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In the present study, we have described the synthesis and characterisation of the theophylline hydrate (Theo hydrate), cocrystal (Theo–Phen·2H2O) and hydrated sodium co-crystal of theophylline (Na–(Theo)2ClO·2H2O), where Theo = theophylline and Phen = 1,10-phenathroline. SC-XRD characterisation revealed strong interactions such as N⋯H⋯O, CO⋯H and C–H⋯π within the crystal structures. Studies revealed that Na–(Theo)2ClO·2H2O presented higher thermal stability (in the solid form) and improved solubility (when in contact with a polar solvent). The detailed analyses of Hirshfeld surface and fingerprint plots provided insight into the nature of non-covalent interactions in the compounds. In the case of Na–(Theo)2ClO·2H2O, solubility measurements indicated that Theo's solubility in water increases by a magnitude of four with respect to pure Theo. Biological studies showed that the high solubility of Theo as well as ClO4− is responsible for the distinct capabilities to inhibit the growth of different microorganisms, including Gram-positive (Staphylococcus epidermidis) and Gram-negative (Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae) bacteria and fungi (Candida albicans and Candida tropicalis). Our results pointed out that cocrystals containing theophylline–phenanthroline can be explored to get promising molecules to be used to combat infections caused by clinically relevant (multi)drug-resistant bacteria and fungi.

The chemical composition and biological properties of citronella essential oil were modified by enzymatic esterification reaction of the major monoterpenic alcohols with cinnamic acid. The almost complete conversion of geraniol and citronellol present in the citronella (Cymbopogon winterianus) essential oil, into geranyl (99%) and citronellyl (98%) cinnamates was obtained after 48 hours of reaction using a molar ratio of 3:1 (cinnamic acid/alcohol), lipase concentration (Novozym 435) of 15% (w/w) and 70 °C. The esterified oil showed higher antimicrobial activity against Staphylococcus aureus bacteria resistant to oxacillin and penicillin and also greater larvicidal activity against Aedes aegypti larvae compared to unesterified oil. The results concerning the evaluation of toxicity against Artemia salina and cytotoxicity against monkey kidney epithelial cells also showed the superiority of the esterified oil.

Multi-walled carbon nanotubes functionalized with naringenin have been developed as new drug carriers to improve the performance of lung cancer treatment. The nanocarrier was characterized by Transmission Electron Microscopy (TEM), Fourier-Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy, Raman Spectroscopy, and Differential Scanning Calorimetry (DSC). Drug release rates were determined in vitro by the dialysis method. The cytotoxic profile was evaluated using the MTT assay, against a human skin cell line (hFB) as a model for normal cells, and against an adenocarcinomic human alveolar basal epithelial (A569) cell line as a lung cancer in vitro model. The results demonstrated that the functionalization of carbon nanotubes with naringenin occurred by non-covalent interactions. The release profiles demonstrated a pH-responsive behavior, showing a prolonged release in the tumor pH environment. The naringenin-functionalized carbon nanotubes showed lower cytotoxicity on non-malignant cells (hFB) than free naringenin, with an improved anticancer effect on malignant lung cells (A549) as an in vitro model of lung cancer., This work was supported by the Banco do Nordeste (grant FUNDECI/2016.0015), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (Fapitec) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Eliana B. Souto would like to acknowledge the contributions from the Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) for the projects M-ERA-NET/0004/2015-PAIRED and UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

Background: The objective of this work has been the development of silver nanoparticles (AgNPs) coated with humic acid (HA) and their incorporation into an enamel for antifungal activity. AgNPs were synthesized by chemical reduction using sodium borohydride (NaBH4) as a reducing agent and coated with HA. Uncoated AgNPs were synthesized as control. AgNPs and HAAgNPs were characterized by ultravioletvisible spectrophotometry (UVVis), differential scanning calorimetry (DSC), dynamic light scattering (DLS), Fouriertransform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and Xray diffraction (XRD). Size of AgNPs and HAAgNPs was recorded within the nanorange, showing HAAgNPs higher stability than noncoated particles by presenting a single plasmatic band around 400 nm. Results Thermal analysis showed conjugated endothermic peaks, which confirms the compatibility HAAgNPs. FTIR depicted absorptions between 1300 and 1000 cm1 (C = C, Ar H, respectively) demonstrating that HA is adsorbed onto AgNPs. TGA showed that HA does not alter the reduction in mass loss of AgNPs, while it was found by XRD that adding HA promoted the formation of more amorphous AgNPs. The effectiveness of HAAgNPs was evaluated against three different fungal species. Minimum inhibitory concentration (MIC) assays showed that ca. 0.5mM of AgNPs were able to inhibit dermatophyte species growth. HAAgNPs were incorporated into a commercial enamel at a concentration of 8% and their organoleptic characteristics, drying time, centrifugation test and thermal stress, were evaluated. Enamels with AgNPs kept their physicochemical properties over 21days of storage. Conclusion HAAgNPs nail polish is thus proposed as an innovative material for onychomycosis infections., This research was financed by the Coordenação Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Sergipe (FAPITEC) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). EBS wishes to acknowledge the sponsorship of the project UIDB/04469/2020 (strategic fund) from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, co-financed by FEDER, under Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

