The Cytotoxic and Anti-Migratory Properties of Caesalpinia sappan and Ficus septica, in Combination with Doxorubicin on 4T1 TNBC Cells with Nephroprotective Potential.

OBJECTIVE: To evaluate the anti-cancer properties of Caesalpinia sappan and Ficus septica in combination with doxorubicin on 4T1 cells, confirm their nephroprotective activities, and predict the molecular targets of the underlying mechanisms. METHODS: The cytotoxic activities of all extracts and doxorubicin were determined by MTT assay followed by cell cycle and apoptosis analysis using flow cytometry. Immunoblotting was used to determine the protein expressions. The proteins involved in the cell proliferation and migration were analyzed through bioinformatics approaches, whereas, the interaction between compounds and protein targets was observed through molecular docking. Furthermore, the effect of the extracts on cell migration was analyzed by scratch wound healing assay. The intracellular ROS after treatment with extracts was observed using DCFDA staining flow cytometry. RESULTS: Both ECS and EFS performed cytotoxic properties and significantly enhanced doxorubicin's cytotoxic effects against 4T1 cells. However, these cytotoxic activities did not correlate with the cell cycle progression. On the contrary, the combination treatment caused apoptosis that may correlate with the decreasing of IκBα phosphorylation, indicating that all agents targeted the inhibition of NF-κB activation. The combination treatments also inhibited cell migration and decreased MMP-9 expression. TNBC proliferation and metastasis needed at least 54 proteins to be activated, some of them are related to NF-κB activation. The inhibitory effect of ECS correlated with the interaction of brazilin and brazilein to IKK, a kinase protein that plays a role in IκBα phosphorylation. In addition, ECS and EFS reduced ROS expression in Vero cells caused by doxorubicin. CONCLUSION: In conclusion, ECS and EFS effectively enhanced the cytotoxic effect of doxorubicin and inhibit cell migration on 4T1 cells and these activities may correlate to the inhibitory effect of NF-κB activation. ECS and EFS also exhibit ROS suppressing effect on Vero cells that may be beneficent to reduce nephrotoxicity of chemotherapeutic treatment.

Biologically active polyphenolic compounds from Lespedeza bicolor.

We investigated the ability of six prenylated prerocarpans, stilbenoid, and a new dimeric flavonoid, lespebicolin B, from stem bark as well as two 3-O-rutinosides and a mixture of 3-O-ß-D-glucosides of quercetin and kaempferol from flowers of Lespedeza bicolor to inhibit HSV-1 replication in Vero cells. Pretreatment of HSV-1 with polyphenolic compounds (direct virucidal effect) showed that pterocarpans lespedezol A2 (1), (6aR,11aR)-6a,11a-dihydrolespedezol A2 (2), (6aR,11aR)-2-isoprenyldihydrolespedezol A2 (4), and (6aR,11aR,3'R)-dihydrolespedezol A3 (5) significantly inhibited viral replication, with a selective index (SI) ≥10. Compound 4 possessed the lowest 50% - inhibiting concentration (IC50) and the highest SI values (2.6 µM and 27.9, respectively) in this test. (6aR,11aR)-2-Isoprenyldihydrolespedezol A2 (4) also had a moderate effect under simultaneous treatment of Vero cells with the tested compound and virus (IC50 and SI values were 5.86 µM and 12.4, respectively). 3-O-rutinosides of quercetin and kaempferol and a mixture of 3-O-ß-D-glucosides of quercetin and kaempferol (10 and 12) also showed significant virucidal activity, with SI values of 12.5, 14.6, and 98.2, respectively, and IC50 values of 8.6, 12.2, and 3.6, respectively. We also performed a quantitative structure-activity relationship (QSAR) analysis of data on the virucidal activity of polyphenolics with 4 < pIC50 < 6. It was found that the virucidal activity of these compounds depended on both the structure of the aromatic part and the conformation of geranyl and isoprenyl side chains of their molecules. These findings are correlated with the largest value of the principal moment of inertia (pmi) descriptor describing the geometry of molecules.

