Intracellular Forms Research Articles

Microwave‐assisted synthesis of 2‐styrylquinoline‐4‐carboxylic acid derivatives to improve the toxic effect against Leishmania (Leishmania) amazonensis

The identification of new compounds is urgent to develop safe and efficacious candidates for leishmaniasis treatment, especially from natural products as a potential source of active molecules against neglected tropical parasite diseases. Inspired by the efficacious quinoline alkaloid microbial effects, we have previously reported the synthesis and biological activity of 2‐phenylquinoline‐4‐carboxylic acids and poly‐substituted quinolines against parasites. In this work, a series of eighteen 2‐styryl‐4‐quinolinecarboxylic acids were synthesized under microwave irradiation settings obtaining from good to excellent yields (60%‐90%), shorter reaction times (2 minutes), and eco‐friendly experimental conditions. All these products were evaluated against infective forms of Leishmania (Leishmania) amazonensis, such as promastigotes and intracellular amastigotes, based on cytotoxicity assays, including host macrophage infection assays. Compounds 4 and 5 possessing a 2‐chloro or 4‐chlorostyryl moiety, respectively, were considered the most promising antileishmanial agents due to the parasite killing effect in intracellular forms inside infected macrophages. Thus, our results revealed that the 2‐styryl‐4‐quinolinecarboxylic acid backbone structure was essential for the activity against intracellular pathogens like L. (L.) amazonensis.

Cell-Wall-Deficient Bacteria in Oral Biofilm: Association with Periodontitis

Cell-wall-deficient bacteria are those that lack cell walls and live in a pleomorphic state. The genus Mycoplasma and L-form bacteria are both members of this group. The aim of this study was to search cellwall-deficient bacteria in periodontal biofilm and link their presence to periodontal disease. Eighty-nine individuals were recruited and divided into three groups: periodontally healthy individuals, individuals with chronic periodontitis, and those with aggressive periodontitis. The presence of cell-wall-deficient bacteria was detected in freshly collected biofilm by light microscopy, transmission electron microscopy (TEM) with and without electron microscopy in situ hybridization, atomic force microscopy and DNA stain (Hoechst). A new dichotomic index of classification for prevalence and morphologic variants was developed to classify cell-wall-deficient bacteria in periodontal biofilm. Cell-wall-deficient bacteria were found in periodontal biofilm and classified into protoplastic, everted, filament and intracellular forms, the last one mainly associated with aggressive periodontitis. We also assessed the prevalence of periodontopathic bacteria by means of polymerase chain-reaction (PCR) and found no clear, statistically significant, correlation among periodontal pathogens tested (except T. denticola) that allowed individuals with chronic periodontitis to be distinguished from those with aggressive periodontitis. Association between cell-walldeficient bacteria and periodontal condition was: periodontally healthy, 3.3% (1/30); individuals with chronic periodontitis, 30.6% (11/36); and those with aggressive periodontitis, 100% (23/23). Cell-walldeficient bacteria were detected in periodontal biofilm and linked to aggressive periodontitis.

Development and cathodic iontophoretic permeation evaluation of liquid crystalline systems provided of retinoic acid microparticles.

The intracellular form of action of retinoids requires these agents to reach deeper layers of the skin with viable cells to ensure therapeutic efficiency. However, studies on swine skin models show that some retinoids have low skin permeability. Thus, the association of innovative formulations with technological strategies involving physical permeation promoters can be employed to increase the permeability of this active, ensuring a targeting effect of the active. In this study, it was proposed the development and evaluation of the release and permeation profile of liquid crystalline systems with retinoic acid polymeric microparticles under passive or iontophoretic diffusional conditions. For this study, release, permeation, and diffusional characterization assays were employed using the Franz diffusion cell model, associating or not the cathodic iontophoresis. Retinoic acid was considered stable in front of the electric current of 0.5mA/cm2 , because it did not show significant degradation (with maintenance from 96.03% to 98.57%), indicating the viability of such agents to be applied with iontophoresis. Controlled release profile was evidenced for microencapsulated systems. Comparatively, formulations F1, F3, and F5 presented a significantly higher RA release profile when compared to formulations with retinoic acid microencapsulated. A significant increase was observed in the absolute amount of RA retained in the skin with cathodic iontophoresis in all proposed formulations (P<.01). The increase was up to two times in relation to the passive condition. The combination of iontophoresis technique with application of retinoic acid and microencapsulated retinoic acid allows the penetration of the active ingredient to deeper layers of the skin.

