In vitro evaluation of the selective cytotoxicity and genotoxicity of three synthetic ortho-nitrobenzyl derivatives in human cancer cell lines, with and without metabolic activation.

Although the presence of nitro groups in chemicals can be recognized as structural alerts for mutagenicity and carcinogenicity, nitroaromatic compounds have attracted considerable interest as a class of agents that can serve as source of potential new anticancer agents. In the present study, the in vitro cytotoxicity, genotoxicity, and mutagenicity of three synthetic ortho-nitrobenzyl derivatives (named ON-1, ON-2 and ON-3) were evaluated by employing human breast and ovarian cancer cell lines. A series of biological assays was carried out with and without metabolic activation. Complementarily, computational predictions of the pharmacokinetic properties and druglikeness of the compounds were performed in the Swiss ADME platform. The MTT assay showed that the compounds selectively affected selectively the cell viability of cancer cells in comparison with a nontumoral cell line. Additionally, the metabolic activation enhanced cytotoxicity, and the compounds affected cell survival, as demonstrated by the clonogenic assay. The comet assay, the cytokinesis-block micronucleus assay, and the immunofluorescence of the γ-H2AX foci formation assay have that the compounds caused chromosomal damage to the cancer cells, with and without metabolic activation. The results obtained in the present study showed that the compounds assessed were genotoxic and mutagenic, inducing double-strand breaks in the DNA structure. The high selectivity indices observed for the compounds ON-2 and ON-3, especially after metabolic activation with the S9 fraction, must be highlighted. These experimental biological results, as well as the theoretical properties predicted for the compounds have shown that they are promising anticancer candidates to be exploited in additional studies.

Toxicological effects of hydroxychloroquine sulfate and chloroquine diphosphate substances on the early-life stages of fish in the COVID-19 pandemic context.

Hydroxychloroquine sulfate (HCQ) and chloroquine diphosphate (CQ) have been used at increased rates to treat COVID-19 but can constitute a potential environmental risk. The objective was to evaluate the toxicity of sublethal concentrations of HCQ and CQ in zebrafish embryos/larvae. The 50% lethal concentrations (LC50) of HCQ and CQ at 96 h post-fertilization (hpf) were calculated by testing various concentrations on 2,160 embryos. The LC50 obtained were 560 and 800 µM for HCQ and CQ, respectively. Next, the embryotoxicity assay was performed, where 1,200 embryos were subjected to sublethal concentrations of HCQ and CQ. The hatching and heart rates were recorded. After euthanasia, photomicrographs of all larvae were taken to measure the total length, pericardial and yolk sac areas. The embryos exposed to sublethal concentrations of HCQ and CQ showed delayed hatching at 72 hpf, as well as an increase in the heart rate, larger pericardial and yolk sac areas, and body malformations at 96 hpf. The findings show that HCQ and CQ are toxic to fish in the early development phases. Understanding the mechanisms of toxicity will help extrapolate the effects of 4-aminoquinoline derivatives when they reach the aquatic environment in the context of the COVID-19 pandemic.

Antileishmanial activity evaluation of thiazolidine-2,4-dione against Leishmania infantum and Leishmania braziliensis.

Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC50 = 23.45 ± 4.54 µM), whereas 2a is the most potent against L. braziliensis (EC50 = 44.16 ± 5.77 µM). This result suggests that lipophilic substituents on either-meta and/or-para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE50 = 49.22 ± 7.71 µM) presented the highest selectivity index.

Brazilian malaria molecular targets (BraMMT): selected receptors for virtual high-throughput screening experiments.

BACKGROUND: Owing to increased spending on pharmaceuticals since 2010, discussions about rising costs for the development of new medical technologies have been focused on the pharmaceutical industry. Computational techniques have been developed to reduce costs associated with new drug development. Among these techniques, virtual high-throughput screening (vHTS) can contribute to the drug discovery process by providing tools to search for new drugs with the ability to bind a specific molecular target. OBJECTIVES: In this context, Brazilian malaria molecular targets (BraMMT) was generated to execute vHTS experiments on selected molecular targets of Plasmodium falciparum. METHODS: In this study, 35 molecular targets of P. falciparum were built and evaluated against known antimalarial compounds. FINDINGS: As a result, it could predict the correct molecular target of market drugs, such as artemisinin. In addition, our findings suggested a new pharmacological mechanism for quinine, which includes inhibition of falcipain-II and a potential new antimalarial candidate, clioquinol. MAIN CONCLUSIONS: The BraMMT is available to perform vHTS experiments using OCTOPUS or Raccoon software to improve the search for new antimalarial compounds. It can be retrieved from www.drugdiscovery.com.br or download of Supplementary data.

