The Synthetic Peptide GA-Hecate and Its Analogs Inhibit Multiple Steps of the Chikungunya Virus Infection Cycle In Vitro.

Chikungunya virus (CHIKV) belongs to the Alphavirus genus and is responsible for significant outbreaks worldwide. Currently, there is no approved antiviral therapy against CHIKV. Bioactive peptides have great potential for new drug development. Here, we evaluated the antiviral activity of the synthetic peptide GA-Hecate and its analogs PSSct1905 and PSSct1910 against CHIKV infection. Initial screening showed that all three peptides inhibited the CHIKV replication cycle in baby hamster kidney fibroblast cells (BHK-21) and human hepatocarcinoma epithelial cells (Huh-7). GA-Hecate and its analog PSSct1905 were the most active, demonstrating suppression of viral infection by more than 91%. The analog PSSct1905 exhibited a protective effect in cells against CHIKV infection. We also observed that the analogs PSSct1905 and PSSct1910 affected CHIKV entry into both cell lines, inhibiting viral attachment and internalization. Finally, all tested compounds presented antiviral activity on the post-entry steps of CHIKV infection in all cells evaluated. In conclusion, this study highlights the potential of the peptide GA-Hecate and its analogs as novel anti-CHIKV compounds targeting different stages of the viral replication cycle, warranting the development of GA-Hecate-based compounds with broad antiviral activity.

Dereplication of Lantana trifolia L. leaves and fruits by UFLC-DAD-(+)-ESI-MS/MS and its antifungal and cytotoxic activities.

INTRODUCTION: Lantana trifolia L. (Verbenaceae) is a shrubby plant. In folk medicine, its leaves are used in the form of infusions and syrups to treat angina, coughs, and colds; they are also applied as tranquilizer. Previous studies have reported the antimicrobial potential of the compounds present in L. trifolia leaves. OBJECTIVES: To report the anti-Candida activities of the fractions obtained from the fruits and leaves of two L. trifolia specimens. METHODS: The L. trifolia fractions were submitted to UFLC-DAD-(+)-ESI-MS/MS, and the data were analyzed by using multivariate statistical tools (PCA, PLS-DA) and spectral similarity analyses based on molecular networking, which aided dereplication of the bioactive compounds. Additionally, NMR analyses were performed to confirm the chemical structure of some of the major compounds in the fractions. RESULTS: The ethyl acetate fractions presented MIC values lower than 100 µg mL-1 against the three Candida strains evaluated herein (C. albicans, C. tropicalis, and C. glabrata). Fractions FrPo AcOEt, FrPe AcOEt, and FrPe nBut had MIC values of 1.46, 2.93, and 2.93 µg mL-1 against C. glabrata, respectively. These values resembled the MIC value of amphotericin B, the positive control (0.5-1.0 µg mL-1), against this same strain. Cytotoxicity was measured and used to calculate the selectivity index. CONCLUSION: On the basis of our data, the most active fractions in the antifungal assay were more selective against C. glabrata than against non-infected cells. The analytical approach adopted here allowed us to annotate 29 compounds, nine of which were bioactive (PLS-DA results) and belong to the class of phenolic compounds.

The Dimeric Peptide (KKYRYHLKPF)2K Shows Broad-Spectrum Antiviral Activity by Inhibiting Different Steps of Chikungunya and Zika Virus Infection.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are important disease-causing agents worldwide. Currently, there are no antiviral drugs or vaccines approved to treat these viruses. However, peptides have shown great potential for new drug development. A recent study described (p-BthTX-I)2K [(KKYRYHLKPF)2K], a peptide derived from the Bothropstoxin-I toxin in the venom of the Bothrops jararacussu snake, showed antiviral activity against SARS-CoV-2. In this study, we assessed the activity of this peptide against CHIKV and ZIKV and its antiviral action in the different stages of the viral replication cycle in vitro. We observed that (p-BthTX-I)2K impaired CHIKV infection by interfering with the early steps of the viral replication cycle, reducing CHIKV entry into BHK-21 cells specifically by reducing both the attachment and internalization steps. (p-BthTX-I)2K also inhibited the ZIKV replicative cycle in Vero cells. The peptide protected the cells against ZIKV infection and decreased the levels of the viral RNA and the NS3 protein of this virus at viral post-entry steps. In conclusion, this study highlights the potential of the (p-BthTX-I)2K peptide to be a novel broad-spectrum antiviral candidate that targets different steps of the replication cycle of both CHIKV and ZIKV.

