Identification and characterization of the anti-SARS-CoV-2 activity of cationic amphiphilic steroidal compounds.

The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.

Upregulation of p38 pathway accelerates proliferation and migration of MDA-MB-231 breast cancer cells.

Tumor cells capture the signaling pathways used by normal tissue to promote their own survival and dissemination and among them, the NF-κB and MAPK pathways (ERK, JNK and p38). MAPK activation has ambiguous effects on tumor cell fate depending on cell type, cancer stage and the engaged MAPK isoforms. A synthetic peptide named LyeTx II, derived from the venom of the Brazilian spider Lycosa erythrognatha, was capable of increasing MDA-MB-231 aggressive breast cancer cell proliferation as indicated by MTT and BrdU (5-bromo-2'-deoxyuridine) incorporation assay and cell migration. A correlation has been established between the accelerated proliferation and migration observed in the presence of LyeTx II and the upregulation of p38 MAPK phosphorylation. The use of the selective inhibitor of p38α/ß (SB203580) abrogated the peptide effect in MDA-MB-231 cells. Besides, an augment of the canonical NF-κB pathway activation considered as crucial in cancer progression was noted after cell incubation with LyeTx II. Importantly, activation of p38 and NF-κB pathways was dependent on TAK1 activity. Together, these data suggest that TAK1-p38 pathway may represent an interesting target for treatment of aggressive breast cancers.

Differential use of TLR2 and TLR9 in the regulation of immune responses during the infection with Trypanosoma cruzi.

Pathogens express ligands for several TLRs that may play a role in the induction or control of the inflammatory response during infection. Concerning Trypanosoma cruzi, the agent of Chagas disease, we have previously characterized glycosylphosphatidylinositol (GPI) anchored mucin-like glycoproteins (tGPI-mucin) and unmethylated CpG DNA sequences as TLR2 and TLR9 agonists, respectively. Here we sought to determine how these TLRs may modulate the inflammatory response in the following cell populations: F4/80(+)CD11b(+) (macrophages), F4/80(low)CD11b(+) (monocytes) and MHCII(+)CD11c(high) (dendritic cells). For this purpose, TLR2(-/-) and TLR9(-/-) mice were infected with Y strain of T. cruzi and different immunological parameters were evaluated. According to our previous data, a crucial role of TLR9 was evidenced in the establishment of Th1 response, whereas TLR2 appeared to act as immunoregulator in the early stage of infection. More precisely, we demonstrated here that TLR2 was mainly used by F4/80(+)CD11b(+) cells for the production of TNF-α. In the absence of TLR2, an increased production of IL-12/IL-23p40 and IFN-γ was noted suggesting that TLR2 negatively controls the Th1 response. In contrast, TLR9 was committed to IL-12/IL-23p40 production by MHCII(+)CD11c(high) cells that constitute the main source of IL-12/IL-23p40 during infection. Importantly, a down-regulation of TLR9 response was observed in F4/80(+)CD11b(+) and F4/80(low)CD11b(+) populations that correlated with the decreased TLR9 expression level in these cells. Interestingly, these cells recovered their capacity to respond to TLR9 agonist when MHCII(+)CD11c(high) cells were impeded from producing IL-12/IL-23p40, thereby indicating possible cross-talk between these populations. The differential use of TLR2 and TLR9 by the immune cells during the acute phase of the infection explains why TLR9- but not TLR2-deficient mice are susceptible to T. cruzi infection.