Evaluation of molecular mechanisms of riboflavin anti-COVID-19 action reveals anti-inflammatory efficacy rather than antiviral activity.

BACKGROUND: Riboflavin (vitamin B2) is one of the most important water-soluble vitamins and a coenzyme involved in many biochemical processes. It has previously been shown that adjuvant therapy with flavin mononucleotide (a water-soluble form of riboflavin) correlates with normalization of clinically relevant immune markers in patients with COVID-19, but the mechanism of this effect remains unclear. Here, the antiviral and anti-inflammatory effects of riboflavin were investigated to elucidate the molecular mechanisms underlying the riboflavin-induced effects. METHODS: Riboflavin was evaluated for recombinant SARS-CoV-2 PLpro inhibition in an enzyme kinetic assay and for direct inhibition of SARS-CoV-2 replication in Vero E6 cells, as well as for anti-inflammatory activity in polysaccharide-induced inflammation models, including endothelial cells in vitro and acute lung inflammation in vivo. RESULTS: For the first time, the ability of riboflavin at high concentrations (above 50 µM) to inhibit SARS-CoV-2 PLpro protease in vitro was demonstrated; however, no inhibition of viral replication in Vero E6 cells in vitro was found. At the same time, riboflavin exerted a pronounced anti-inflammatory effect in the polysaccharide-induced inflammation model, both in vitro, preventing polysaccharide-induced cell death, and in vivo, reducing inflammatory markers (IL-1ß, IL-6, and TNF-α) and normalizing lung histology. CONCLUSIONS: It is concluded that riboflavin reveals anti-inflammatory rather than antiviral activity for SARS-CoV-2 infection. GENERAL SIGNIFICANCE: Riboflavin could be suggested as a promising compound for the therapy of inflammatory diseases of broad origin.

Lysine oxidase exposes a dependency on the thioredoxin antioxidant pathway in triple-negative breast cancer cells.

PURPOSE: Transformed cells are vulnerable to depletion of certain amino acids. Lysine oxidase (LO) catalyzes the oxidative deamination of lysine, resulting in lysine depletion and hydrogen peroxide production. Although LO has broad antitumor activity in preclinical models, the cytotoxic mechanisms of LO are poorly understood. METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control media, lysine-free media or control media supplemented with LO and examined for cell viability, caspase activation, induction of reactive oxygen species (ROS) and antioxidant signaling. To determine the role of nuclear factor erythroid 2-related factor 2 (NRF2) and thioredoxin reductase-1 (TXNRD1) in LO-induced cell death, NRF2 and TXNRD1 were individually silenced by RNAi. Additionally, the pan-TXNRD inhibitor auranofin was used in combination with LO. RESULTS: LO activates caspase-independent cell death that is suppressed by necroptosis and ferroptosis inhibitors, which are inactive against lysine depletion, pointing to fundamental differences between LO and lysine depletion. LO rapidly induces ROS with a return to baseline levels within 24 h that coincides temporally with induction of TXNRD activity, the rate-limiting enzyme in the thioredoxin antioxidant pathway. ROS induction is required for LO-mediated cell death and NRF2-dependent induction of TXNRD1. Silencing NRF2 or TXNRD1 enhances the cytotoxicity of LO. The pan-TXNRD inhibitor auranofin is synergistic with LO against transformed breast epithelial cells, but not untransformed cells, underscoring the tumor-selectivity of this strategy. CONCLUSIONS: LO exposes a redox vulnerability of TNBC cells to TXNRD inhibition by rendering tumor cells dependent on the thioredoxin antioxidant pathway for survival.

Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking.

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

Potato mop-top virus: the coat protein-encoding RNA and the gene for cysteine-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana.

Full-length genomic cDNA clones of the Swedish isolate of Potato mop-top virus (PMTV) were transcribed in vitro using T7 RNA polymerase. The combination of RNA 1, 2 and 3 synthesized in the presence of m(7)GpppG cap analogue was infectious when inoculated onto Nicotiana benthamiana plants. Also, the combination of RNA 1 (encodes the viral replicase) with RNA 3 [encodes the triple gene block proteins and a small cysteine-rich protein (CRP)] was infectious and both RNAs moved systemically in N. benthamiana plants in the absence of RNA 2, which encodes the coat protein (CP). However, the yellow mosaic symptoms that typically developed following PMTV infection with all three RNAs were not observed in plants infected with RNA 1+RNA 3. Site-directed mutagenesis experiments revealed that expression of the putative CRP was not required for systemic infection and symptom induction in N. benthamiana. These data show that PMTV represents an example of a multipartite virus capable of establishing systemic infection without the CP-encoding RNA, and also without the putative CRP.