Influence of the Structural Features of Carrageenans from Red Algae of the Far Eastern Seas on Their Antiviral Properties.

The structural diversity and unique physicochemical properties of sulphated polysaccharides of red algae carrageenans (CRGs), to a great extent, determine the wide range of their antiviral properties. This work aimed to compare the antiviral activities of different structural types of CRGs: against herpes simplex virus type 1 (HSV-1) and enterovirus (ECHO-1). We found that CRGs significantly increased the resistance of Vero cells to virus infection (preventive effect), directly affected virus particles (virucidal effect), inhibited the attachment and penetration of virus to cells, and were more effective against HSV-1. CRG1 showed the highest virucidal effect on HSV-1 particles with a selective index (SI) of 100. CRG2 exhibited the highest antiviral activity by inhibiting HSV-1 and ECHO-1 plaque formation, with a SI of 110 and 59, respectively, when it was added before virus infection. CRG2 also significantly reduced the attachment of HSV-1 and ECHO-1 to cells compared to other CRGs. It was shown by molecular docking that tetrasaccharides-CRGs are able to bind with the HSV-1 surface glycoprotein, gD, to prevent virus-cell interactions. The revealed differences in the effect of CRGs on different stages of the lifecycle of the viruses are apparently related to the structural features of the investigated compounds.

An approach to chimeric subunit immunogen provides efficient protection against toxicity, type III and type v secretion systems of Shigella.

OBJECTIVES: Shigellosis is one of the infectious diseases causing severe intestinal illness in human beings. Development of an effective vaccine against Shigella is a key to deal with this bacterium. The present study aimed at evaluation of the antibody response as well as the protection of the recombinant chimeric protein containing IpaD, IpaB, StxB, and VirG against Shigella dysentery and flexneri. METHODS: Chimeric protein was expressed and purified by Ni-NTA resin. The identity of the protein was determined by Western blot analysis. Mouse groups were immunized with the recombinant protein and the humoral immune response was measured by Enzyme-Linked Immunosorbent Assay (ELISA). Additionally, neutralization of the bacterial toxin by antibody was assessed by MTT assay. Animal challenge against S.dysentery and S. flexneri was evaluated, as well. RESULTS: Protein expression and purification were confirmed by SDS-PAGE and western blotting. Analysis of the immune responses demonstrated that the antibody responses were higher in the sera of the subcutaneously immunized mice compared to those immunized intraperitoneally. In vitro neutralization analysis indicated that the 1:10000 dilution of the sera had a high ability to neutralize 0.25 ng/µl (CD50) of the toxin on the Vero cell line. Furthermore, the results of the animal challenge showed that the immunized mice were completely protected against 50 LD50 of the bacterial toxin. Immunization also protected 80% of the mice from 10 LD50 by S. flexneri and S.dysentery. In addition, passive immunization conferred 60% protection in the mice against S. flexneri and S.dysentery. Organ burden studies also revealed a significant reduction in infection among the immunized mice. CONCLUSION: This study revealed that the chimeric protein produced inE. colicould be a promising chimeric immunogen candidate against Shigella.

Indole alkaloids inhibit zika and chikungunya virus infection in different cell lines.

