SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

Directly Bridging Indoles to 3,3'-Bisindolylmethanes by Using Carboxylic Acids and Hydrosilanes under Mild Conditions.

A straightforward Lewis acid-promoted protocol for 3,3'-bisindolylmethanes (BIMs) synthesis by reductive alkylation of indoles at the C3 position with carboxylic acids in the presence of hydrosilane was developed for the first time. Instead of aldehydes, more readily available, stable, and easy-to-handle carboxylic acids have been employed as alternative alkylating agents. As an efficient organocatalyst, B(C6 F5 )3 enables the reductive alkylation of various substituted indole derivatives with carboxylic acids with up to 98 % yield at room temperature and under neat conditions. This metal-free strategy offers an alternative approach for the direct functionalization of indoles to BIMs with carboxylic acids and such protocol allows selective reduction of carboxylic acid to aldehyde in combination with C-C bond formation.

Diosgenin inhibits tumor necrosis factor-induced tissue factor activity and expression in THP-1 cells via down-regulation of the NF-κB, Akt, and MAPK signaling pathways.

AIM: To investigate whether diosgenin could modulate tissue factor (TF) procoagulation activity, expression, and related signal transduction pathways. METHODS: Human THP-1 monocytic cells were exposed to tumor necrosis factor-α (TNF-α, 10 ng·mL(-1)) with or without diosgenin (0.01, 0.1, and 1 µmol · L(-1)) for 2 h or 5 h to induce TF procoagulant activity and expression, which were determined by the simplified chromogenic assay, reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR, and Western blotting assays. In addition, the activation of the NF-κB, Akt, and MAPK signaling pathways were also measured by Western blotting. RESULTS: Diosgenin significantly inhibited TNF-α-induced TF procoagulant activity at concentrations of 0.01 to 1 µmol · L(-1) with IC50 of 0.25 µmol · L(-1). It also reduced protein expression and mRNA accumulation of TF dose-dependently in activated THP-1 cells. TNF-α stimulated significantly phosphorylation on Ser536 of NF-κB/p65, Ser473 of Akt at 5-15 min, and activations of IKK-ß and ERK at 15-30 min. Diosgenin (1 µmol · L(-1)) could inhibit the phosphorylation of NF-κB/p65, IKK-ß, Akt, ERK, and JNK, but had no remarkable effects on IκB and p38 phosphorylation in THP-1 cells. CONCLUSION: Diosgenin inhibits TNF-α-induced TF activity and expression in monocytes, partly due to its down-regulation of the phosphorylation of NF-κB/p65, IKK-ß, Akt, ERK, and JNK.

Hypoxia-inducible factor-1α dependent pathways mediate the renoprotective role of acetazolamide against renal ischemia-reperfusion injury.

BACKGROUND/AIMS: Acute kidney injury (AKI) is a major complication of kidney transplantation, resulting in early graft dysfunction. Since diuretic acetazolamide (AZA) has been shown to improve contrast induced AKI, we hypothesized that AZA also protected against ischemia-reperfusion (I/R) caused AKI. METHODS: An in vivo mouse renal I/R injury model and an in vitro H2O2 stimulated HK-2 cell injury model were utilized to examine the renoprotective effect of AZA. Renal injury and blood flow were measured. Nitric oxide synthase (eNOS)/Nitric oxide (NO), cell apoptosis and hypoxia-inducible factor-1α (HIF-1α) changes were analyzed. RESULTS: AZA reduced kidney injury scores and improved renal function by decreasing serum creatinine and BUN levels after I/R. Impaired renal blood flow was restored by increasing eNOS activities and NO production, as indicated by Laser Doppler imaging. TUNEL staining presented that AZA reduced apoptotic cells due to attenuated caspase activation and increased Bcl-2/Bax ratio. Furthermore, HIF-1α induction by AZA was demonstrated. AZA also enhanced in vitro NO production, reduced cell apoptosis and increased HIF-1α expression. Knockdown of HIF-1α by RNAi confirmed that AZA exerted its protective role depending on HIF-1α. AZA's effects were significantly reduced by Akt inhibitor LY294002. CONCLUSIONS: The present study demonstrated that AZA exerted a renoprotective role against I/R induced AKI through activating HIF-1α and downstream pathways.

Roles of vimentin and 14-3-3 zeta/delta in the inhibitory effects of heparin on PC-3M cell proliferation and B16-F10-luc-G5 cells metastasis.

AIM: To investigate the inhibitory effects of heparin on PC-3M cells proliferation in vitro and B16-F10-luc-G5 cells metastasis in Balb/c nude mice and identify the protein expression patterns to elucidate the action mechanism of heparin. METHODS: Human prostate cancer PC-3M cells were incubated with heparin 0.5 to 125 µg/mL for 24 h. The proliferation of PC-3M cells was assessed by MTS assay. BrdU incoporation and Ki67 expression were detected using a high content screening (HCS) assay. The cell cycle and apoptosis of PC-3M cells were tested by flow cytometry. B16-F10-luc-G5 cardinoma cells were injected into the lateral tail vein of 6-week old male Balb/c nude mice and heparin 30 mg/kg was administered iv 30 min before and 24 h after injection. The metasis of B16-F10-luc-G5 cells was detected by bioluminescence assay. Activated partial thromboplastin time (APTT) and hemorheological parameters were measured on d 14 after injection of B16-F10-luc-G5 carcinoma cells in Balb/c mice. The global protein changes in PC-3M cells and frozen lung tissues from mice burdened with B16-F10-luc-G5 cells were determined by 2-dimensional gel electrophoresis and image analysis. The protein expression of vimentin and 14-3-3 zeta/delta was measured by Western blot. The mRNA transcription of vimentin, transforming growth factor (TGF)-ß, E-cadherin, and α(v)-integrin was measured by RT-PCR. RESULTS: Heparin 25 and 125 µg/mL significantly inhibited the proliferation, arrested the cells in G(1) phase, and suppressed BrdU incorporation and Ki67 expression in PC-3M cells compared with the model group. But it had no significant effect on apoptosis of PC-3M cells. Heparin 30 mg/kg markedly inhibits the metastasis of B16-F10-luc-G5 cells on day 8. Additionally, heparin administration maintained relatively normal red blood hematocrit but had no influence on APTT in nude mice burdened with B16-F10-luc-G5 cells. Thirty of down-regulated protein spots were identified after heparin treatment, many of which are related to tumor development, extracellular signaling, energy metabolism, and cellular proliferation. Vimentin and 14-3-3 zeta/delta were identified in common in PC-3M cells and the lungs of mice bearing B16-F10-luc-G5 carcinoma cells. Heparin 25 and 125 µg/mL decreased the protein expression of vimentin and 14-3-3 zeta/delta and the mRNA expression of α(v)-integrin. Heparin 125 µg/mL decreased vimentin and E-cadherin mRNA transcription while increased TGF-ß mRNA transcription in the PC-3M cells, but the differences were not significant. Transfection of vimentin-targeted siRNA for 48 h significantly decreased the BrdU incoporation and Ki67 expression in PC-3M cells. CONCLUSION: Heparin inhibited PC-3M cell proliferation in vitro and B16-F10-luc-G5 cells metastasis in nude mice by inhibition of vimentin, 14-3-3 zeta/delta, and α(v)-integrin expression.