Larvicidal activity of vegetable oils and esterified compounds against Culex quinquefasciatus (Diptera: Culicidae).

Control of Culex quinquefasciatus using chemical insecticides may result in the selection of resistant mosquito strains. Thus, the use of plant-derived products has been studied as alternative for the mosquito control. Fatty acid methyl esters (FAMEs) obtained by transesterification of vegetable oils may result in compounds with larvicidal potential against C. quinquefasciatus. However, little is known about the morphological, physiological or biochemical effects that these FAMEs may have on mosquito larvae. The present study reports the effects of these FAMEs in mosquito larvae. The FAMEs were obtained by transesterification of canola, corn, sunflower, and soybean oils with acid catalysis and the determination of FAMEs composition was done by gas chromatography-mass spectrometry (GC-MS). Larvae of C. quinquefasciatus were exposed to different concentrations of the vegetable oils and FAMEs. Thereby, different FAMEs showed LC50 values ranging from 42.32 to 196.27mg/L against C. quinquefasciatus larvae. The methyl ester obtained from sunflower oil showed the lowest LC50. Histology of C. quinquefasciatus larvae exposed to LC50 of FAMEs was performed and changes in the midgut and fat body morphology were identified. Therefore, larval mortality and changes in the internal organs suggested that FAMEs might be a promising new class of larvicidalcompounds. Cytotoxicity of FAMEs compounds was assessed with the HeLa human cell line and no effect was observed.

The Effect of Melatonin Adsorbed to Polyethylene Glycol Microspheres on the Survival of MCF-7 Cells.

Although melatonin exhibits oncostatic properties such as antiproliferative effects, the oral bioavailability of this hormone is less than 20%. Modified drug release systems have been used to improve the pharmacological efficiency of drugs. These systems can change the pharmacokinetics and biodistribution of the associated drugs. Thus, this study investigated the effect of melatonin adsorbed to polyethylene glycol (PEG) microspheres on MCF-7 human breast cancer cells. The MCF-7 cells were obtained from the American Type Culture Collection. MCF-7 cells were preincubated for 24 h with or without melatonin (100 ng/ml), PEG microspheres or melatonin adsorbed to PEG microspheres (100 ng/ml). Viability, intracellular calcium release and apoptosis in MCF-7 cells were determined by flow cytometry. MCF-7 cells incubated with melatonin adsorbed to PEG microspheres showed a lower viability rate (40.0 ± 8.3 with melatonin adsorbed to PEG microspheres compared to 54.1 ± 7.3 with melatonin; 81.8 ± 12.5 with PEG microsphere and 92.7 ± 4.1 with medium), increased spontaneous intracellular Ca2+ release (27.0 ± 8.6 with melatonin adsorbed to PEG microspheres compared to 21.5 ± 13.4 with melatonin; 10.1 ± 5.4 with PEG microsphere and 9.1 ± 5.6 with medium) and increased apoptosis index (51.2 ± 2.7 with melatonin adsorbed to PEG microspheres compared to 36.0 ± 2.1 with melatonin; 4.9 ± 0.5 with PEG microsphere and 3.1 ± 0.6 with medium). The results indicate that melatonin adsorbed to PEG microspheres exerts antitumor effects on human MCF-7 breast cancer cells. However, clinical tests must be performed to confirm the use of melatonin adsorbed to PEG microspheres as an alternative therapy against cancer.

Revealing the Binding Process of New 3-Alkylpyridine Marine Alkaloid Analogue Antimalarials and the Heme Group: An Experimental and Theoretical Investigation.

Synthetic 3-alkylpyridine marine alkaloid (3-APA) analogues have shown good antimalarial activity against Plasmodium falciparum. However, despite their structural originality, their molecular target was unknown. Herein, we report a proposal for the antimalarial mechanism of action of 3-APA analogues through interference with the process of hemozoin (Hz) formation. The interaction between 3-APA analogues and heme groups was investigated employing an in silico approach and biophysical techniques such as ultraviolet-visible light (UV-vis) titration and electrospray ionization-mass spectrometry (ESI-MS). The in silico approach was performed based on pure ab initio electronic structure methods in order to obtain insights at the molecular level concerning the binding process of antimalarial drugs at their target site, the heme group. In silico results showed that the formation of heme:3-APA complexes at a molecular ratio of 2:1 are more stable than 1:1 complexes. These results were further confirmed by experimental techniques, such as UV-vis and high-resolution mass spectrometry (ESI-TOF), for two of the most active 3-APA analogues.