BACKGROUND: In recent years, an increase in the occurrence of illnesses caused by two clinically- important arboviruses has been reported: Zika virus (ZIKV) and Chikungunya virus (CHIKV). There is no licensed antiviral treatment for either of the two abovementioned viruses. Bearing in mind that the antiviral effect of indole alkaloids has been reported for other arboviral models, the present study proposed to evaluate the antiviral in vitro and in silico effects of four indole alkaloids on infections by these two viruses in different cell lines. METHODS: The antiviral effects of voacangine (VOAC), voacangine-7-hydroxyindolenine (VOAC-OH), rupicoline and 3-oxo voacangine (OXO-VOAC) were evaluated in Vero, U937 and A549 cells using different experimental strategies (Pre, Trans, Post and combined treatment). Viral infection was quantified by different methodologies, including infectious viral particles by plating, viral genome by RT-qPCR, and viral protein by cell ELISA. Moreover, molecular docking was used to evaluate the possible interactions between structural and nonstructural viral proteins and the compounds. The results obtained from the antiviral strategies for each experimental condition were compared in all cases with the untreated controls. Statistically significant differences were identified using a parametric Student's t-test. In all cases, p values below 0.05 (p < 0.05) were considered statistically significant. RESULTS: In the pre-treatment strategy in Vero cells, VOAC and VOAC-OH inhibited both viral models and OXO-VOAC inhibited only ZIKV; in U937 cells infected with CHIKV/Col, only VOAC-OH inhibited infection, but none of the compounds had activity in A549 cells; in U937 cells and A549 cells infected with ZIKV/Col, the three compounds that were effective in Vero cells also had antiviral activity. In the trans-treatment strategy, only VOAC-OH was virucidal against ZIKV/Col. In the post-treatment strategy, only rupicoline was effective in the CHIKV/Col model in Vero and A549 cells, whereas VOAC and VOAC-OH inhibited ZIKV infection in all three cell lines. In the combined strategy, VOAC, VOAC-OH and rupicoline inhibited CHIKV/Col and ZIKV/Col, but only rupicoline improved the antiviral effect of ZIKV/Col-infected cultures with respect to the individual strategies. Molecular docking showed that all the compounds had favorable binding energies with the structural proteins E2 and NSP2 (CHIKV) and E and NS5 (ZIKV). CONCLUSIONS: The present study demonstrates that indole alkaloids are promising antiviral drugs in the process of ZIKV and CHIKV infection; however, the mechanisms of action evaluated in this study would indicate that the effect is different in each viral model and, in turn, dependent on the cell line.

Effects of Colchicum baytopiorum leaf extract on cytotoxicity and cell death pathways in C-4 I and Vero cell lines.

PURPOSE: The present study aimed to research the cytotoxic effects of Colchicum baytopiorum extract on normal and cancerous cells and reveal the cell death mechanisms in cancerous cells triggered by this effect. METHODS: Within this framework, the cells' index values obtained with an xCELLigence Real Time Cell Analysis DP device, selectivity index (SI), apoptotic index (AI) based on a DAPI application and time-related activities of caspase 3,7 and 9 with a spectrofluorometer were inspected. The expressions of apoptosis/autophagy/entosis/necroptosis/anoikis-related genes were researched with qRT-PCR. RESULTS: It was determined that C. baytopiorum extract had displayed a high selectivity [(SI)=4], increased AI (p<0.01) and activation of caspases 3,7 and 9 (p<0.05). It was observed that the mRNA expressions of Atg12, Atg16, Atg5, Atg7, bad, bak, bax, bcl-xL, Beclin1, caspase3, FLIP, Puma, LC3, mcl-1, Bit1, Rho, RIP1, ROCK and TRAF2 genes in C-4 I cells to which the plant extract was applied had increased significantly in comparison with the control group (FC≥1.5). A lowering was detected in the mRNA levels of IAP, SRC kinase and TNF. CONCLUSION: Consequently, it was revealed that the plant extract used had increased the gene expressions in the autophagic cell death pathway in C-4 I cells along with apoptosis and thus, it could be a promising candidate for cervix carcinoma treatment.

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

Identification of 3,3'-O-dimethylellagic acid and apigenin as the main antiplasmodial constituents of Endodesmia calophylloides Benth and Hymenostegia afzelii (Oliver.) Harms.