Effect of proteins isolated from Brazilian snakes on enterovirus A71 replication cycle: An approach against hand, foot and mouth disease.

Enterovirus A71 (EVA71) belongs to the Picornaviridae family and is the main etiological agent of hand, foot, and mouth disease (HFMD). There is no approved antiviral against EVA71, and therefore the search for novel anti-EVA71 therapeutics is essential. In this context, the antiviral activity of proteins isolated from snake venoms has been reported against a range of viruses. Here, the proteins CM10 and CM14 isolated from Bothrops moojeni, and Crotamin and PLA2CB isolated from Crotalus durissus terrificus were investigated for their antiviral activity against EVA71 infection. CM14 and Crotamin possessed a selective index (SI) of 170.8 and 120.4, respectively, while CM10 and PLA2CB had an SI of 67.4 and 12.5, respectively. CM14 inhibited all steps of viral replication (protective effect: 76 %; virucidal: 99 %; and post-entry: 99 %). Similarly, Crotamin inhibited up to 99 % of three steps. In contrast, CM10 and PLA2CB impaired one or two steps of EVA71 replication, respectively. Further dose-response assays using increasing titres of EVA71 were performed and CM14 and Crotamin retained functionality with high concentrations of EVA71 (up to 1000 TCID50). These data demonstrate that proteins isolated from snake venom are potent inhibitors of EVA71 and could be used as scaffolds for future development of novel antivirals.

An update on the development of antiviral against Mayaro virus: from molecules to potential viral targets.

Mayaro virus (MAYV), first isolated in 1954 in Trinidad and Tobago islands, is the causative agent of Mayaro fever, a disease characterized by fever, rashes, headaches, myalgia, and arthralgia. The infection can progress to a chronic condition in over 50% of cases, with persistent arthralgia, which can lead to the disability of the infected individuals. MAYV is mainly transmitted through the bite of the female Haemagogus spp. mosquito genus. However, studies demonstrate that Aedes aegypti is also a vector, contributing to the spread of MAYV beyond endemic areas, given the vast geographical distribution of the mosquito. Besides, the similarity of antigenic sites with other Alphavirus complicates the diagnoses of MAYV, contributing to underreporting of the disease. Nowadays, there are no antiviral drugs available to treat infected patients, being the clinical management based on analgesics and non-steroidal anti-inflammatory drugs. In this context, this review aims to summarize compounds that have demonstrated antiviral activity against MAYV in vitro, as well as discuss the potentiality of viral proteins as targets for the development of antiviral drugs against MAYV. Finally, through rationalization of the data presented herein, we wish to encourage further research encompassing these compounds as potential anti-MAYV drug candidates.

Insights into the role of the cobalt(III)-thiosemicarbazone complex as a potential inhibitor of the Chikungunya virus nsP4.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disease that can result in disability. Until now, there is no antiviral treatment against CHIKV, demonstrating that there is a need for development of new drugs. Studies have shown that thiosemicarbazones and their metal complexes possess biological activities, and their synthesis is simple, clean, versatile, and results in high yields. Here, we evaluated the mechanism of action (MOA) of a cobalt(III) thiosemicarbazone complex named [CoIII(L1)2]Cl based on its in vitro potent antiviral activity against CHIKV previously evaluated (80% of inhibition on replication). Furthermore, the complex has no toxicity in healthy cells, as confirmed by infecting BHK-21 cells with CHIKV-nanoluciferase in the presence of the compound, showing that [CoIII(L1)2]Cl inhibited CHIKV infection with the selective index of 3.26. [CoIII(L1)2]Cl presented a post-entry effect on viral replication, emphasized by the strong interaction of [CoIII(L1)2]Cl with CHIKV non-structural protein 4 (nsP4) in the microscale thermophoresis assay, suggesting a potential mode of action of this compound against CHIKV. Moreover, in silico analyses by molecular docking demonstrated potential interaction of [CoIII(L1)2]Cl with nsP4 through hydrogen bonds, hydrophobic and electrostatic interactions. The evaluation of ADME-Tox properties showed that [CoIII(L1)2]Cl presents appropriate lipophilicity, good human intestinal absorption, and has no toxicological effect as irritant, mutagenic, reproductive, and tumorigenic side effects.

