compounds represent inspiring scaffolds for drug design studies. In this work, the antimicrobial activity of a fully synthetic chalcone derivative (r4MB) was determined on a clinical panel of 34 MDR Gram-positive bacteria, mostly constituted by E. faecalis and E. faecium, along with Staphylococcus spp., amongst others. Compound r4MB showed activity against 100% of the tested strains, with the minimum inhibitory concentration (MIC) in the range of 5-20 µM. The lethal action of the compound was evaluated using different fluorescent-based assays. The compound showed a time-dependent permeabilisation of the membrane of a vancomycin-resistant E. faecalis, detected by the fluorescent probe SYTOX Green, and digital fluorescent microscopy corroborated the spectrofluorimetric analysis within 6 min of incubation. Flow cytometry analysis of the membrane electric potential demonstrated a significant depolarization, confirming the target of the compound towards the bacterial membrane. No cytotoxic haemolysis was observed with mammalian erythrocytes, and a 99% cytotoxic concentration of 118 µM on NCTC cells demonstrated a promising antimicrobial selectivity. In silico studies using SwissADME and ADMETLabs servers suggest that compound r4MB displayed adequate ADME properties, with no alerts for pan-assay interference compounds (PAINS). Future hit-to-lead optimization of this chalcone derivative can contribute to developing a more potent derivative against infections caused by MDR enterococci.

Chalcones with potential antibacterial and antibiofilm activities against periodontopathogenic bacteria.

OBJECTIVES: Periodontitis is a pathology resulting from complex interaction of microorganisms in the dental biofilm with the host's immune system. Increased use of antibiotics associated with their inappropriate use has increased resistance levels in anaerobic bacteria. Therefore, identifying new antimicrobial compounds, such as chalcones, is urgent. This study evaluates the antibacterial activity and the antibiofilm activity of 15 chalcones against the periodontopathogenic bacteria Prevotella nigrescens (ATCC 33563), P. oralis (ATCC 33269), Peptostreptococcus anaerobius (ATCC 27337), Actinomyces viscosus (ATCC 43146), Porphyromonas asaccharolytica (ATCC 25260), and Fusobacterium nucleatum (ATCC 25586). METHODS: The compounds were evaluated by minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) tests. RESULTS: Compounds 1-6 showed good antibacterial and antibiofilm activities against most of the evaluated bacteria: MIC was lower than or equal to 6.25 µg/mL, biofilm biomass was reduced by 95%, and the compounds at concentrations between 0.78 and 100 µg/mL totally inhibited cell viability. Among the tested chalcones, 3 stood out: it was effective against all the bacteria, as revealed by the MIC and MBIC results. CONCLUSIONS: Our results have consolidated a base for the development of new studies on the effects of the tested chalcones as agents to combat and to prevent periodontitis.

Antibacterial Activity of Isobavachalcone (IBC) Is Associated with Membrane Disruption.

Isobavachalcone (IBC) is a natural prenylated chalcone with a broad spectrum of pharmacological properties. In this work, we newly synthesized and investigated the antibacterial activity of IBC against Gram-positive, Gram-negative and mycobacterial species. IBC was active against Gram-positive bacteria, mainly against Methicillin-Susceptible Staphylococcus aureus (MSSA) and Methicillin-Resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) values of 1.56 and 3.12 µg/mL, respectively. On the other hand, IBC was not able to act against Gram-negative species (MIC > 400 µg/mL). IBC displayed activity against mycobacterial species (MIC = 64 µg/mL), including Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii. IBC was able to inhibit more than 50% of MSSA and MRSA biofilm formation at 0.78 µg/mL. Its antibiofilm activity was similar to vancomycin, which was active at 0.74 µg/mL. In order to study the mechanism of the action by fluorescence microscopy, the propidium iodide (PI) and SYTO9 fluorophores indicated that IBC disrupted the membrane of Bacillus subtilis. Toxicity assays using human keratinocytes (HaCaT cell line) showed that IBC did not have the capacity to reduce the cell viability. These results suggested that IBC is a promising antibacterial agent with an elucidated mode of action and potential applications as an antibacterial drug and a medical device coating.

Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro.

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

Antibiofilm and cytotoxic effect of 3,3'-dihydroxycurcumin (DHC) as photosensitizer agent in antimicrobial photodynamic therapy for endodontic purposes.