In Vitro and In Vivo Evaluation of an Adamantyl-Based Phenyl Sulfonyl Acetamide against Cutaneous Leishmaniasis Models of Leishmania amazonensis.

Phenotypic assay against Leishmania amazonensisin vitro and in vivo led to identification of an adamantyl-based phenyl sulfonyl acetamide (compound 1) as a promising antileishmanial agent. Compound 1 inhibited the growth of intracellular forms of L. amazonensis (50% inhibitory concentration [IC50] = 4 μM) and exhibited low toxicity to host cells, with a selectivity index (SI) of >125. However, in a cutaneous leishmaniasis (CL) mouse model, compound 1 did not reduce lesions and parasite load when administered as monotherapy or when given simultaneously with a suboptimal dose of miltefosine.

Visfatin: A Possible Role in Cardiovasculo-Metabolic Disorders.

Visfatin/NAMPT (nicotinamide phosphoribosyltransferase) is an adipocytokine with several intriguing properties. It was first identified as pre-B-cell colony-enhancing factor but turned out to possess enzymatic functions in nicotinamide adenine dinucleotide biosynthesis, with ubiquitous expression in skeletal muscles, liver, cardiomyocytes, and brain cells. Visfatin exists in an intracellular (iNAMPT) and extracellular (eNAMPT) form. Intracellularly, visfatin/iNAMPT plays a regulatory role in NAD+ biosynthesis and thereby affects many NAD-dependent proteins such as sirtuins, PARPs, MARTs and CD38/157. Extracellularly, visfatin is associated with many hormone-like signaling pathways and activates some intracellular signaling cascades. Importantly, eNAMPT has been associated with several metabolic disorders including obesity and type 1 and 2 diabetes. In this review, a brief overview about visfatin is presented with special emphasis on its relevance to metabolic diseases. Visfatin/NAMPT appears to be a unique molecule with clinical significance with a prospective promising diagnostic, prognostic, and therapeutic applications in many cardiovasculo-metabolic disorders.

Evaluating the Effect of Cinnarizine on Promastigotes and Amastigotes forms of Leishmania major.

In this experimental study, four concentrations of the drug (5, 10, 15 and 20 μg/ml) were added to Leishmania major cultures at 24, 48 and 72 hours intervals. MTT assays were performed to determine parasite viability and drug toxicity. Leishmania major promastigotes were augmented to the in vitro cultured macrophages (J774 cells) and then incubated for 72 hours. Half maximal inhibitory concentration (IC50) was ascertained by counting parasites. The inhibitory effect of the drug was compared with that of Glucantime. Flow-cytometry was performed to investigate apoptosis. Each test was repeated thrice. The IC50 values of Cinnarizine after 72 hours were calculated to be 34.76 μg/ml and 23.73 μg/ml for promastigotes and amastigotes, respectively. The results of MTT assays showed 48 % promastigote viability after 72 hour-exposure to Cinnarizine at 20 μg/ml concentration. Programmed cell death in promastigote- and amastigote-infected macrophages was quantified to be 13.66 % and 98.7 %, respectively. Flow- cytometry analysis indicated that Cinnarizine induced early and late apoptosis in parasites. All treatments produced results which differed significantly from control group (P<0.05). Cinnarizine showed low toxicity with anti-leishmanial and apoptosis effects on both promastigote and intracellular amastigote forms. Therefore, we may suggest further assessment on animal models of this drug as candidates for cutaneous leishmaniasis therapy.