BACKGROUND: Endodesmia calophylloides and Hymenostegia afzelii belong to the Guttiferae and Caesalpiniaceae plant families with known uses in African ethno-medicine to treat malaria and several other diseases. This study aimed at identifying antiplasmodial natural products from selected crude extracts from H. afzelii and E. calophylloides and to assess their cytotoxicity. METHODS: The extracts from H. afzelii and E. calophylloides were subjected to bioassay-guided fractionation to identify antiplasmodial compounds. The hydroethanol and methanol stem bark crude extracts, fractions and isolated compounds were assessed for antiplasmodial activity against the chloroquine-sensitive 3D7 and multi-drug resistant Dd2 strains of Plasmodium falciparum using the SYBR green I fluorescence-based microdilution assay. Cytotoxicity of active extracts, fractions and compounds was determined on African green monkey normal kidney Vero and murine macrophage Raw 264.7 cell lines using the Resazurin-based viability assay. RESULTS: The hydroethanolic extract of H. afzelii stem bark (HasbHE) and the methanolic extract of E. calophylloides stem bark (EcsbM) exhibited the highest potency against both Pf3D7 (EC50 values of 3.32 ± 0.15 µg/mL and 7.40 ± 0.19 µg/mL, respectively) and PfDd2 (EC50 of 3.08 ± 0.21 µg/mL and 7.48 ± 0.07 µg/mL, respectively) strains. Both extracts showed high selectivity toward Plasmodium parasites (SI > 13). The biological activity-guided fractionation led to the identification of five compounds (Compounds 1-5) from HasbHE and one compound (Compound 6) from EcsbM. Of these, Compound 1 corresponding to apigenin (EC50 Pf3D7, of 19.01 ± 0.72 µM and EC50 PfDd2 of 16.39 ± 0.52 µM), and Compound 6 corresponding to 3,3'-O-dimethylellagic acid (EC50 Pf3D7 of 4.27 ± 0.05 µM and EC50 PfDd2 of 1.36 ± 0.47 µM) displayed the highest antiplasmodial activities. Interestingly, both compounds exhibited negligible cytotoxicity against both Vero and Raw 264.7 cell lines with selectivity indices greater than 9. CONCLUSIONS: This study led to the identification of two potent antiplasmodial natural compounds, 3,3'-O-dimethylellagic acid and apigenin that could serve as starting points for further antimalarial drug discovery.

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

Bacillus spp. metabolites are effective in eradicating Aedes aegypti (Diptera: Culicidae) larvae with low toxicity to non-target species.

The growing spread of dengue, chikungunya and Zika viruses demand the development of new and environmentally safe control methods for their vector, the mosquito Aedes aegypti. This study aims to find novel larvicidal agents from mutualistic (endophytic and rhizospheric) or edaphic bacteria that have no action against non-target organisms. Eleven out of the 254 bacterial strains tested were able to kill Ae. aegypti larvae. Larvicidal activity did not depend on presence of cells, since culture supernatants or crude lipopeptide extracts (CLEs) killed the larvae. Bacillus safensis BacI67 and Bacillus paranthracis C21 supernatants were the best performing supernatants, displaying the lowest lethal concentrations (LC50 = 31.11 µL/mL and 45.84 µL/mL, respectively). Bacillus velezensis B64a and Bacillus velezensis B15 produced the best performing CLEs (LC50 = 0.11 mg/mL and 0.12 mg/mL, respectively). Mass spectrometry analysis of CLEs detected a mixture of surfactins, iturins, and fengycins. The samples tested were weakly- or non-toxic to mammalian cells (RAW 264.7 macrophages and VERO cells) and non-target organisms (Caenorhabditis elegans, Galleria mellonella, Scenedesmus obliquus, and Tetrahymena pyriformis) - especially B. velezensis B15 CLE. The biosynthetic gene clusters related to secondary metabolism identified by whole genome sequencing of the four best performing bacteria strains revealed clusters for bacteriocin, beta-lactone, lanthipeptide, non-ribosomal peptide synthetases, polyketide synthases (PKS), siderophores, T3PKS, type 1 PKS-like, terpenes, thiopeptides, and trans-AT-PKS. Purification of lipopeptides may clarify the mechanisms by which these extracts kill Ae. aegypti larvae.