BACKGROUND: Curcuminoids have been designed not only to improve chemical and metabolic stability of curcumin (CUR), but also to increase its antimicrobial activity, without effecting its ability as photosensitizer agent in antimicrobial photodynamic therapy (aPDT) with light emitting diode (LED). This study evaluated the antimicrobial and antibiofilm action of curcumin analog DHC (or 3,3'-dihydroxycurcumin), submitted or not to LED irradiation, on microorganisms of endodontic importance and its influence on fibroblasts viability. METHODS: DHC was synthetized by modified Pablon's methodology and the experiments were conducted under irradiation or not with indium gallium nitride-based LED (440-480nm, 100 mW/cm2, 0.78 cm2,60 s). The antimicrobial activity of CUR and DHC were determined by the Minimum Inhibitory and Bactericidal Concentration assays against Gram-positive and Gram-negative bacteria and the effect of both compounds on fibroblast viability was tested using colorimetric assays. They were also evaluated on 72h and 7days single-species biofilms and on 14 days multispecies biofilms formed inside dentin tubules by bacterial colonies counts and confocal microscopy, respectively. Data were analyzed statistically considering p<0.05. RESULTS: DHC had bactericidal effect against all bacteria tested higher than CUR, in planktonic conditions. CUR and DHC (at 39 and 19 µg/mL, respectively) were cytocompatible and LED irradiation reduced fibroblast viability, regardless of compound. CUR and DHC reduced the growth of single-species biofilms and the effect of aPDT was bacteria dependent. DHC reduced more than 70% of microorganisms from multispecies biofilms, superior to CUR effect. CONCLUSIONS: DHC showed low cytotoxicity and antibiofilm effect similar to curcumin, when submitted or not to aPDT, and could be further explored as a bioactive compound for endodontic purposes.

Development, Characterization and Cell Viability Inhibition of PVA Spheres Loaded with Doxorubicin and 4'-Amino-1-Naphthyl-Chalcone (D14) for Osteosarcoma.

Chalcones (1,3-diaryl-2-propen-1-ones) are naturally occurring polyphenols with known anticancer activity against a variety of tumor cell lines, including osteosarcoma (OS). In this paper, we present the preparation and characterization of spheres (~2 mm) from polyvinyl alcohol (PVA) containing a combination of 4'-Amino-1-Naphthyl-Chalcone (D14) and doxorubicin, to act as a new polymeric dual-drug anticancer delivery. D14 is a potent inhibitor of osteosarcoma progression and, when combined with doxorubicin, presents a synergetic effect; hence, physically crosslinked PVA spheres loaded with D14 and doxorubicin were prepared using liquid nitrogen and six freeze-thawing cycles. Physical-chemical characterization using a scanning electron microscope (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) presented that the drugs were incorporated into the spheres via weak interactions between the drugs and the polymeric chains, resulting in overall good drug stability. The cytotoxicity activity of the PVA spheres co-encapsulating both drugs was tested against the U2OS human osteosarcoma cell line by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay, and compared to the spheres carrying either D14 or doxorubicin alone. The co-delivery showed a cytotoxic effect 2.6-fold greater than doxorubicin alone, revealing a significant synergistic effect with a coefficient of drug interaction (CDI) of 0.49. The obtained results suggest this developed PVA sphere as a potential dual-drug delivery system that could be used for the prominent synergistic anticancer activity of co-delivering D14 and doxorubicin, providing a new potential strategy for improved osteosarcoma treatment.

Cytotoxicity and effects of curcumin and cinnamaldehyde hybrids on biofilms of oral pathogens.

The objective of the study was to evaluate the cytotoxicity and effect of curcumin-cinnamaldehyde hybrids (CCHs) on the biofilm of oral pathogens. Of the 18 hybrids tested, nine had an inhibitory effect on at least one of the bacterial species tested, with minimal inhibitory and bactericidal concentrations ranging from 9 to 625 µg ml-1. CCH 7 promoted a potent inhibitory effect against all the bacterial species tested and better compatibility than chlorhexidine (CHX). CCH 7 also presented a similar or improved effect over that of CHX, causing a reduction in bacterial metabolism and viability in single and dual-species biofilms. CCH 7 reduced by 86% and 34% the viability of multispecies biofilms formed by collection and clinical strains. It can be concluded that CCH 7 was cytocompatible at the minimal inhibitory concentration, presented anti-biofilm action against oral pathogens, and could act as an antimicrobial agent for application in endodontics.

2-Hydroxychalcone as a Potent Compound and Photosensitizer Against Dermatophyte Biofilms.