Antiprotozoal Activity of Xanthone Derivatives

A series of xanthone derivatives containing different side chains, including ω-bromo and ω-aminoalkoxylxanthones (with linear alkoxy chains of 3, 4 and 5 carbon atoms and methyl, propyl, tert -butylamino and piperidinyl moieties), synthesized from the natural xanthone lichexanthone were tested for their antiprotozoal activities against Leishamania braziliensis , L. major and Trypanosoma cruzi (extra and intracellular forms). The ω-aminoalkoxylxanthones showed good antileishmanial activity, with IC 50 ranging from 62.8 to 0.1 µM for promastigotes and 119.3 to 2.4 µM for amastigotes. In general, compounds with longer alkyl chains and tert -butylamino moiety showed better activity. The cytotoxicity on VERO cells was also described for some derivatives. Compound 15 ( tert -butylaminobutyloxy side chain) was the most active on promastigote forms (IC 50 0.1 µM), compound 16 ( tert -butylaminopentyloxy side chain) sowed the best activity for amastigotes (IC 50 2.4 µM) and derivative 13 (dipropylaminopentyloxy side chain) was the most selective (selectivity index of 9.4). Regarding trypanocidal activity, 16 and other groups of derivatives, (ω-bromoalkoxylxanthones, prenyl and epoxyl side chains) had moderate to good activity on T. cruzi trypomastigotes, with IC 50 ranging from 30.6 to 4.1 µM, while only two ω-aminoalkoxylxanthones ( 10 and 11 ) were weakly active against T. cruzi amastigotes. DOI: http://dx.doi.org/10.17807/orbital.v12i3.1460

In vitro inhibitory effect of aluminum phthalocyanine tetrasulfonate chloride against Leishmania (Viannia) Peruviana and Leishmania (Viannia) Braziliensis.

To evaluate the in vitro photodynamic activity of aluminum phthalocyanine tetrasulfonate chloride (AlPcClS4) on promastigotes and amastigotes of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis. The activity of photodynamic therapy using AlPcClS4 on Leishmania promastigote and amastigotes was determined by the Methyl Thiazole Tetrazolium (MTT) colorimetric method and quantitative PCR, respectively. Photodynamic treatment showed an inhibitory effect on promastigotes, particularly on Leishmania (V.) peruviana, to a lesser extent on Leishmania (V.) braziliensis and also on intracellular forms of both species. At 24 hours post-radiation, using concentrations of 200 μM and 350 μM, the inhibitory effect on Leishmania (V.) peruviana was 72.9% and 73.9% respectively; at 96 hours the inhibitory effect was of 78.8% and 80.6%, respectively. Regarding intracellular forms, the inhibitory effect on Leishmania (V.) peruviana amastigotes was 57.8% at 72 hours post-treatment, using a concentration of 200 μM. The IC50 was 56.5, 50, 44 and 39.7 μM, at 24, 48, 72 and 96 hours post-radiation, respectively. Photodynamic therapy using AlPcClS4 against Leishmania species showed encouraging results, mainly on Leishmania (V.) peruviana, suggesting a potential use as an alternative or complement to the usual treatment of tegumentary leishmaniasis. However, new trials are still required to determine the selectivity index for the intracellular form of the parasite, and to develop methods to facilitate the efficient entry of the molecule into the host cell and the parasite.

Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3’ UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.

Molecular interactions and functions of IL-32.

IL-32 is a multifaceted cytokine associated with several diseases and inflammatory conditions. Its expression is induced in response to cellular stress such as hypoxia, infections, and pro-inflammatory cytokines. IL-32 can be secreted from cells and can induce the production of pro-inflammatory cytokines from several cell types but are also described to have anti-inflammatory functions. The intracellular form of IL-32 is shown to play an important role in various cellular processes, including the defense against intracellular bacteria and viruses and in modulation of cell metabolism. In this review, we discuss current literature on molecular interactions of IL-32 with other proteins. We also review data on the role of intracellular IL-32 as a metabolic regulator and its role in antimicrobial host defense.

Biodiverse Histoplasma Species Elicit Distinct Patterns of Pulmonary Inflammation following Sublethal Infection.