Leishmaniasis is one of the most relevant neglected tropical diseases in the world, affecting 14 million people. Despite the high morbidity, mortality and socio-economic impact, few therapeutic options are available for this disease. To make matters worse, the available molecules have several limitations such as limited efficacy, high cost, side effects and increased resistance. In this context, our group previously synthesized new compounds with anti-leishmania potential being the bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 (complex 4) the most promising one. Therefore, this present work revealed some morphological and physiological changes promoted by complex 4 on Leishmania amazonensis promastigotes as well as it was evidenced its potential against intramacrophage amastigotes. Complex 4 promoted a progressive reduction on the promastigotes size and a remarkable increase on the granularity/complexity as judged by flow cytometry. Transmission electron microscopy (TEM) analysis revealed extensive mitochondrial and plasma membrane alterations, although plasma membrane integrity remained. The mitochondrial changes observed by TEM were accompanied by a decrease in the activity of mitochondrial dehydrogenases with increased production of reactive oxygen species. Interestingly, promastigotes also showed changes in lipid metabolism. Besides the very low toxicity to macrophages, complex 4 had a great effect on intramacrophage amastigotes, displaying an IC50 of 3.91 μM. Collectively, the data presented here reinforce the potential of aminopyridyl compounds complexed to zinc against L. amazonensis. Thus, our work serves as a basis for in vivo assays to be designed or even the synthesis of more selective/effective compounds with lower cost.

Amphotericin B deoxycholate (AmB) or Fungizone is a well-known antifungal drug belonging to the polyene class, which was originally isolated from Streptomyces nodosus. Interestingly, AmB also presents antiparasitic properties, including anti-Leishmania action. Thinking into the drug repurposing strategy, AmB has been administered as an alternative treatment for leishmaniasis since the 1960s due to the increasing parasite resistance to the classical antimonials (e.g., glucantime), presenting good clinical outcomes. Different mechanisms of action have already been described for AmB, such as membrane sterol binding, ergosterol sequestration, and production of reactive oxygen species. However, AmB also able to interact with cholesterol in human plasma membranes, promoting a number of frequent and serious side effects, which are limiting factors in the administration of this drug. The use of AmB in clinical settings is related to different side effects such as vomiting, nephrotoxicity, nausea, and anemia, which often result in treatment failures. In order to reduce the adverse side effects and to increase effectiveness, the field of AmB nanotechnology-based controlled release systems has been widely studied and explored over the last years. Nanoparticles are polymeric colloidal particles that can encapsulate a wide variety of drugs with different physicochemical characteristics, and they have an efficient system of control and targeted drug delivery to infected cells, for example. Studies on the development of AmB nanoparticles with anti-Leishmania action indicate that these formulations have reduced toxicity rates and increased therapeutic efficacy. Therefore, this review aims to present the recent developments of different AmB carrier nanoparticles and their effectiveness as an anti-Leishmania agent.