Potential in vitro anti-periodontopathogenic, anti-Chikungunya activities and in vivo toxicity of Brazilian red propolis.

Bacterial and viral infections are serious public health issue. Therefore, this study aimed to evaluate the antibacterial, antibiofilm and antiviral potential of the Brazilian Red Propolis (BRP) crude hydroalcoholic extract, fractions, and isolated compounds, as well as their in vivo toxicity. The antibacterial activity was evaluated by determining the Minimum Inhibitory Concentration and the antibiofilm activity by determining the Minimum Inhibitory Concentration of Biofilm (MICB50). The viable bacteria count (Log10 UFC/mL) was also obtained. The antiviral assays were performed by infecting BHK-21 cells with Chikungunya (CHIKV) nanoluc. The toxicity of the BRP was evaluated in the Caenorhabditis elegans animal model. The MIC values for the crude hydroalcoholic extract sample ranged from 3.12 to 100 µg/mL, while fractions and isolated compounds the MIC values ranged from 1.56 to 400 µg/mL.The BRP crude hydroalcoholic extract, oblongifolin B, and gutiferone E presented MICB50 values ranging from 1.56 to 100 µg/mL against monospecies and multispecies biofilms. Neovestitol and vestitol inhibited CHIKV infection by 93.5 and 96.7%, respectively. The tests to evaluate toxicity in C. elegans demonstrated that the BRP was not toxic below the concentrations 750 µg/mL. The results constitute an alternative approach for treating various infectious diseases.

In vitro antiviral activity of piperidine alkaloids from Senna spectabilis flowers on Chikungunya virus infection.

BACKGROUND: Chikungunya fever is an endemic disease caused by the Chikungunya virus (CHIKV). To date there is no antiviral treatment against this infection or licensed vaccine to prevent it. Our study aims to evaluate whether (-)-cassine (1) and (-)-spectaline (2), the main alkaloids of Senna spectabilis, display anti-CHIKV activity. Both compounds have been described to be biologically active against neglected tropical diseases, including malaria, leishmaniasis, and schistosomiasis, which emphasizes that these molecules could be repurposed for chikungunya fever treatment. METHODS: The structures of the isolated compounds 1 and 2 were identified by NMR and HRESIMS analyses, and their antiviral activity against CHIKV was assessed by a dose-response assay employing BHK-21 cells and CHIKV-nanoluc, a recombinant virus carrying the nanoluciferase gene reporter. RESULTS: Compound 1 presented CC50 of 126.5 µM and EC50 of 14.9 µM, while compound 2 presented CC50 of 91.9 µM and EC50 of 8.3 µM. The calculated selectivity index (SI) was 8.5 for 1 and 11.3 for 2. CONCLUSION: The data presented herein show that compounds 1 and 2 have potential for being repurposed as anti-CHIKV drug. Our promising in vitro results encourage further in vitro and in vivo assays. This is the first description of the antiviral activity of compounds 1 and 2 against CHIKV infection, which can impact the development of antiviral drug candidates against chikungunya fever, which sometimes can be debilitating.

Chikungunya virus entry is strongly inhibited by phospholipase A2 isolated from the venom of Crotalus durissus terrificus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2CB (PLA2CB), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2CB on the CHIKV replicative cycle, and suggest that PLA2CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells.

Is the ADP ribose site of the Chikungunya virus NSP3 Macro domain a target for antiviral approaches?