Histoplasma is an endemic dimorphic fungus that can cause disease in healthy and immunocompromised individuals after the transition of inhaled spores into the facultative intracellular yeast form. There is substantial diversity among Histoplasma species, but it is not clear how this heterogeneity impacts the progression of pathology and cellular immune responses during acute respiratory infection, which represents the vast majority of histoplasmosis disease burden. After inoculating mice intranasally with a sublethal inoculum, we characterized the immune response to Histoplasma capsulatum (strain G186A) and Histoplasma ohiense (strain G217B) using comprehensive flow cytometric and single-cell analyses. Within 8 days after inoculation, H. ohiense induced a significantly higher infiltration of neutrophils and inflammatory monocytes into the lung compared to H. capsulatum Microscopic analysis of infected lung tissue revealed that although the total number of fungi was similar within inflamed lung lesions, we observed different species-dependent intracellular yeast distribution patterns. Inoculation with gfp-expressing strains indicated that H. ohiense, but not H. capsulatum, was associated primarily with alveolar macrophages early after infection. Interestingly, we observed a significant reduction in the total number of alveolar macrophages 12 to 16 days after H. ohiense, but not H. capsulatum infection, despite similar intracellular growth dynamics within AMJ2-C11 alveolar macrophages in vitro Together, our data suggest that H. ohiense, but not H. capsulatum, preferentially interacts with alveolar macrophages early after infection, which may lead to a different course of inflammation and resolution despite similar rates of fungal clearance.IMPORTANCE Acute pulmonary histoplasmosis in healthy individuals comprises most of the disease burden caused by the fungal pathogen Histoplasma Fungal pneumonia is frequently delayed in diagnosis and treatment due to a prolonged period of quiescence early during infection. In this study, we used a murine respiratory model of histoplasmosis to investigate how different Histoplasma species modulate lung inflammation throughout the complete course of infection. We propose that a relatively low, sublethal inoculum is ideal to model acute pulmonary histoplasmosis in humans, primarily due to the quiescent stage of fungal growth that occurs in the lungs of mice prior to the initiation of inflammation. Our results reveal the unique course of lung immunity associated with divergent species of Histoplasma and imply that the progression of clinical disease is considerably more heterogeneous than previously recognized.

Preparation and characterization of a synthetic curcumin analog inclusion complex and preliminary evaluation of in vitro antileishmanial activity.

The aim of this work was to prepare and characterize inclusion complexes between a synthetic curcumin analog (dibenzalacetone, DBA) and beta-cyclodextrin (β-CD); and to evaluate their in vitro antileishmanial activity. DBA was synthetized and characterized by spectroscopic methods and the inclusion complexes were obtained by kneading and lyophilization (LIO) in 1:1 and 1:2 stoichiometries. Phase solubility and dissolution assays showed a 40-fold increase in the aqueous solubility of DBA and its complete dissolution from LIO 1:1 formulation after 120min respectively. Solid-state characterization by differential scanning calorimetry and near infrared spectroscopy demonstrated the inclusion of DBA in the β-CD cavity at the molar ratios tested, with LIO 1:1 formulation being the most stable. Using nuclear magnetic resonance experiments, the protons inside the cavity of β-CD were the most affected after the inclusion of DBA molecule. The cellular viability of THP-1 macrophage cells treated with plain DBA, β-CD and DBA/CD inclusion complexes showed that the plain DBA and DBA/CD at 1:2 stoichiometry presented toxicity, while β-CD alone and DBA/CD at 1:1 stoichiometry showed no toxicity up to 640μgmL-1. The in vitro assay with free-living promastigotes demonstrated that plain DBA and β-CD had IC50 of < 10 and > 320μgmL-1 respectively, while only inclusion complexes with 1:1 stoichiometry showed antiproliferative activity with IC50=51.3μgmL-1. Using the amastigote intracellular forms, there was also a difference between the plain and β-CD complexed DBA with complexes of 1:1 and 1:2 stoichiometry presenting EC50=66.3μgmL-1 and 58.9μgmL-1 respectively. The study concluded that DBA/CD at 1:1molar ratio has the potential to decrease the intrinsic toxicity of plain DBA towards Leishmania host cells, which may be a therapeutic advantage in the application of these compounds.