Reaction of benzohydroxamic acid (Bha), 2-pyridinehydroxamic acid (2-pyha), 2-amino-phenylhydroxamic acid (2-NH2-pha) and salicylhydroxamic acid (Sha) with SbCl3 in ethanol gave the corresponding novel hydroxamato Sb(iii) complexes, [Sb(Bha-1H)2Cl] 1, [SbCl2(2-pyha-1H)] 2, [Sb(2-NH2-pha-1H)(2-NH3-pha-1H)]Cl23 and [SbCl(Sha-1H)2] 4. In all cases the hydroxamic acids coordinate to the Sb centres in the typical bidentate hydroxamato (O,O') coordination mode, via the carbonyl oxygen and deprotonated hydroxyl group. Reaction of the histone deacetylase inhibitor (HDACi) suberoylanilidehydroxamic acid (SAHA) with Sb(OEt)3 gave the Sb(iii) hydroxamato/hydroximato complex, [Sb(SAHA-1H)(SAHA-2H)] 6. All test complexes significantly inhibited the promastigote proliferation of Leishmania amazonensis and L. chagasi and induced substantial changes in the general morphology of the parasites, including reduction in size and loss of flagellum, when compared to the untreated promastigotes. A dose-response approach using the test complexes showed a decreased in plasma membrane permeability and the mitochondrial dehydrogenase activities of the Leishmania species. [Sb(Bha-1H)2Cl] exhibited the best activity and was superior to the Sb HDACi complex 6. Though 1 exhibited noteworthy anti-leishmanial activity, the selectivity indexes determined suggest that [Sb(2-NH2-pha-1H)(2-NH3-pha-1H)]Cl23 is the test complex that merits further investigation as a potential anti-leishmanial agent.

• Some aspects of T. cruzi epimastigote's physiology and biochemistry are demonstrated.

Since the discovery of the28 first drugs used in leishmaniasis treatment up to now, the search for compounds with anti-Leishmania activity without toxic effects and able to overcome the emergency of resistant strains remains a major goal to combat this neglected disease. With this in mind, in the present work, we evaluated the effects of the calpain inhibitor MDL28170 on the interaction process of Leishmania amazonensis promastigote forms with murine peritoneal macrophages and on the intracellular amastigotes. Our results showed that the calpain inhibitor MDL28170 at 15 and 30μM significantly reduced the interaction process of promastigotes with macrophages by 16% and 41%, respectively. The inhibitor was also able to drastically reduce the number of infected macrophages in a time- and dose-dependent manner: after only 24h, MDL28170 was able to significantly diminish the infection rate, presenting an IC50 value of 18.2μM for amastigotes. The treatment with MDL28170 did not alter the nitric oxide production, but the production of TNF-α was significantly raised. Altogether, the results presented here contribute to the search of new proteolytic inhibitors able to act in a selective and effective manner against the diseases caused by trypanosomatids.

The introduction of the HIV aspartic peptidase inhibitors (HIV-PIs) has revolutionized the medical arena, since they have drastically reduced the number and the severity of opportunistic infections, including the protozoal diseases that afflict the HIV-infected individuals worldwide. HIV-PIs rapidly and profoundly diminish the viral load, which is paralleled by increase in the CD4+ T lymphocyte counts and stimulation of the survival and activation of neutrophil, monocyte and endothelial cells, culminating in a vigorous reduction in the number of deaths due to the AIDS, in the number of new cases of AIDS and in the number of hospitalization days. Many research groups around the globe are trying to decipher both the in vitro and in vivo antiprotozoal mechanisms behind the use of HIVPIs. These studies have been supported by the urgent need to discover novel active compounds able to treat incurable parasitoses, including three major neglected diseases: malaria, leishmaniasis and Chagas’ disease. The present review summarizes the recent advances on the effects of HIV-PIs against Plasmodium spp., Leishmania spp. and Trypanosoma cruzi.

Trypanosoma cruzi and Leishmania amazonensis are the causative agents of Chagas' disease and leishmaniasis, respectively. These conditions affect millions of people worldwide, especially in developing countries. As such, there is an urgent need for novel, efficient and cost-effective treatments for these diseases, given the growing resistance and side-effects of current therapies. This work details the synthesis and evaluation of the anti-parasitic activity of novel amino- and iminopyridyl metal chelators, their glycosylated derivatives and some of their metal complexes. Our results revealed the potent and metal-dependent activity for the aminopyridyl compounds: Cu(ii) complexes were most effective against T. cruzi trypomastigotes, while Zn(ii) complexes presented excellent activity against L. amazonensis promastigotes. In addition, the compounds showed excellent selectivity indexes and very low relative toxicity as judged by in vitro and in vivo studies, respectively, using RAW macrophages and Galleria mellonella larvae model.

