RESUMO
Pathologic hyperreactive inflammatory responses occur when there is excessive activation of a proinflammatory NF-κB pathway and a reduced cytoprotective NRF2 cascade. The noncytotoxic, highly selective COX-2 inhibitory flavonol-enriched butanol fraction (UaB) from Uvaria alba (U. alba) was investigated for its inflammatory modulating potential by targeting NF-κB activation and NRF2 activity. Enzyme-linked immunosorbent assay was initially performed to measure levels of proinflammatory mediators [nitric oxide (NO), prostaglandin E2, and reactive oxygen species (ROS)] and cytokines [tumor necrosis factor-alpha (TNF-α), IL-1ß, and IL-6], followed by reverse transcription-polymerase chain reaction and western blotting to determine mRNA and protein expression, respectively. Using immunofluorescence staining combined with western blot analysis, the activation of NF-κB was further investigated. NRF2 activity was also measured using a luciferase reporter assay. UaB abrogated protein and mRNA expressions of inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1ß, and IL-6 in RAW 264.7 macrophages, thereby suppressing the production of proinflammatory mediators and cytokines. This was further validated when a concentration-dependent decrease in NO and ROS production was observed in zebrafish (Danio rerio) larvae. UaB also increased NRF2 activity in HaCaT/ARE cell line and attenuated NF-κB activation by inhibiting the nuclear translocation of transcription factor p65 in RAW 264.7 macrophages. Nontargeted LC-MS analysis of UaB revealed the presence of the flavonols quercitrin (1), quercetin (2), rutin (3), kaempferol (4), and kaempferol 3-O-rutinoside (5). Molecular docking indicates that major flavonol aglycones have high affinity toward COX-2 NSAID-binding sites, TNF-α, and TNF-α converting enzyme, while the glycosylated flavonoids showed strong binding toward iNOS and IKK-all possessing dynamic stability when performing molecular dynamics simulations at 140 ns. This is the first report to have elucidated the mechanistic anti-inflammatory potential of the Philippine endemic plant U. alba.
RESUMO
The novel coronavirus SARS-CoV2, the causative agent of the pandemic disease COVID-19, emerged in December 2019 forcing lockdown of communities in many countries. The absence of specific drugs and vaccines, the rapid transmission of the virus, and the increasing number of deaths worldwide necessitated the discovery of new substances for anti-COVID-19 drug development. With the aid of bioinformatics and computational modelling, ninety seven antiviral secondary metabolites from fungi were docked onto five SARS-CoV2 enzymes involved in viral attachment, replication, post-translational modification, and host immunity evasion infection mechanisms followed by molecular dynamics simulation and in silico ADMET prediction (absorption, distribution, metabolism, excretion and toxicity) of the hit compounds. Thus, three fumiquinazoline alkaloids scedapin C (15), quinadoline B (19) and norquinadoline A (20), the polyketide isochaetochromin D1 (8), and the terpenoid 11a-dehydroxyisoterreulactone A (11) exhibited high binding affinities on the target proteins, papain-like protease (PLpro), chymotrypsin-like protease (3CLpro), RNA-directed RNA polymerase (RdRp), non-structural protein 15 (nsp15), and the spike binding domain to GRP78. Molecular dynamics simulation was performed to optimize the interaction and investigate the stability of the top-scoring ligands in complex with the five target proteins. All tested complexes were found to have dynamic stability. Of the five top-scoring metabolites, quinadoline B (19) was predicted to confer favorable ADMET values, high gastrointestinal absorptive probability and poor blood-brain barrier crossing capacities.Communicated by Ramaswamy H. Sarma.