Complement receptor 3 mediates ruffle-like, actin-rich aggregates during phagocytosis of Leishmania infantum metacyclics

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes.

Effect of essential oils on Leishmania amazonensis: a systematic review.

This systematic review investigated the evidence for the therapeutic potential of essential oils (EOs) against Leishmania amazonensis. We searched available scientific publications from 2005 to 2019 in the PubMed and Web of Science electronic databases, according to PRISMA statement. The search strategy utilized descriptors and free terms. The EOs effect of 35 species of plants identified in this systematic review study, 45.7% had half of the maximal inhibitory concentration (IC50) 10 < IC50 ⩽ 50 μg mL-1 and 14.3% had a 10 < IC50μg mL-1 for promastigote forms of L. amazonensis. EOs from Cymbopogon citratus species had the lowest IC50 (1.7 μg mL-1). Among the plant species analyzed for activity against intracellular amastigote forms of L. amazonensis, 39.4% had an IC50 10 < IC50 ⩽ 50 μg mL-1, and 33.3% had an IC50 10 < IC50μg mL-1. Aloysia gratissima EO showed the lowest IC50 (0.16 μg mL-1) for intracellular amastigotes. EOs of Chenopodium ambrosioides, Copaifera martii and Carapa guianensis, administered by the oral route, were effective in reducing parasitic load and lesion volume in L. amazonensis-infected BALB/c mice. EOs of Bixa orellana and C. ambrosioides were effective when administered intraperitoneally. Most of the studies analyzed in vitro and in vivo for the risk of bias showed moderate methodological quality. These results indicate a stimulus for the development of new phytotherapy drugs for leishmaniasis treatment.

EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

Mast Cell Deficiency Protects Susceptible BALB/c Mice from Progressive Murine Cutaneous Leishmaniasis

Cutaneous leishmaniasis is a parasitic skin disease with ∼1 million new cases and about 30,000 annual deaths (Sacks and Noben-Trauth, 2002). During blood feeding, sand flies inoculate parasites into host skin where they are ingested by phagocytes such as macrophages or neutrophils. Later, intracellular life forms of the parasites are ingested by dendritic cells, which then migrate to draining lymph nodes and release IL-12p40. This in turn activates IFNγ-producing CD8+ Tc1 and CD4+ T helper (Th) 1 T cells, enabling infected macrophages to kill intracellular parasites (Sacks and Noben-Trauth, 2002).

Tolnaftate inhibits ergosterol production and impacts cell viability of Leishmania sp.

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. The treatment of all forms of leishmaniasis relies on first-line drug, pentavalent antimonial, and in cases of drug failure, the second-line drug amphotericin B has been used. Besides the high toxicity of drugs, parasites can be resistant to antimonial in some areas of the World, making it necessary to perform further studies for the characterization of new antileishmanial agents. Thus, the aim of the present work was to evaluate the leishmanicidal activity of tolnaftate, a selective reversible and non-competitive inhibitor of the fungal enzyme squalene epoxidase, which is involved in the biosynthesis of ergosterol, essential to maintain membrane physiology in fungi as well as trypanosomatids. Tolnaftate eliminated promastigote forms of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum (EC50 ~ 10 μg/mL and SI ~ 20 for all leishmanial species), and intracellular amastigote forms of all studied species (EC50 ~ 23 μg/mL in infections caused by dermatotropic species; and 11.7 μg/mL in infection caused by viscerotropic species) with high selectivity toward parasites [SI ~ 8 in infections caused by dermatotropic species and 17.4 for viscerotropic specie]. Promastigote forms of L. (L.) amazonensis treated with the EC50 of tolnaftate displayed morphological and physiological changes in the mitochondria and cell membrane. Additionally, promastigote forms treated with tolnaftate EC50 reduced the level of ergosterol by 5.6 times in comparison to the control parasites. Altogether, these results suggest that tolnaftate has leishmanicidal activity towards Leishmania sp., is selective, affects the cell membrane and mitochondria of parasites and, moreover, inhibits ergosterol production in L. (L.) amazonensis.