Acai seed extracts are reported for containing polyphenols with pharmacological properties against inflammation, hypertension, and metabolic syndrome. However, presently, those seeds accumulate as waste, causing environmental problems. Therefore, we aimed to characterize the polyphenol profile of acai seed extracts to investigate its composition and biological effects. Multiple techniques were used for the identification of the extract's metabolites, such as HPLC, mass spectrometry, and mass spectrometry imaging (MALDI-IMS). The extract was majorly composed of B-type and A-type oligomeric procyanidins with a mean degree of polymerization of 11.4 (>3000 Da), formed by catechin and epicatechin as starter and extension subunits, respectively. MALDI-IMS confirmed, for the first time, the presence of A-type procyanidins in the seed's tegument. The extract was not cytotoxic to THP1 (macrophage cell line) and LLC-MK2 (epithelial cell) and, moreover, it efficiently protected macrophages from oxidative stress, which correlated to its high antioxidant capacity, confirmed by DPPH, TEAC and ORAC methods. Additionally, the extract presented antimicrobial activity against gram-positive bacterial strains and Candida albicans. These data indicate the potential use of seed extracts for cosmetic and pharmaceutical formulations.

Biosurfactants have potential applications in the remediation of petroleum-contaminated sites. Several strategies can be used to reduce the production costs of these surfactants and make the process more environmentally friendly. In this study, we combined some of these strategies to produce the rhamnolipid-type biosurfactant, including the use of the genetically modified strain Pseudomonas aeruginosa-estA, an industrial coproduct as a carbon source, a simple and low-cost medium, and a simple downstream process. The process resulted in a high yield (17.6 g L−1), even using crude glycerin as the carbon source, with substrate in product conversion factor (YRML/s) of 0.444. The cell-free supernatant (CFS) was not toxic to Artemia salina and selected mammalian cell lineages, suggesting that it can be used directly in the environment without further purification steps. Qualitative analysis showed that CFS has excellent dispersion in the oil-displacement test, emulsifying (IE24 = 65.5 % ), and tensoactive properties. When salinity, temperature and pressure were set to seawater conditions, the values for interfacial tension between crude oil and water were below 1.0 mN m−1. Taken together, these results demonstrate that it is possible to obtain a nontoxic crude rhamnolipid product, with high productivity, to replace petroleum-based surfactants in oil spill cleanups and other environmental applications.

Hydrazide ligand, (Z)-N'-(6-oxo-1,10-phenanthrolin-5(6H)-ylidene)isonicotinohydrazide, 1 forms from a 1:1 Schiff base condensation reaction between isoniazid (INH) and 1,10-phenanthroline-5,6-dione (phendione). Ag+ and Mn2+ complexes with 1:2 metal:ligand stoichiometry are prepared: [Ag(1)2]NO3, [Ag(1)2]BF4 and [Mn(1)2](NO3)2. Polymeric {[Ag(1)(NO3)]}n has 1:1 stoichiometry and forms upon infusion of CH2Cl2 into a DMSO solution of [Ag(1)2]NO3. {[Ag(1)(NO3)]}n was structurally characterized using X-ray crystallography. Metal-free 1 and its 1:2 complexes exhibit very good, broad-spectrum antimicrobial activity and are not excessively toxic to mammalian cells (A549 lineage).

BACKGROUND Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms. OBJECTIVES/METHODS The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy. FINDINGS After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy. MAIN CONCLUSIONS The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.

Objective The low investment in research, diagnosis and treatment are factors that contribute to the continuity of Chagas’ disease as a neglected tropical diseases (NTDs). In this context, the repositioning of drugs represents a useful strategy, in the search for new chemotherapeutic approaches for NTDs. HIV aspartic peptidase inhibitors (HIV IPs) are good candidates for drug repurposing. Here, we modeled the three dimensional structure of an aspartyl peptidase of Trypanosoma cruzi, the causative agent of Chagas’ disease, aligned it to the HIV aspartyl peptidase and performed docking binding assays with the HIV PIs. Results The 3D structure confirmed the presence of acid aspartic residues, which are critical to enzyme activity. The docking experiment revealed that HIV IPs bind to the active site of the enzyme, being ritonavir and lopinavir the ones with greater affinity. Benznidazole presented the worst binding affinity, this drug is currently used in Chagas’ disease treatment and was included as negative control. These results together with previous data on the trypanocidal effect of the HIV PIs support the hypothesis that a T. cruzi aspartyl peptidase can be the intracellular target of these inhibitors. However, the direct demonstration of the inhibition of T. cruzi aspartyl peptidase activity by HIV PIs is still a goal to be persuaded. Electronic supplementary material The online version of this article (10.1186/s13104-018-3927-z) contains supplementary material, which is available to authorized users.

