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We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6′-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses.

BACKGROUND AND AIMS EPO resistance and iron deficiency in CKD-related anemia are associated with an increase in inflammatory cytokines associated with the innate immune response, such as IL-6, IL-1 and TNF-α. Recently (Bandach et al. Sci Reports 2021) we reported that kidney fibrosis and anemia of CKD can be worsened or relieved when IL1 effects are enhanced or inhibited respectively. The Water lily (Nuphar lutea) plant has been widely used as a traditional remedy for the treatment of rheumatism, scars, pain and more. Gopas, Golan-Goldhirsh et al. (Cancer Biology Therapy 2009) extracted an active ingredient from the family of nupharidines: 6,6′-Dihydroxythiobinupharidine (DTBN), which showed anti-inflammatory properties, mainly through the inhibition of NF-κB. The purpose of this study was to test the effects of DTBN in a mouse model of CKD-associated anemia. METHOD 8 weeks old male C57BL/6 mice were divided into three groups: control, CKD (induced by adenine diet) and CKD-DTBN. CKD groups were injected with saline-DMSO or 30 µg DTBN, every 2 days and sacrificed after 3 weeks from dietary intervention. RESULTS Serum urea and kidney TGFβ mRNA were significantly decreased in CKD- DTBN versus CKD. Kidney histology in CKD mice showed macrophage infiltration and renal fibrosis, which was ameliorated in CKD-DTBN. A significant improvement in inflammation indices (blood lymphocytes, kidney and liver IL-6 and p-STAT3, liver CRP), as well as kidney immunoreactive NF-κB and F4\80, which were increased in CKD, were decreased in CKD-DTBN. CKD anemia indices (hemoglobin, hematocrit and red blood cell (RBC) count) significantly improved in CKD-DTBN. Kidney HIF-2α and EPO mRNA were significantly decreased in both uremic groups, but was unchanged in CKD- DTBN versus CKD. However, serum iron and transferrin saturation significantly increased in CKD-DTBN versus CKD. CONCLUSION Uremic mice treated with DTBN show improvement in kidney fibrosis, inflammation and anemia indices. Thus, DTBN may be a novel therapeutic alternative for CKD and its complications.