Dual Inhibition of Vacuolar-ATPase and TMPRSS2 Is Required for Complete Blockade of SARS-CoV-2 Entry into Cells.

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using nonspecific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high-throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

High-Level Oxygen Reduction Catalysts Derived from the Compounds of High-Specific-Surface-Area Pine Peel Activated Carbon and Phthalocyanine Cobalt.

Non-platinum carbon-based catalysts have attracted much more attention in recent years because of their low cost and outstanding performance, and are regarded as one of the most promising alternatives to precious metal catalysts. Activated carbon (AC), which has a large specific surface area (SSA), can be used as a carrier or carbon source at the same time. In this work, stable pine peel bio-based materials were used to prepare large-surface-area activated carbon and then compound with cobalt phthalocyanine (CoPc) to obtain a high-performance cobalt/nitrogen/carbon (Co-N-C) catalyst. High catalytic activity is related to increasing the number of Co particles on the large-specific-area activated carbon, which are related with the immersing effect of CoPc into the AC and the rational decomposed temperature of the CoPc ring. The synergy with N promoting the exposure of CoNx active sites is also important. The Eonset of the catalyst treated with a composite proportion of AC and CoPc of 1 to 2 at 800 °C (AC@CoPc-800-1-2) is 1.006 V, higher than the Pt/C (20 wt%) catalyst. Apart from this, compared with other AC/CoPc series catalysts and Pt/C (20 wt%) catalyst, the stability of AC/CoPc-800-1-2 is 87.8% in 0.1 M KOH after 20,000 s testing. Considering the performance and price of the catalyst in a practical application, these composite catalysts combining biomass carbon materials with phthalocyanine series could be widely used in the area of catalysts and energy storage.

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.