Chikungunya virus (CHIKV) is a mosquito-transmitted virus of special concern as it causes Chikungunya fever, characterized by an acute febrile illness, rash, and arthralgia that can progress to chronic and debilitating arthritic symptoms. The effects of climate change on the geographic distribution of the mosquito vector has the potential to expose more of the globe to this virus. No antiviral agents or vaccines are currently available against CHIKV infection and the development of novel therapies that may lead to a future treatment is therefore necessary. In this context, the ADP-ribose binding site of the CHIKV nsP3 macro domain has been reported as a potential target for the development of antivirals. Mutations in the ADP-ribose binding site demonstrated decreased viral replication in cell culture and reduced virulence. In this study, 48,750 small molecules were screened in silico for their ability to bind to the ADP-ribose binding site of the CHIKV nsP3 macro domain. From this in silico analysis, 12 molecules were selected for in vitro analysis using a CHIKV subgenomic replicon in Huh-7 cells. Cell viability and CHIKV replication were evaluated and molecules C5 and C13 demonstrated 53 and 66% inhibition of CHIKV replication, respectively. By using a CHIKV-Dual luciferase replicon contain two reporter genes, we also demonstrated that the treatment with either compounds are probably interfering in the early replication rather than after RNA replication has occurred.

Antivirals against Chikungunya Virus: Is the Solution in Nature?

The worldwide outbreaks of the chikungunya virus (CHIKV) in the last years demonstrated the need for studies to screen antivirals against CHIKV. The virus was first isolated in Tanzania in 1952 and was responsible for outbreaks in Africa and Southwest Asia in subsequent years. Between 2007 and 2014, some cases were documented in Europe and America. The infection is associated with low rates of death; however, it can progress to a chronic disease characterized by severe arthralgias in infected patients. This infection is also associated with Guillain-Barré syndrome. There is no specific antivirus against CHIKV. Treatment of infected patients is palliative and based on analgesics and non-steroidal anti-inflammatory drugs to reduce arthralgias. Several natural molecules have been described as antiviruses against viruses such as dengue, yellow fever, hepatitis C, and influenza. This review aims to summarize the natural compounds that have demonstrated antiviral activity against chikungunya virus in vitro.

Organometallic Complex Strongly Impairs Chikungunya Virus Entry to the Host Cells.

Chikungunya fever is a disease caused by the Chikungunya virus (CHIKV) that is transmitted by the bite of the female of Aedes sp. mosquito. The symptoms include fever, muscle aches, skin rash, and severe joint pains. The disease may develop into a chronic condition and joint pain for months or years. Currently, there is no effective antiviral treatment against CHIKV infection. Treatments based on natural compounds have been widely studied, as many drugs were produced by using natural molecules and their derivatives. Alpha-phellandrene (α-Phe) is a naturally occurring organic compound that is a ligand for ruthenium, forming the organometallic complex [Ru2Cl4(p-cymene)2] (RcP). Organometallic complexes have shown promising as candidate molecules to a new generation of compounds that presented relevant biological properties, however, there is a lack of knowledge concerning the anti-CHIKV activity of these complexes. The present work evaluated the effects of the RcP and its precursors, the hydrate ruthenium(III) chloride salt (RuCl3⋅xH2O) (Ru) and α-Phe, on CHIKV infection in vitro. To this, BHK-21 cells were infected with CHIKV-nanoluciferase (CHIKV-nanoluc), a viral construct harboring the nanoluciferase reporter gene, at the presence or absence of the compounds for 16 h. Cytotoxicity and impact on infectivity were analyzed. The results demonstrated that RcP exhibited a strong therapeutic potential judged by the selective index > 40. Antiviral effects of RcP on different stages of the CHIKV replicative cycle were investigated; the results showed that it affected early stages of virus infection reducing virus replication by 77% at non-cytotoxic concentrations. Further assays demonstrated the virucidal activity of the compound that completely blocked virus infectivity. In silico molecular docking calculations suggested different binding interactions between aromatic rings of RcP and the loop of amino acids of the E2 envelope CHIKV glycoprotein mainly through hydrophobic interactions. Additionally, infrared spectroscopy spectral analysis indicated interactions of RcP with CHIKV glycoproteins. These data suggest that RcP may act on CHIKV particles, disrupting virus entry to the host cells. Therefore, RcP may represent a strong candidate for the development of anti-CHIKV drugs.