Dermatophytes, fungi that cause dermatophytosis, can invade keratinized tissues in humans and animals. The biofilm-forming ability of these fungi was described recently, and it may be correlated with the long treatment period and common recurrences of this mycosis. In this study, we evaluated the anti-dermatophytic and anti-biofilm activity of 2-hydroxychalcone (2-chalcone) in the dark and photodynamic therapy (PDT)-mediated and to determine its mechanism of action. Trichophyton rubrum and Trichophyton mentagrophytes strains were used in the study. The antifungal susceptibility test of planktonic cells, early-stage biofilms, and mature biofilms were performed using colorimetric methods. Topographies were visualized by scanning electron microscopy (SEM). Human skin keratinocyte (HaCat) monolayers were also used in the cytotoxicity assays. The mechanisms of action of 2-chalcone in the dark and under photoexcitation were investigated using confocal microscopy and the quantification of ergosterol, reactive oxygen species (ROS), and death induction by apoptosis/necrosis. All strains, in the planktonic form, were inhibited after treatment with 2-chalcone (minimum inhibitory concentration (MIC) = 7.8-15.6 mg/L), terbinafine (TRB) (MIC = 0.008-0.03 mg/L), and fluconazole (FLZ) (1-512 mg/L). Early-stage biofilm and mature biofilms were inhibited by 2-chalcone at concentrations of 15.6 mg/L and 31.2 mg/L in all tested strains. However, mature biofilms were resistant to all the antifungal drugs tested. When planktonic cells and biofilms (early-stage and mature) were treated with 2-chalcone-mediated PDT, the inhibitory concentrations were reduced by four times (2-7.8 mg/L). SEM images of biofilms treated with 2-chalcone showed cell wall collapse, resulting from a probable extravasation of cytoplasmic content. The toxicity of 2-chalcone in HaCat cells showed higher IC50 values in the dark than under photoexcitation. Further, 2-chalcone targets ergosterol in the cell and promotes the generation of ROS, resulting in cell death by apoptosis and necrosis. Overall, 2-chalcone-mediated PDT is a promising and safe drug candidate against dermatophytes, particularly in anti-biofilm treatment.

Chalcones and their B-aryl analogues as myeloperoxidase inhibitors: In silico, in vitro and ex vivo investigations.

In the present study, a series of chalcones and their B-aryl analogues were prepared and evaluate as inhibitors of myeloperoxidase (MPO) chlorinating activity, using in vitro and ex vivo assays. Among these, B-thiophenyl chalcone (analogue 9) demonstrated inhibition of in vitro and ex vivo MPO chlorinating activity, exhibiting IC50 value of 0.53 and 19.2 µM, respectively. Potent ex vivo MPO inhibitors 5, 8 and 9 were not toxic to human neutrophils at 50 µM, as well as displayed weak 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH•) and hypochlorous acid (HOCl) scavenger abilities. Docking simulations indicated binding mode of MPO inhibitors, evidencing hydrogen bonds between the amino group at 4'position (ring A) of chalcones with Gln91, Asp94, and Hys95 MPO residues. In this regard, the efficacy and low toxicity promoted aminochalcones and arylic analogues to the rank of hit compounds in the search for new non-steroidal anti-inflammatory compounds.

Flavonoids from Pterogyne nitens as Zika virus NS2B-NS3 protease inhibitors.

Although the widespread epidemic of Zika virus (ZIKV) and its neurological complications are well-known there are still no approved drugs available to treat this arboviral disease or vaccine to prevent the infection. Flavonoids from Pterogyne nitens have already demonstrated anti-flavivirus activity, although their target is unknown. In this study, we virtually screened an in-house database of 150 natural and semi-synthetic compounds against ZIKV NS2B-NS3 protease (NS2B-NS3p) using docking-based virtual screening, as part of the OpenZika project. As a result, we prioritized three flavonoids from P. nitens, quercetin, rutin and pedalitin, for experimental evaluation. We also used machine learning models, built with Assay Central® software, for predicting the activity and toxicity of these flavonoids. Biophysical and enzymatic assays generally agreed with the in silico predictions, confirming that the flavonoids inhibited ZIKV protease. The most promising hit, pedalitin, inhibited ZIKV NS2B-NS3p with an IC50 of 5 µM. In cell-based assays, pedalitin displayed significant activity at 250 and 500 µM, with slight toxicity in Vero cells. The results presented here demonstrate the potential of pedalitin as a candidate for hit-to-lead (H2L) optimization studies towards the discovery of antiviral drug candidates to treat ZIKV infections.

