A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity.

Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

The Importance of Epigallocatechin as a Scaffold for Drug Development against Flaviviruses.

Arboviruses such as Dengue, yellow fever, West Nile, and Zika are flaviviruses vector-borne RNA viruses transmitted biologically among vertebrate hosts by blood-taking vectors. Many flaviviruses are associated with neurological, viscerotropic, and hemorrhagic diseases, posing significant health and socioeconomic concerns as they adapt to new environments. Licensed drugs against them are currently unavailable, so searching for effective antiviral molecules is still necessary. Epigallocatechin molecules, a green tea polyphenol, have shown great virucidal potential against flaviviruses, including DENV, WNV, and ZIKV. The interaction of EGCG with the viral envelope protein and viral protease, mainly identified by computational studies, describes the interaction of these molecules with viral proteins; however, how the viral NS2B/NS3 protease interacts with epigallocatechin molecules is not yet fully deciphered. Consequently, we tested the antiviral potential of two epigallocatechin molecules (EGC and EGCG) and their derivative (AcEGCG) against DENV, YFV, WNV, and ZIKV NS2B/NS3 protease. Thus, we assayed the effect of the molecules and found that a mixture of the molecules EGC (competitive) and EGCG (noncompetitive) inhibited the virus protease of YFV, WNV, and ZIKV more effectively with IC50 values of 1.17 ± 0.2 µM, 0.58 ± 0.07 µM, and 0.57 ± 0.05 µM, respectively. As these molecules fundamentally differ in their inhibitory mode and chemical structure, our finding may open a new line for developing more effective allosteric/active site inhibitors to combat flaviviruses infection.

Riboflavin, a Potent Neuroprotective Vitamin: Focus on Flavivirus and Alphavirus Proteases.

Several neurotropic viruses are members of the flavivirus and alphavirus families. Infections caused by these viruses may cause long-term neurological sequelae in humans. The continuous emergence of infections caused by viruses around the world, such as the chikungunya virus (CHIKV) (Alphavirus genus), the zika virus (ZIKV) and the yellow fever virus (YFV) (both of the Flavivirus genus), warrants the development of new strategies to combat them. Our study demonstrates the inhibitory potential of the water-soluble vitamin riboflavin against NS2B/NS3pro of ZIKV and YFV and nsP2pro of CHIKV. Riboflavin presents a competitive inhibition mode with IC50 values in the medium µM range of 79.4 ± 5.0 µM for ZIKV NS2B/NS3pro and 45.7 ± 2.9 µM for YFV NS2B/NS3pro. Against CHIKV nsP2pro, the vitamin showed a very strong effect (93 ± 5.7 nM). The determined dissociation constants (KD) are significantly below the threshold value of 30 µM. The ligand binding increases the thermal stability between 4 °C and 8 °C. Unexpectedly, riboflavin showed inhibiting activity against another viral protein; the molecule was also able to inhibit the viral entry of CHIKV. Molecular dynamics simulations indicated great stability of riboflavin in the protease active site, which validates the repurposing of riboflavin as a promising molecule in drug development against the viruses presented here.

Design of D-Amino Acids SARS-CoV-2 Main Protease Inhibitors Using the Cationic Peptide from Rattlesnake Venom as a Scaffold.

The C30 endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and, hence, is a promising drug target. Molecules isolated from animals, insects, plants, or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus, has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial, and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

Promising Natural Compounds against Flavivirus Proteases: Citrus Flavonoids Hesperetin and Hesperidin.

Ubiquitous in citrus plants, Hesperidin and Hesperetin flavanones possess several biological functions, including antiviral activity. Arbovirus infections pose an ever-increasing threat to global healthcare systems. Among the severe arboviral infections currently known are those caused by members of the Flavivirus genus, for example, Dengue Virus-DENV, Yellow Fever Virus-YFV, and West Nile Virus-WNV. In this study, we characterize the inhibitory effect of Hesperidin and Hesperetin against DENV2, YFV, and WNV NS2B/NS3 proteases. We report the noncompetitive inhibition of the NS2B/NS3pro by the two bioflavonoids with half maximal inhibitory concentration (IC50) values <5 µM for HST and <70 µM for HSD. The determined dissociation constants (KD) of both flavonoids is significantly below the threshold value of 30 µM. Our findings demonstrate that a new generation of anti-flavivirus drugs could be developed based on selective optimization of both molecules.

