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, genusAlphavirus), dengue virus (DENV, genusFlavivirus), and zika virus (ZIKV, genusFlavivirus). 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/NS3^proof ZIKV and nsP2^proof CHIKV and, Hesperidin (HSD) against nsP2^proof CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC₅₀values are in low µM range for HST against ZIKV NS2B/NS3^pro(12.6 ± 1.3 µM) and against CHIKV nsP2^pro(2.5 ± 0.4 µM). The IC₅₀for HSD against CHIKV nsP2^prowas 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.

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 (3CL^pro). 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 3CL^pro 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 3CL^pro. 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.

The C30 endopeptidase (3C-like protease; 3CL^pro) 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 3CL^pro.

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/NS3^pro by the two bioflavonoids with half maximal inhibitory concentration (IC₅₀) values <5 µM for HST and <70 µM for HSD. The determined dissociation constants (K_D) 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.