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Chagas' disease is one of the most impactful and prevalent neglected tropical diseases in the Americas, specially affecting the poor and underdeveloped areas in Latin America. Aggravating this scenario, the medicines used in the current chemotherapy are old, toxic and present a low efficacy to treat the chronic stage of this disease. In addition, resistant strains of Trypanosoma cruzi, the etiological agent, are frequently reported. So, there is an imperative requirement for novel chemotherapeutic options to treat this debilitating disease. In this context, peptidases have emerged as potential targets and, consequently, proteolytic inhibitors have confirmed to be valuable drugs against several human pathologies. In this line of thinking, T. cruzi produces a major multifunctional cysteine peptidase, named cruzipain, which directly and/or indirectly orchestrates several physiological and pathological processes, which culminate in a successful parasitic infection. Taken together, these findings point out that cruzipain is one of the most important targets for driving a chemotherapy approach against the human pathogen T. cruzi. The present review summarizes some of the recent advances and failures in this area, with particular emphasis on recently published studies.

Flavonoids are one of the most important and diversified phenolic groups among products of natural origin. An important property of this metabolite class is the antioxidant action. This study evaluated the antioxidant and cytotoxic activities and oxidative stress of transesterification products of the flavonoid rutin, catalyzed by Novozym® 435. The presence of monoacetate and diacetate was confirmed by quantitative evaluation of the retention times (rutin, 15.68 min; rutin monoacetate, 18.14 min; and rutin diacetate, 18.57 min) and by the data from LC-MS and NMR 1H and 13C. The experiment showed excellent conversion values of 96% in total acetates (rutin monoacetate and diacetate). These results confirmed that rutin derivatives have antioxidant potential, as evaluated by the ORAC method (rutin standard: 0.53 ± 0.08 μM Trolox/g and rutin derivatives: 2.33 ± 1.08 μM Trolox/g) and also show low cytotoxicity in human and animal cells. Rutin derivatives reduced the production of reactive oxygen species in RAW macrophages as well. Many qualities attributed to rutin derivatives make them promising potential candidates for use as nutraceuticals, including their high amounts of antioxidants, biological potential and low toxicity, which contribute to the reduction of oxidative stress.

The development of HIV aspartyl peptidase inhibitors (HIV-PIs) and their introduction into AIDS therapy preceded a significant decrease in the incidence, morbidity and mortality of relevant protozoan co-infections. However, few data are available about how HIV-PIs act on pathogenic parasites, such as Trypanosoma cruzi, the etiological agent of Chagas disease. Therefore, the aim of the present work was to evaluate different physiological aspects of the treatment of the infective trypomastigote forms of T. cruzi with the HIV-PIs, nelfinavir and lopinavir. At the LD50/4 h doses, both HIV-PIs significantly reduced the trypomastigote size and markedly increased the granularity/complexity. Transmission electron microscopy analysis associated to biochemical assays permitted definition of the main HIV-PIs targets in the parasite. Lopinavir and nelfinavir induced (i) plasma membrane shedding, particularly in the flagellar region, which drastically affected parasite integrity; (ii) strong mitochondrial swelling with rare matrix fragmentation, which were linked to severely reduced hydrolytic activity of dehydrogenases and organelle membrane depolarization; (iii) increased generation of reactive oxygen species (ROS); (iv) dilation of both nuclear envelope (without DNA disruption) and endoplasmic reticulum (with formation of autophagosomes), and (v) accumulation of intracellular lipid droplets, revealing a typical lipid metabolism disorder. Collectively, our study demonstrated that nelfinavir and lopinavir target vital cellular structures of trypomastigotes, culminating in irreversible metabolic injuries that lead to T. cruzi death.