The Repurposed Drugs Suramin and Quinacrine Cooperatively Inhibit SARS-CoV-2 3CLpro In Vitro.

Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) the WHO reported more than 148 million confirmed cases and 3.1 million losses globally up to now. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide, resulting in a pandemic of unprecedented magnitude. To date, several clinically safe and efficient vaccines (e.g., Pfizer-BioNTech, Moderna, Johnson & Johnson, and AstraZeneca COVID-19 vaccines) as well as drugs for emergency use have been approved. However, increasing numbers of SARS-Cov-2 variants make it imminent to identify an alternative way to treat SARS-CoV-2 infections. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is repurposing clinically developed drugs, e.g., antiparasitic drugs. The results described in this study demonstrated the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules presented a competitive and noncompetitive inhibition mode, respectively, with IC50 values in the low micromolar range. Surface plasmon resonance (SPR) experiments demonstrated that quinacrine and suramin alone possessed a moderate or weak affinity with SARS-CoV-2 3CLpro but suramin binding increased quinacrine interaction by around a factor of eight. Using docking and molecular dynamics simulations, we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin, in combination with quinacrine, showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. We suppose that the identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease and repurposable drug candidates offer fast therapeutic breakthroughs, mainly in a pandemic moment.

In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases.

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

Binding studies of a putative C. pseudotuberculosis target protein from Vitamin B12 Metabolism.

Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.

Interactions of Lipid Polar Headgroups with Carbendazim Fungicide.

Carbendazim (MBC) is a fungicide widely used in agriculture which allows the high productivity of several cultures, a necessary condition considering the growing of the world population. Moreover, MBC has environmental impact mainly on the soil and water sources, and consequently, on animal and human lives. However, even though the toxicity of fungicides is well established, their action mechanism in cell membranes are not completely understood. Herein, we investigate the interaction of different polar headgroups: dimethyldioctadecylammonium bromide (DODAB), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG); and different chain unsaturation degrees DPPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with MBC. Lipid monolayers at the air/water interface were applied as mimetic systems of cell membranes to investigate the interaction with MBC dissolved in the ultrapure water subphase. It was found that the interaction is driven preferably by electrostatic forces of the headgroups, with higher affinity for DODAB (cationic), intermediate for DPPC (zwitterionic), and absent for DPPG (anionic), considering the monolayer in the condensed phase. DODAB-MBC electrostatic interaction was consistent with FTIR (cast films). We also investigated giant unilamellar vesicles (GUVs) of zwitterionic lipids (DPPC, POPC, and DOPC) with distinct chain unsaturations in the presence of MBC by confocal microscopy and molecular dynamic (MD) simulations. The results indicate that, unlike the chain unsaturation, the polar headgroups play key role on the lipid-MBC interaction.

The polyanions heparin and suramin impede binding of free adenine to a DNA glycosylase from C. pseudotuberculosis.

Currently no effective treatment is available to combat infections caused by Corynebacterium pseudotuberculosis in livestock. Survival of this Gram-positive bacterium in rapidly-growing pathogens in hostile environments is strongly dependent on the existence of a robust DNA repair system to prevent DNA mutations and contribute to bacterial colonization and virulence. The adenine/guanine-specific DNA glycosylase (MutY) is evolutionarily conserved and has been well characterized due to its central role in the prevention of mutagenesis and DNA repair. The aim of this study was the characterization of the target protein interaction with free adenine, suramin, and heparin, as well as the binding competition characterization between the molecules. The dissociation constant for free adenine interaction with Corynebacterium pseudotuberculosis MutY (Cp-MutY) was determined, 86 ±â€¯2.5 µM. NMR competition experiments demonstrated, that the polyanions heparin and suramin compete with adenine for the protein active site. The determined dissociation constant for the heparin/Cp-MutY interaction was 5.9 ±â€¯1.0 µM and for suramin was 16 ±â€¯1.5 µM. Docking of both polyanions with Cp-MutY revealed a possible mode of interaction and indicates that these molecules can interfere with the protein interaction with damaged DNA or prevent the binding of the adenine base in the enzyme active site.

Zika virus NS2B/NS3 proteinase: A new target for an old drug - Suramin a lead compound for NS2B/NS3 proteinase inhibition.

