The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

Inflammatory responses by the innate and adaptive immune systems protect against infections and are essential to health and survival. Many diseases including atherosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, and obesity involve persistent chronic inflammation. Currently available anti-inflammatory agents, including non-steroidal anti-inflammatory drugs, steroids, and biologics, are often unsafe for chronic use due to adverse effects. The development of effective non-toxic anti-inflammatory agents for chronic use remains an important research arena. We previously reported that oral administration of Oxy210, a semi-synthetic oxysterol, ameliorates non-alcoholic steatohepatitis (NASH) induced by a high-fat diet in APOE*3-Leiden.CETP humanized mouse model of NASH and inhibits expression of hepatic and circulating levels of inflammatory cytokines. Here, we show that Oxy210 also inhibits diet-induced white adipose tissue inflammation in APOE*3-Leiden.CETP mice, evidenced by the inhibition of adipose tissue expression of IL-6, MCP-1, and CD68 macrophage marker. Oxy210 and related analogs exhibit anti-inflammatory effects in macrophages treated with lipopolysaccharide in vitro, mediated through inhibition of toll-like receptor 4 (TLR4), TLR2, and AP-1 signaling, independent of cyclooxygenase enzymes or steroid receptors. The anti-inflammatory effects of Oxy210 are correlated with the inhibition of macrophage polarization. We propose that Oxy210 and its structural analogs may be attractive candidates for future therapeutic development for targeting inflammatory diseases.

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has motivated the clinical development of Smoothened (Smo) antagonists, such as vismodegib and sonidegib. However, Smo antagonists have failed to benefit patients suffering from Hh pathway-dependent solid tumors, such as pancreatic, colorectal, or ovarian cancer. Hh-dependent cancers are often driven by activating mutations that occur downstream of Smo and directly activate the transcription factors known as glioma-associated oncogenes (Gli1-3). Hence, the direct targeting of Gli could be a more effective strategy for achieving disease modification compared to Smo antagonism. In this study, we report on the biological and pharmacological evaluation of Oxy186, a semisynthetic oxysterol analogue, as a novel inhibitor of Hh signaling acting downstream of Smo, with encouraging drug-like properties. Oxy186 exhibits strong inhibition of ligand-induced Hh signaling in NIH3T3-E1 fibroblasts, as well as in constitutively activated Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells. Oxy186 also inhibits Gli1 transcriptional activity in NIH3T3-E1 cells expressing exogenous Gli1 and Gli-dependent reporter constructs. Furthermore, Oxy186 suppresses Hh signaling in PANC-1 cells, a human pancreatic ductal adenocarcinoma (PDAC) tumor cell line, as well as PANC-1 cell proliferation in vitro, and in human lung cancer cell lines, A549 and H2039.

Non-Small Cell Lung Cancer (NSCLC) is a common malignancy and leading cause of death by cancer. Metastasis and drug resistance are serious clinical problems encountered in NSCLC therapy. Aberrant activation of the Transforming Growth Factor beta (TGFβ) and Hedgehog (Hh) signal transduction cascades often associate with poor prognosis and aggressive disease progression in NSCLC, as these signals can drive cell proliferation, angiogenesis, metastasis, immune evasion and emergence of drug resistance. Therefore, simultaneous inhibition of TGFβ and Hh signaling, by a single agent, or in combination with other drugs, could yield therapeutic benefits in NSCLC and other cancers. In the current study, we report on the biological and pharmacological evaluation of Oxy210, an oxysterol-based dual inhibitor of TGFβ and Hh signaling. In NSCLC cells, Oxy210 inhibits proliferation, epithelial-mesenchymal transition (EMT) and invasive activity. Combining Oxy210 with Carboplatin (CP) increases the anti-proliferative response to CP and inhibits TGFβ-induced resistance to CP in A549 NSCLC cells. In addition, Oxy210 displays encouraging drug-like properties, including chemical scalability, metabolic stability and oral bioavailability in mice. Unlike other known inhibitors, Oxy210 antagonizes TGFβ and Hh signaling independently of TGFβ receptor kinase inhibition and downstream of Smoothened, respectively.

Background and aims: We previously reported that Oxy210, an oxysterol-based drug candidate, exhibits antifibrotic and anti-inflammatory properties. We also showed that, in mice, it ameliorates hepatic hallmarks of non-alcoholic steatohepatitis (NASH), including inflammation and fibrosis, and reduces adipose tissue inflammation. Here, we aim to investigate the effects of Oxy210 on atherosclerosis, an inflammatory disease of the large arteries that is linked to NASH in epidemiologic studies, shares many of the same risk factors, and is the major cause of mortality in people with NASH. Methods: Oxy210 was studied in vivo in APOE*3-Leiden.CETP mice, a humanized mouse model for both NASH and atherosclerosis, in which symptoms are induced by consumption of a high fat, high cholesterol “Western” diet (WD). Oxy210 was also studied in vitro using two cell types that are important in atherogenesis: human aortic endothelial cells (HAECs) and macrophages treated with atherogenic and inflammatory agents. Results: Oxy210 reduced atherosclerotic lesion formation by more than 50% in hyperlipidemic mice fed the WD for 16 weeks. This was accompanied by reduced plasma cholesterol levels and reduced macrophages in lesions. In HAECs and macrophages, Oxy210 reduced the expression of key inflammatory markers associated with atherosclerosis, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), vascular cell adhesion molecule-1 (VCAM-1), and E-Selectin. In addition, cholesterol efflux was significantly enhanced in macrophages treated with Oxy210. Conclusions: These findings suggest that Oxy210 could be a drug candidate for targeting both NASH and atherosclerosis, as well as chronic inflammation associated with the manifestations of metabolic syndrome.