Aspartyl-type peptidases are promising chemotherapeutic targets in protozoan parasites. In the present work, we identified an aspartyl peptidase activity from the soluble extract ofLeishmania amazonensispromastigotes, which cleaved the fluorogenic peptide 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D-Arg-amide (cathepsin D substrate) under acidic pH conditions at 37°C, showing aKMof 0.58 μM andVmaxof 129.87 fluorescence arbitrary units/s mg protein. The leishmanial aspartyl peptidase activity was blocked by pepstatin A (IC50= 6.8 μM) and diazo-acetyl-norleucinemetilester (IC50= 10.2 μM), two classical aspartyl peptidase inhibitors. Subsequently, the effects of 6 asymmetric peptidomimetics, containingL-tartaric acid core, were tested on both aspartyl peptidase and growth ofL.amazonensispromastigotes. The peptidomimetics named 88, 154 and 158 promoted a reduction of 50% on the leishmanial aspartyl peptidase activity at concentrations ranging from 40 to 85 μM, whereas the peptidomimetic 157 was by far the most effective, presenting IC50of 0.04 μM. Furthermore, the peptidomimetics 157 and 154 reduced the parasite proliferation in a dose-dependent manner, displaying IC50values of 33.7 and 44.5 μM, respectively. Collectively, the peptidomimetic 157 was the most efficient compound able to arrest both aspartyl peptidase activity and leishmanial proliferation, which raises excellent perspectives regarding its use against this human pathogenic protozoan.

The current treatments for leishmaniasis bump into several obstacles, including low efficacy, high costs, long monitoring, and several/severe side effects. Consequently, the search for promising compounds is a tangible need. Recently, we reported the anti-Leishmania amazonensis action of asymmetric peptidomimetic compounds containing tartaric acid as core, especially the 157 derivative that contains valine/leucine substituents in its structure. Herein, we decipher the multiple effects of 157 on the L. amazonensis physiology and on the interaction process with macrophages. The peptidomimetic 157 induced significant changes on the morphometric (internal granularity reduction as judged by flow cytometer) and on the ultrastructural (round-shaped parasites, presence of plasma membrane blebs and flagellum loss as visualized by scanning electron microscopy) aspects of treated promastigotes compared to untreated ones. The alteration on the plasma membrane permeability was confirmed by the passive incorporation of propidium iodide in 157-treated promastigotes. In parallel, the low viability of promastigotes was also associated to the perturbation of mitochondrial transmembrane electric potential. These combined results demonstrated that 157 induced irreversible metabolic damages that led to L. amazonensis death. The pre-treatment of promastigotes with 157 inhibited the association index with macrophages in a typically dose-dependent manner. Additionally, 157 significantly reduced the number of intramacrophage amastigotes after 72 h of drug contact, presenting an IC50 value of 30.2 μM. Under our experimental conditions, 157 showed higher toxicity to promastigotes and amastigotes when compared to RAW cells, resulting in good selective indexes. Therefore, 157 can be considered as an interesting candidate for further optimization, since its synthesis is simple and cheap.

There is an urgent need to implement new strategies and to search for new chemotherapeutic targets to combat Chagas' disease. In this context, repositioning of clinically approved drugs appears as a viable tool to combat this and several other neglected pathologies. An example is the use of aspartic peptidase inhibitors (PIs) currently applied in human immunodeficiency virus (HIV) treatment against different infectious agents. Therefore, the main objective of this work was to verify the effects of the HIV-PIs nelfinavir and lopinavir against Trypanosoma cruzi using in vitro models of infection. Cytotoxicity assays with LLC-MK2 epithelial cells and RAW macrophages allowed an evaluation of the effects of HIV-PIs on the interaction between trypomastigotes and these cells as well as the survival of intracellular amastigotes. Pre-treatment of trypomastigotes with nelfinavir and lopinavir inhibited the association index with LLC-MK2 cells and RAW macrophages in a dose- and time-dependent manner. In addition, nelfinavir and lopinavir also significantly reduced the number of intracellular amastigotes in both mammalian cell lineages, particularly when administered in daily doses. Both compounds had no effect on nitric oxide production in infected RAW macrophages. These results open the possibility for the use of HIV-PIs as a tangible alternative in the treatment of Chagas' disease. However, the main mechanism of action of nelfinavir and lopinavir has yet to be elucidated, and more studies using in vivo models must be conducted.