Leishmanicidal Activity of an In Silico-Screened Novel Inhibitor against Ascorbate Peroxidase of Leishmania donovani.

Peroxidases are a heterogeneous family of enzymes that have diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defense system of Leishmania In view of developing new and novel therapeutics, we performed in silico studies in order to search for a ligand library and identify new drug candidates and their physiological roles against promastigotes and intracellular amastigotes of Leishmania donovani Our results demonstrated that the selected inhibitor ZINC96021026 has significant antileishmanial effect and effectively killed both free and intracellular forms of the parasite. ZINC96021026 was found to be identical to ML-240, a selective inhibitor of valosin-containing protein (VCP), or p97, a member of the AAA-ATPase protein family which was derived from the scaffold of N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), targeting the D2-ATPase domain of the enzyme. ZINC96021026 (ML-240) thus has a broad range of cellular functions, thought to be derived from its ability to unfold proteins or disassemble protein complexes, besides inhibiting the ascorbate peroxidase activity. ML-240 may inhibit the parasite's ascorbate peroxidase, leading to extensive apoptosis and inducing generation of reactive oxygen species. Taken together, our results demonstrated that ML-240 could be an attractive therapeutic option for treatment against leishmaniasis.

Inactivation of antibiotic-resistant bacteria and antibiotic resistance genes by electrochemical oxidation/electro-Fenton process.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment are of great concern due to their potential risk to human health. The effluents from wastewater treatment plants and livestock production are major sources of ARB and ARGs. Chlorination, UV irradiation, and ozone disinfection cannot remove ARGs completely. In this study, the potential of electrochemical oxidation and electro-Fenton processes as alternative treatment technologies for inactivation of ARB and ARGs in both intracellular and extracellular forms was evaluated. Results showed that the electrochemical oxidation process was effective for the inactivation of selected ARB but not for the removal of intracellular ARGs or extracellular ARGs. The electro-Fenton process was more effective for the removal of both intracellular and extracellular ARGs. The removal efficiency after 120 min of electro-Fenton treatment under 21.42 mA/cm2 was 3.8 logs for intracellular tetA, 4.1 logs for intracellular ampC, 5.2 logs for extracellular tetA, and 4.8 logs for extracellular ampC, respectively in the presence of 1.0 mmol/L Fe2+. It is suggested that electrochemical oxidation is an effective disinfection method for ARB and the electro-Fenton process is a promising technology for the removal of both intracellular and extracellular ARGs in wastewater.

Recent Advances in NAMPT Inhibitors: A Novel Immunotherapic Strategy.

Nicotinamide adenine dinucleotide (NAD) is a cofactor of many enzymatic reactions as well as being a substrate for a number of NAD-consuming enzymes (e.g., PARPS, sirtuins, etc). NAD can be synthesized de novo starting from tryptophan, nicotinamide, nicotinic acid, or nicotinamide riboside from the diet. On the other hand, the nicotinamide that is liberated by NAD-consuming enzymes can be salvaged to re-form NAD. In this former instance, nicotinamide phosphoribosyltransferase (NAMPT) is the bottleneck enzyme. In the many cells in which the salvage pathway is predominant, NAMPT, therefore, represents an important controller of intracellular NAD concentrations, and as a consequence of energy metabolism. It is, therefore, not surprising that NAMPT is over expressed by tumoral cells, which take advantage from this to sustain growth rate and tumor progression. This has led to the initiation of numerous medicinal chemistry programs to develop NAMPT inhibitors in the context of oncology. More recently, however, it has been shown that NAMPT inhibitors do not solely target the tumor but also have an effect on the immune system. To add complexity, this enzyme can also be secreted by cells, and in the extracellular space it acts as a cytokine mainly through the activation of Toll like Receptor 4 (TLR4), although it has not been clarified yet if this is the only receptor responsible for its actions. While specific small molecules have been developed only against the intracellular form of NAMPT, growing evidences sustain the possibility to target the extracellular form. In this contribution, the most recent evidences on the medicinal chemistry of NAMPT will be reviewed, together with the key elements that sustain the hypothesis of NAMPT targeting and the drawbacks so far encountered.