Antiviral Role of Phenolic Compounds against Dengue Virus: A Review.

Phenolic compounds have been related to multiple biological activities, and the antiviral effect of these compounds has been demonstrated in several viral models of public health concern. In this review, we show the antiviral role of phenolic compounds against dengue virus (DENV), the most widespread arbovirus globally that, after its re-emergence, has caused multiple epidemic outbreaks, especially in the last two years. Twenty phenolic compounds with anti-DENV activity are discussed, including the multiple mechanisms of action, such as those directed against viral particles or viral proteins, host proteins or pathways related to the productive replication viral cycle and the spread of the infection.

NS1-based DNA vaccination confers mouse protective immunity against ZIKV challenge.

The recent pandemic of Zika virus (ZIKV) infections highlight the urgent need for the development of a safe and efficacious ZIKV vaccine. We previously demonstrated that robust humoral and cellular immunity was elicited in BALB/c mice by ZIKV DNA vaccine encoding the precursor membrane (prM) and envelope (E) proteins while the protective efficacies were not evaluated against ZIKV challenge. To further explore the protective immunity elicited by various targets of ZIKV, we constructed a novel DNA-based vaccine expressing nonstructural protein 1 (NS1), named as VRC-NS1, and evaluated and compared immune responses and protective efficacies of three ZIKV DNA vaccine candidates (VRC-prME, VRC-NS1, and VRC-prME+VRC-NS1) using an A129 (Ifnar-/-) murine challenge model. The results showed that each of DNA vaccine candidates induced robust antigen-specific humoral immunity and conferred protection against ZIKV-SMGC-1 with two doses (20 µg per dose) of homologous intramuscularly (i.m.) immunizations via in vivo electroporation. All DNA vaccine candidates induced significant protection against infection-associated weight loss in addition to preventing viral replication in blood, brain and spleen tissue following in vivo viral challenge. Notably, NS1-based DNA vaccination alone was capable of conferring mouse protective immunity to reduce viremia and viral burden in tissues against ZIKV challenge, even though it did not induce neutralizing antibodies. These data demonstrated that VRC-NS1 and VRC-prME are highly promising vaccine candidates for ZIKV control. Furthermore, our results highlight an alternative strategy (DNA vaccine based on non-neutralizing antigen NS1) for designing novel flaviviral vaccines (including for ZIKV) and provide a foundation for the development of a safe and effective NS1-based vaccine against ZIKV infection.

MoS2-Calix[4]arene Catalyzed Synthesis and Molecular Docking Study of 2,4,5-Trisubstituted Imidazoles As Potent Inhibitors of Mycobacterium tuberculosis.

A MoS2-supported-calix[4]arene (MoS2-CA4) nanocatalyst was used for efficient synthesis of 2,4,5-trisubstituted imidazole derivatives from 1-(4-nitrophenyl)-2-(4-(trifluoromethyl)phenyl)ethane-1,2-dione, aldehydes and ammonium acetate under solvent-free conditions. Reusability of the catalyst up to five cycles without any significant loss in the yields of the product is the unique feature of this heterogeneous solid catalysis. Furthermore, the noteworthy highlights of this method are safe reaction profiles, broad substrate scope, excellent yields, economical, solvent-free, and simple workup conditions. All synthesized compounds were evaluated for their in vitro antitubercular (TB) activity against Mycobacterium tuberculosis (Mtb) H37Rv. Among the screened compounds 3c, 3d, 3f, 3m, and 3r had MIC values of 2.15, 2.78, 5.75, 1.36, and 0.75 µM, respectively, and exhibited more potency than the reference drugs pyrazinamide (MIC: 3.12 µM), ciprofloxacin (MIC: 4.73 µM), and ethambutol (7.61 µM). Besides, potent compounds (3c, 3d, 3f, 3m, and 3r) have been tested for inhibition of MabA (ß-ketoacyl-ACP reductase) enzyme and cytotoxic activity against mammalian Vero cell line. A molecular docking study was carried out on the MabA (PDB ID: 1UZN) enzyme to predict the interactions of the synthesized compounds. The results of the in vitro anti-TB activity and docking study showed that synthesized compounds have a strong anti-TB activity and can be adapted and produced more effectively as a lead compound.