Zika virus infection is the focus of much research due to the medical and social repercussions. Due the role of the viral NS2B/NS3 proteinase in maturation of the viral proteins, it had become an attractive antiviral target. Numerous investigations on viral epidemiology, structure and function analysis, vaccines, and therapeutic drugs have been conducted around the world. At present, no approved vaccine or even drugs have been reported. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified the polyanion suramin, an approved antiparasitic drug with antiviral properties, as a potential inhibitor of Zika virus complex NS2B/NS3 proteinase with IC50 of 47 µM. Using fluorescence spectroscopy results we could determine a kd value of 28 µM and had shown that the ligand does not affect the thermal stability of the protein. STD NMR spectroscopy experiments and molecular docking followed by molecular dynamics simulation identified the binding epitopes of the molecule and shows the mode of interaction, respectively. The computational analysis showed that suramin block the Ser135 residue and interact with the catalytically histidine residue.

Modeling and molecular dynamics indicate that snake venom phospholipase B-like enzymes are Ntn-hydrolases.

Phospholipase-B-like (SVPLB-like) enzymes are present in relatively small amounts in a number of venoms, however, their biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated by homology modeling based on the crystal structures of bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation by autocatalytic cleavage results in the removal of 25 amino acids which increases accessibility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide bond.

Inhibition of thioredoxin A1 from Corynebacterium pseudotuberculosis by polyanions and flavonoids.

In pathogens, the thioredoxin system forms part of the defense against oxidative stress and ensures the formation of the proper disulfide bonds to ensure protein function. In Corynebacterium pseudotuberculosis, the role and mechanism of TrxA1 has not been elucidated, but, the significant homology among different Trxs and the conservation of the residues that form their active sites underline the importance of the Trx systems. Proteins involved in redox metabolism and low molecular weight thiols, which might interact with them, become attractive targets to modulate the activity of pathogens. The activity of the protein was investigated using a turbidimetric assay system. The influence of different pH and low molecular weight thiols were tested. Additionally, this assay was used to investigate the inhibitory potential of ligands from different molecular families, such as, polyanions (suramin and heparin) and flavonoids (hesperetin and hesperidin). All four compounds showed inhibition of the protein activity by approximately 80%. The interactions between these compounds and Cp-TrxA1 were investigated using CD spectroscopy, NMR, molecular docking and dynamics. Our results demonstrate that suramin and hesperetin can serve as lead molecules for the development of specific inhibitors for the C. pseudotuberculosis TrxA1.

Interaction of Artepillin C with model membranes.

Green propolis, a mixture of beeswax and resinous compounds processed by Apis mellifera, displays several pharmacological properties. Artepillin C, the major compound in green propolis, consists of two prenylated groups bound to a phenyl group. Several studies have focused on the therapeutic effects of Artepillin C, but there is no evidence that it interacts with amphiphilic aggregates to mimic cell membranes. We have experimentally and computationally examined the interaction between Artepillin C and model membranes composed of dimyristoylphosphatidylcholine (DMPC) because phosphatidylcholine (PC) is one of the most abundant phospholipids in eukaryotic cell membranes. PC is located in both outer and inner leaflets and has been used as a simplified membrane model and a non-specific target to study the action of amphiphilic molecules with therapeutic effects. Experimental results indicated that Artepillin C adsorbed onto the DMPC monolayers. Its presence in the lipid suspension pointed to an increased tendency toward unilamellar vesicles and to decreased bilayer thickness. Artepillin C caused point defects in the lipid structure, which eliminated the ripple phase and the pre-transition in thermotropic chain melting. According to molecular dynamics (MD) simulations, (1) Artepillin C aggregated in the aqueous phase before it entered the bilayer; (2) Artepillin C was oriented along the direction normal to the surface; (3) the negatively charged group on Artepillin C was accommodated in the polar region of the membrane; and (4) thinner regions emerged around the Artepillin C molecules. These results help an understanding of the molecular mechanisms underlying the biological action of propolis.

Microhydration effects on geometric properties and electronic absorption spectra of ortho-aminobenzoic acid.

TD-DFT and a combination of polarized continuum model (PCM) and microhydration methods helped to simulate the optical electronic absorption spectrum of ortho-aminobenzoic acid (o-Abz). The microhydration method involved the use of different numbers, from 1 to 5, of first solvation layer water molecules. We examined how implicit and explicit water affected the energies of the HOMO-LUMO transition in the o-Abz/water systems. Adding until five water molecules, the theoretical spectrum becomes closer to the experimental data. Microhydration combined with the PCM method leads to agreement between the theoretical result for five water molecules and the experimentally measured absorption bands.