Successful application of virtual screening and molecular dynamics simulations against antimalarial molecular targets.

The main challenge in the control of malaria has been the emergence of drug-resistant parasites. The presence of drug-resistant Plasmodium sp. has raised the need for new antimalarial drugs. Molecular modelling techniques have been used as tools to develop new drugs. In this study, we employed virtual screening of a pyrazol derivative (Tx001) against four malaria targets: plasmepsin-IV, plasmepsin-II, falcipain-II, and PfATP6. The receiver operating characteristic curves and area under the curve (AUC) were established for each molecular target. The AUC values obtained for plasmepsin-IV, plasmepsin-II, and falcipain-II were 0.64, 0.92, and 0.94, respectively. All docking simulations were carried out using AutoDock Vina software. The ligand Tx001 exhibited a better interaction with PfATP6 than with the reference compound (-12.2 versus -6.8 Kcal/mol). The Tx001-PfATP6 complex was submitted to molecular dynamics simulations in vacuum implemented on an NAMD program. The ligand Tx001 docked at the same binding site as thapsigargin, which is a natural inhibitor of PfATP6. Compound TX001 was evaluated in vitro with a P. falciparum strain (W2) and a human cell line (WI-26VA4). Tx001 was discovered to be active against P. falciparum (IC50 = 8.2 µM) and inactive against WI-26VA4 (IC50 > 200 µM). Further ligand optimisation cycles generated new prospects for docking and biological assays.

New quinoline derivatives demonstrate a promising antimalarial activity against Plasmodium falciparum in vitro and Plasmodium berghei in vivo.

Malaria continues to be an important public health problem in the world. Nowadays, the widespread parasite resistance to many drugs used in antimalarial therapy has made the effective treatment of cases and control of the disease a constant challenge. Therefore, the discovery of new molecules with good antimalarial activity and tolerance to human use can be really important in the further treatment of the disease. In this study we have investigated the antiplasmodial activity of 10 synthetic compounds derived from quinoline, five of them combined to sulfonamide and five to the hydrazine or hydrazide group. The compounds were evaluated according to their cytotoxicity against HepG2 and HeLa cell lines, their antimalarial activity against CQ-sensitive and CQ-resistant Plasmodium falciparum strains and, finally, their schizonticide blood action in mice infected with Plasmodium berghei NK65. The compounds exhibited no cytotoxic action in HepG2 and HeLa cell lines when tested up to a concentration of 100 µg/mL. In addition, the hydrazine or hydrazide derivative compounds were less cytotoxic against cell lines and more active against CQ-sensitive and CQ-resistant P. falciparum strains, showing high SI (>1000 when SI was calculated using the CC50 from the 3D7 strain as reference). When tested in vivo, the hydrazine derivative 1f compound showed activity against the development of blood parasites similar to that observed with CQ, the reference drug. Interestingly, the 1f compound demonstrated the best LipE value (4.84) among all those tested in vivo. Considering the in vitro and in vivo activities of the compounds studied here and the LipE values, we believe the 1f compound to be the most promising molecule for further studies in antimalarial chemotherapy.

γ-Benzylidene digoxin derivatives synthesis and molecular modeling: Evaluation of anticancer and the Na,K-ATPase activity effect.

Cardiotonic steroids (CS), natural compounds with traditional use in cardiology, have been recently suggested to exert potent anticancer effects. However, the repertoire of molecules with Na,K-ATPase activity and anticancer properties is limited. This paper describes the synthesis of 6 new digoxin derivatives substituted (on the C17-butenolide) with γ-benzylidene group and their cytotoxic effect on human fibroblast (WI-26 VA4) and cancer (HeLa and RKO) cell lines as well as their effect on Na,K-ATPase activity and expression. As digoxin, compound BD-4 was almost 100-fold more potent than the other derivatives for cytotoxicity with the three types of cells used and was also the only one able to fully inhibit the Na,K-ATPase of HeLa cells after 24h treatment. No change in the Na,K-ATPase α1 isoform protein expression was detected. On the other hand it was 30-40 fold less potent for direct Na,K-ATPase inhibition, when compared to the most potent derivatives, BD-1 and BD-3, and digoxin. The data presented here demonstrated that the anticancer effect of digoxin derivatives substituted with γ-benzylidene were not related with their inhibition of Na,K-ATPase activity or alteration of its expression, suggesting that this classical molecular mechanism of CS is not involved in the cytotoxic effect of our derivatives.

Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca²âº-ATPase PfATP6.

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Synthesis and biological evaluation of novel 3-alkylpyridine marine alkaloid analogs with promising anticancer activity.