Synthesis and biological evaluation of 12 novel (-)-camphene-based 1,3,4-thiadiazoles against Mycobacterium tuberculosis.

Aim: To evaluate the activity, cytotoxicity and efflux pumps inhibition of a series of 12 novels (-)-camphene-based 1,3,4-thiadiazoles (TDZs) against Mycobacterium tuberculosis (Mtb). Materials & methods: The minimum inhibitory concentration (MIC), cytotoxicity for three cell lines, ethidium bromide accumulation and checkerboard methods were carried out. Results: Compounds (6a, 6b, 6c, 6g, 6h and 6j) showed significant anti-Mtb activity (MIC 3.9-7.8 µg/ml) and no antagonism with anti-TB drugs already used in the TB treatment. Selectivity index (SI) was also determined, with values reaching 42.9 for H37Rv strain and 97.1 for clinical isolate. Five compounds also showed bacterial efflux pumps inhibition and one showed modulator effect with three drugs. Conclusion: These six TDZs should be considered as new scaffolds to develop anti-TB drugs.

In vitro and in vivo activity of voriconazole and benznidazole combination on trypanosoma cruzi infection models.

Combination therapy has been proposed as an ideal strategy to reduce drug toxicity and improve treatment efficacy in Chagas disease. Previously, we demonstrated potent in vivo anti-Trypanosoma cruzi activity of voriconazole. In this work, we aimed to study the synergistic effect of voriconazole (VCZ) and benznidazole (BZ) both in vitro and in vivo models of T. cruzi infection using the Tulahuen strain. Combining VCZ and BZ at fixed concentrations, the inhibitory concentration 50% (IC50) on amastigotes was lower than the obtained IC50 for BZ alone and the Fractional Inhibitory Concentration Index (∑FIC) suggested an in vitro additive effect on T. cruzi amastigotes inhibition at concentrations devoid of cytotoxic effects. Treatment response in the in vivo model was evaluated by comparing behavior and physical aspects, parasitemia and mortality of mice infected with Tulahuen strain. VCZ and BZ treatments alone or in combination were well tolerated. All treated animals displayed significantly lower mean peak parasitemia and mortality compared to infected non-treated controls (p< 0.05). However, VCZ + BZ combination elicited no additional benefits over BZ monotherapy. VCZ efficacy was not enhanced by combination therapy with BZ at the doses studied, requiring further and astringent non-clinical studies to establish the VCZ efficacy and eventually moving forward to clinical trials.

Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro.

In recent decades, chikungunya virus (CHIKV) has re-emerged, leading to outbreaks of chikungunya fever in Africa, Asia and Central and South America. The disease is characterized by a rapid onset febrile illness with (poly)arthralgia, myalgia, rashes, headaches and nausea. In 30 to 40% of the cases, CHIKV infection causes persistent (poly)arthralgia, lasting for months or even years after initial infection. Despite the drastic re-emergence and clinical impact there is no vaccine nor antiviral compound available to prevent or control CHIKV infection. Here, we evaluated the antiviral potential of tomatidine towards CHIKV infection. We demonstrate that tomatidine potently inhibits virus particle production of multiple CHIKV strains. Time-of -addition experiments in Huh7 cells revealed that tomatidine acts at a post-entry step of the virus replication cycle. Furthermore, a marked decrease in the number of CHIKV-infected cells was seen, suggesting that tomatidine predominantly acts early in infection yet after virus attachment and cell entry. Antiviral activity was still detected at 24 hours post-infection, indicating that tomatidine controls multiple rounds of CHIKV replication. Solasodine and sarsasapogenin, two structural derivatives of tomatidine, also showed strong albeit less potent antiviral activity towards CHIKV. In conclusion, this study identifies tomatidine as a novel compound to combat CHIKV infection in vitro.

Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency.

Accidents with venomous snakes are a major health hazard in tropical countries. Bothrops genus is responsible for almost 80% of snakebites in Brazil. Immunotherapy is the only approved specific treatment against snake toxins and the production of therapeutic antivenoms requires quality control tests to determine their neutralizing potency. Currently, these controls are performed by in vivo lethality neutralization, however, the inhibition of particular events produced by bothropic venoms such as coagulopathy, hemorrhage, edema or cytotoxic effects are also required. The aim of this work is to develop an in vitro alternative assay for antivenom pre-clinical evaluation. In this sense, we designed a cell viability assay using different amounts (0.2-10 µL/well) of low and high potency anti-bothropic sera, previously classified by the traditional in vivo test, for assessing the antivenom capacity to protect the cells against B. jararaca venom cytotoxicity (5xEC50 = 58.95 µg/mL). We found that high potency sera are more effective in neutralizing B. jararaca venom cytotoxicity when compared to low potency sera, which is in accordance to their pre-determined in vivo potency. Considering sera in vitro inhibitory concentration able to prevent 50% cell death (IC50) and their known in vivo potency, a cut-off point was determined to discriminate low and high potency sera. Our data provide insights for the development of an in vitro method which can determine the anti-bothropic antivenom potency during its production.

Glycyrrhizic acid derivatives as Dengue virus inhibitors.

Dengue virus (DENV) is one of the most geographically distributed pathogenic flaviviruses transmitted by mosquitoes Aedes sps. In this study, the structure-antiviral activity relationships of Glycyrrhizic acid (GL) derivatives was evaluated by the inhibitory assays on the cytopathic effect (CPE) and viral infectivity of DENV type 2 (DENV2) in Vero E6 cells. GL (96% purity) had a low cytotoxicity to Vero E6 cells, inhibited DENV2-induced CPE, and reduced the DENV-2 infectivity with the IC50 of 8.1 µM. Conjugation of GL with amino acids or their methyl esters and the introduction of aromatic acylhydrazide residues into the carbohydrate part strongly influenced on the antiviral activity. Among compounds tested GL conjugates with isoleucine 13 and 11-aminoundecanoic acid 17 were found as potent anti-DENV2 inhibitors (IC50 1.2-1.3 µM). Therefore, modification of GL is a perspective way in the search of new antivirals against DENV2 infection.

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

Highly Effective and Safe Polymeric Inhibitors of Herpes Simplex Virus in Vitro and in Vivo.

A series of poly(ethylene glycol)-block-poly(3-(methacryloylamino)propyl trimethylammonium chloride) (PEG-b-PMAPTAC) water-soluble block copolymers consisting of PEG and PMPTAC were obtained by reversible addition-fragmentation chain-transfer (RAFT) polymerization and demonstrated to function as highly effective herpes simplex virus type 1 (HSV-1) inhibitors as shown by in vitro tests (Vero E6 cells) and in vivo experiments (mouse model). Half-maximal inhibitory concentration (IC50) values were determined by quantitative polymerase chain reaction to be 0.36 ± 0.08 µg/mL for the most effective polymer PEG45-b-PMAPTAC52 and 0.84 ± 1.24 µg/mL for the less effective one, PEG45-b-PMAPTAC74. The study performed on the mouse model showed that the polymers protect mice from lethal infection. The polymers are not toxic to the primary human skin fibroblast cells up to the concentration of 100 µg/mL and to the Vero E6 cells up to 500 µg/mL. No systemic or topical toxicity was observed in vivo, even with mice treated with concentrated formulation (100 mg/mL). The mechanistic studies indicated that polymers interacted with the cell and blocked the formation of the entry/fusion complex. Physicochemical and biological properties of PEGx-b-PMAPTACy make them promising drug candidates.