SUMMARYIn this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms ofTrypanosoma cruziisolate Dm28c. MDL28170 at 70 μMpromoted a powerful reduction in the growth rate after 48 h. The IC50value was calculated to be 31·7 μM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect.T. cruziCALPs presented a strong cross-reactivity with anti-Drosophila melanogastercalpain and anti-cytoskeleton-associated protein fromTrypanosoma bruceiantibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TCI), Dm28c (TCII) and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

There is a general lack of effective and non-toxic chemotherapeutic agents against Chagas' disease despite more than a century of research. In this regard, we have verified the impact of human immunodeficiency virus aspartic peptidase inhibitors (HIV-PIs) on the viability and morphology of infective trypomastigote forms of Trypanosoma cruzi as well as on the aspartic peptidase and proteasome activities produced by this parasite. The effects of HIV-PIs on viability were assessed by counting motile parasites in a Neubauer chamber. Morphological alterations were detected by light microscopy of Giemsa-stained smears and scanning electron microscopy. Modulation of aspartic peptidase and proteasome activities by the HIV-PIs was measured by cleavage of fluorogenic peptide substrates. The majority of the HIV-PIs (6/9) were able to drastically decrease the viability of trypomastigotes after 4 h of treatment, with nelfinavir and lopinavir being the most effective compounds presenting LD50 values of 8.6 µM and 10.6 µM, respectively. Additionally, both HIV-PIs were demonstrated to be effective in a time- and cell density-dependent manner. Treatment with nelfinavir and lopinavir caused many morphological/ultrastructural alterations in trypomastigotes; parasites became round in shape, with reduced cell size and flagellar shortening. Nelfinavir and lopinavir were also capable of significantly inhibiting the aspartic peptidase and proteasome activities measured in trypomastigote extracts. These results strengthen the data on the positive effects of HIV-PIs on parasitic infections, possibly by targeting the parasite aspartic peptidase(s) and proteasome(s), opening a new possibility for the use of these clinically approved drugs as an alternative chemotherapy to treat Chagas' disease.

Background: Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas’ disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas’ disease. Methodology and Principal Findings: HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 mM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIVPIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pretreatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages. Conclusions and Significance: The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.

The treatment for both leishmaniasis and trypanosomiasis, which are severe human infections caused by trypanosomatids belonging to Leishmania and Trypanosoma genera, respectively, is extremely limited because of concerns of toxicity and efficacy with the available anti-protozoan drugs, as well as the emergence of drug resistance. Consequently, the urgency for the discovery of new trypanosomatid targets and novel bioactive compounds is particularly necessary. In this context, the investigation of changes in parasite gene expression between drug resistant/sensitive strains and in the up-regulation of virulence-related genes in infective forms has brought to the fore the involvement of calpain-like proteins in several crucial pathophysiological processes performed by trypanosomatids. These studies were encouraged by the publication of the complete genome sequences of three human pathogenic trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which allowed in silico analyses that in turn directed the identification of numerous genes with interesting chemotherapeutic characteristics, including a large family of calpain-related proteins, in which to date 23 genes were assigned as calpains in T. brucei, 40 in T. cruzi and 33 in L. braziliensis. In the present review, we intend to add to these biochemical/biological reports the investigations performed upon the inhibitory capability of calpain inhibitors against human pathogenic trypanosomatids.

Infections caused by resistant microorganisms often fail to respond to conventional therapy, resulting in prolonged illness, increased treatment costs and greater risk of death. Consequently, the development of novel antimicrobial drugs is becoming more demanding every day since the existing drugs either have too many side-effects or they tend to lose effectiveness due to the selection of resistant strains. In view of these facts, a number of new strategies to obstruct vital biological processes of a microbial cell have emerged; one of these is focused on the use of metal-chelating agents, which are able to selectively disturb the essential metal metabolism of the microorganism by interfering with metal acquisition and bioavailability for crucial reactions. The chelation activity is able to inhibit the biological role of metal-dependent proteins (e.g., metalloproteases and transcription factors), disturbing the microbial cell homeostasis and culminating in the blockage of microbial nutrition, growth and development, cellular differentiation, adhesion to biotic (e.g., extracellular matrix components, cell and/or tissue) and abiotic (e.g., plastic, silicone and acrylic) structures as well as controlling the in vivo infection progression. Interestingly, chelating agents also potentiate the activity of classical antimicrobial compounds. The differences between the microorganism and host in terms of the behavior displayed in the presence of chelating agents could provide exploitable targets for the development of an effective chemotherapy for these diseases. Consequently, metal chelators represent a novel group of antimicrobial agents with potential therapeutic applications. This review will focus on the anti-fungal and anti-protozoan action of the most common chelating agents, deciphering and discussing their mode of action.