Cancer continues to be one of the most important health problems worldwide, and the identification of novel drugs and treatments to address this disease is urgent. During recent years, marine organisms have proven to be a promising source of new compounds with action against tumoral cell lines. Here, we describe the synthesis and anticancer activity of eight new 3-alkylpyridine alkaloid (3-APA) analogs in four steps and with good yields. The key step for the synthesis of these compounds is a Williamson etherification under phase-transfer conditions. We investigated the influence of the length of the alkyl chain attached to position 3 of the pyridine ring on the cytotoxicity of these compounds. Biological assays demonstrated that compounds with an alkyl chain of ten carbon atoms (4c and 5c) were the most active against two tumoral cell lines: RKO-AS-45-1 and HeLa. Micronucleus and TUNEL assays showed that both compounds are mutagenic and induce apoptosis. In addition, Compound 5c altered the cellular actin cytoskeleton in RKO-AS-45-1 cells. The results suggest that Compounds 4c and 5c may be novel prototype anticancer agents.

Towards development of new antimalarial compounds through in silico and in vitro assays.

Malaria is one of most widespread infectious disease in world. The antimalarial therapy presents a series of limitations, such as toxicity and the emergence of resistance, which makes the search for new drugs urgent. Thus, it becomes necessary to explore essential and exclusive therapeutic targets of the parasite to achieve selective inhibition. Enoyl-ACP reductase is an enzyme of the type II fatty acid biosynthetic pathway and is responsible for the rate-limiting step in the fatty acid elongation cycle. In this work, we use hierarchical virtual screening and drug repositioning strategies to prioritize compounds for phenotypic assays and molecular dynamics studies. The molecules were tested against chloroquine-resistant W2 strain of Plasmodium falciparum (EC50 between 330.05 and 13.92 µM). Nitrofurantoin was the best antimalarial activity at low micromolar range (EC50 = 13.92 µM). However, a hit compound against malaria must have a biological activity value below 1 µM. A large number of molecules present problems with permeability in biological membranes and reaching an effective concentration in their target's microenvironment. Nitrofurantoin derivatives with inclusions of groups which confer increased lipid solubility (methyl groups, halogens and substituted and unsubstituted aromatic rings) have been proposed. These derivatives were pulled through the lipid bilayer in molecular dynamics simulations. Molecules 14, 18 and 21 presented lower free energy values than nitrofurantoin when crossing the lipid bilayer.

Synthesis and cytotoxic evaluation of heterocyclic compounds by vinylic substitution of ketene dithioacetals.

N-heterocyclic compounds are important molecular scaffolds in the search for new drugs, since most drugs contain heterocyclic moieties in their molecular structure, and some of these classes of heterocycles are able to provide ligands for two or more biological targets. Ketene dithioacetals are important building blocks in organic synthesis and are widely used in the synthesis of N-heterocyclic compounds. In this work, we used double vinylic substitution reactions on ketene dithioacetals to synthesize a small library of heterocyclic derivatives and evaluated their cytotoxic activity in breast and ovarian cancer cells, identifying two benzoxazoles with good potency and selectivity. In silico predictions indicate that the two most active derivatives exhibit physicochemical properties within the range of drug-like compounds and showed potential to interact with HDAC8 and ERK1 cancer-related targets.

Antimalarial activity of 4-metoxychalcones: docking studies as falcipain/plasmepsin inhibitors, ADMET and lipophilic efficiency analysis to identify a putative oral lead candidate.

Herein, we report the antimalarial activity of nine 4-methoxychalcone derivatives 1a-i and an initial analysis of their ADMET properties. All compounds showed potent activity against the P. falciparum chloroquine-resistant clone W2, with IC50 values ranging from 1.96 µM to 10.99 µM, with moderate or low cytotoxicity against the HeLa cell line. The compound 1a (IC50 = 2.06 µM) had the best selectivity index (9.0). All the sulfonamide 4-metychalcone derivatives synthesized had cLogP values between 2 and 5 (mean value 3.79) and molecular weights (MWs) below 500. The substitution of the pyrrolidine group in 1i by a morpholine group in 1a reduced the cLogP value from 3.05 in compound 1i to 2.34 in compound 1a. Indeed, compound 1a had the highest LipE value. The binding free energy of compound 1a showed it to be the most optimal chalcone derivative for plasmepsin-2 (-7.3 Kcal/mol). The physicochemical properties and LipE analysis of the dataset allowed us to establish that compound 1a is the highest quality compound of the series and a potential oral lead candidate.

Antiplasmodial Activity of Hydroalcoholic Extract from Jucá (Libidibia ferrea) Pods.