Chalcone T4, a novel chalconic compound, inhibits inflammatory bone resorption in vivo and suppresses osteoclastogenesis in vitro.

OBJECTIVE: This study aimed to assess the effect of a novel synthetic chalcone, Chalcone T4, on a murine model of periodontitis and on RANKL-induced osteoclastogenesis in vitro. BACKGROUND: Chalcones are natural compounds with anti-inflammatory properties, and its synthetic analogs with enhanced biological effects have potential as therapeutic agents. Periodontitis is characterized by chronic inflammation of the periodontium and alveolar bone resorption. Safe and effective anti-inflammatory agents can have an important additive effect in the treatment in this disease. METHODS: Periodontitis was induced via the installation of a ligature around the first molar. Rats (n = 32) received Chalcone T4 (5 and 50 mg/kg) or distilled water by gavage daily for 15 days. Outcomes assessed were bone resorption (µCT), TNF-α production (ELISA), cellular infiltrate, and collagen content (stereometric analysis, CD45+ cells by immunohistochemistry), and activation of NFATc1 and NF-kB (immunohistochemistry). In vitro, RAW 264.7 were treated with Chalcone T4 and stimulated with RANKL for assessment of osteoclast differentiation (actin ring staining) and activity (pit assay). RESULTS: Chalcone T4 significantly reduced periodontitis-associated bone resorption, as well as the cellular infiltrate, while increasing the collagen content. Production of TNF-α, infiltration of CD45-positive cells, and NF-kB activation were markedly reduced. In vitro, chalcone T4 inhibited both osteoclast differentiation and activity. CONCLUSION: Chalcone T4 significantly inhibited alveolar bone resorption and inflammation in vivo and RANKL-induced osteoclastogenesis in vitro, suggesting a therapeutic role for this compound in the treatment of periodontitis.

Incorporation of Nonyl 3,4-Dihydroxybenzoate Into Nanostructured Lipid Systems: Effective Alternative for Maintaining Anti-Dermatophytic and Antibiofilm Activities and Reducing Toxicity at High Concentrations.

Dermatophytosis is the most common mycosis worldwide, affecting approximately 20 to 25% of the population, regardless of gender, race, color, and age. Most antifungal agents used for the treatment of dermatophytosis belong to the azole and allylamine classes. Dermatophytes are reported to be resistant to most commercial drugs, especially microbial biofilms, in addition to their considerable toxicity. It should be emphasized the importance of looking for new molecules with reduced toxicity, as well as new targets and mechanisms of action. This work aims to incorporate nonyl 3,4-dihydroxybenzoate, a potent fungicide compound against planktonic cells and dermatophyte biofilms in nanostructured lipid systems (NLS), in order to reduce toxicity in high concentrations, improve its solubility and maintain its effectiveness. The compound was incorporated into NLS constituted by cholesterol, mixture of polyoxyethylene (23) lauryl ether (Brij®98) and soybean phosphatidylcholine (Epikuron® 200)], 2: 1 ratio and PBS (phosphate-buffered saline). The characterization of the incorporation was performed. Susceptibility tests were conducted according to document M38-A2 by CLSI (2008). The toxicity of the NLS compound was evaluated in HaCaT cell lines by the sulforhodamine B method and in alternative models Caenorhabditis elegans and zebrafish. Finally, its efficacy was evaluated against the mature Trichophyton rubrum and Trichophyton mentagrophytes biofilms. NLS and nonyl 3,4-dihydroxybenzoate loaded into NLS displayed sizes ranging from 137.8 ± 1.815 to 167.9 ± 4.070 nm; the polydispersity index (PDI) varying from 0.331 ± 0.020 to 0.377 ± 0.004 and zeta potential ranging from -1.46 ± 0.157 to -4.63 ± 0.398 mV, respectively. Polarized light microscopy results confirmed the formation of NLS of the microemulsion type. Nonyl incorporated into NLS showed minimum inhibitory concentration (MIC) values, ranging from 2 to 15.6 mg/L. The toxicity tests presented cell viability higher than 80% in all tested concentrations, as well as, a significantly increased of the survival of Caenorhabditis elegans and zebrafish models. Anti-biofilm tests proved the efficacy of the incorporation. These findings contribute significantly to the search for new antifungals and allow the systemic administration of the compound, since the incorporation can increase the solubility of non-polar compounds, improve bioavailability, effectiveness and reduce toxicity.