Malaria is an infectious and parasitic disease caused by protozoa of the genus Plasmodium, which affects millions of people in tropical and subtropical areas. Recently, there have been multiple reports of drug resistance in Plasmodium populations, leading to the search for potential new active compounds against the parasite. Thus, we aimed to evaluate the in vitro antiplasmodial activity and cytotoxicity of the hydroalcoholic extract of Jucá (Libidibia ferrea) in serial concentrations. Jucá was used in the form of a freeze-dried hydroalcoholic extract. For the cytotoxicity assay, the(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method with the WI-26VA4 human cell line was used. For the antiplasmodial activity, Plasmodium falciparum synchronized cultures were treated with serial concentrations (0.2 to 50 µg/mL) of the Jucá extract. In terms of the chemical composition of the Jucá extract, gas chromatography coupled to mass spectrometry measurements revealed the main compounds as ellagic acid, valoneic acid dilactone, gallotannin, and gallic acid. The Jucá hydroalcoholic extract did not show cytotoxic activity per MTT, with an IC50 value greater than 100 µg/mL. Regarding the antiplasmodial activity, the Jucá extract presented an IC50 of 11.10 µg/mL with a selective index of nine. Because of its antiplasmodial activity at the tested concentrations and low toxicity, the Jucá extract is presented as a candidate for herbal medicine in the treatment of malaria. To the best of our knowledge, this is the first report of antiplasmodial activity in Jucá.

Antiplasmodial activity of coumarins isolated from Polygala boliviensis: in vitro and in silico studies.

Polygala boliviensis is found in the Brazilian semiarid region. This specie is little chemically and biologically studied. Polygala spp. have different metabolites, especially coumarins. Studies indicate that coumarins have antimalarial potential, denoting the importance of researching new active compounds from plants, since the resistance of Plasmodium strains to conventional therapy has increased. The present study aimed to evaluate the antiplasmodial activity of auraptene and poligalen against a chloroquine-resistant strain of Plasmodium falciparum. Coumarins were isolated from P. boliviensis by open column chromatography and identified by Nuclear Magnetic Resonance Spectroscopy. A cytotoxicity assay was carried out using MTT test, and the in vitro antiplasmodial activity was evaluated using the W2 strain. The antiplasmodial activity results found were IC50=0.171 ± 0.016 for auraptene and 0.164 ± 0.012 for poligalen; the selectivity indexes were 78.71 and 609.76, respectively. Inverse virtual screening in the BRAMMT database by OCTOPUS 1.2 was applied to coumarins to find potential P. falciparum targets and showed higher affinity energy of auraptene for purine nucleoside phosphorylase (PfPNP) and of poligalen for dihydroorotate dehydrogenase (PfDHODH). Molecular Dynamics studies (MD and MM-GBSA) approach were applied to calculate binding energies against selected P. falciparum targets and showed that all coumarins were stable at the binding site during simulations. Furthermore, energies were favorable for complexation. This is the first report of auraptene in P. boliviensis species and of in vitro antiplasmodial activity of auraptene and poligalen. In silico studies indicated that the mechanism of action of coumarins is the inhibition of PfPNP and PfDHODH.Communicated by Ramaswamy H. Sarma.

Design and synthesis of new chacones substituted with azide/triazole groups and analysis of their cytotoxicity towards HeLa cells.

A series of new chalcones substituted with azide/triazole groups were designed and synthesized, and their cytotoxic activity was evaluated in vitro against the HeLa cell line. O-Alkylation, Claisen-Schmidt condensation and Cu(I)-catalyzed cycloaddition of azides with terminal alkynes were applied in key steps. Fifteen compounds were tested against HeLa cells. Compound 8c was the most active molecule, with an IC50 value of 13.03 µM, similar to the value of cisplatin (7.37 µM).

COVID-19 pandemic: multilevel dental technical guidelines based on new scientific evidence.

The COVID-19 pandemic imposed restrictive measures on dentistry in different regions of the world, ranging from stoppage of care to only permission for urgent and emergency dental services. Thus, new biosafety guidelines for resuming activities, whether in single dental offices, large clinics or dental education activities, are urgently required. In this sense, herein, guidelines that incorporate common points of the main protocols found in the literature for the resumption of dental activities at their different levels, whether in the scope of care or education, are presented. Furthermore, we present the incorporation of measures that allow an increase in the level of biosafety, such as the control of the dental team, the inclusion in the history of conjunctivitis as a possible alert for COVID-19, and the use of the pulse oximeter to assess the risk of silent hypoxemia, which may indicate a complication of COVID-19. In addition, new perspectives for directing research and innovation for biosafety in dentistry are discussed.