The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

In December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines.In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2’ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication.Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.

Hyperfibrinolytic situations can lead to lifethreatening bleeding, especially during cardiac surgery. The approved antifibrinolytic agents such as tranexamic acid, εaminocaproic acid, 4-aminomethylbenzoic acid, and aprotinin were developed in the 1960s without the structural insight of their respective targets. Crystal structures of the main antifibrinolytic targets, the lysine binding sites on plasminogen's kringle domains, and plasmin's serine protease domain greatly contributed to the structure-based drug design of novel inhibitor classes. Two series of ligands targeting the lysine binding sites have been recently described, which are more potent than the most-widely used antifibrinolytic agent, tranexamic acid. Furthermore, four types of promising active site inhibitors of plasmin have been developed: tranexamic acid conjugates targeting the S1 pocket and primed sites, substrate-analogue linear homopiperidylalanine-containing 4amidinobenzylamide derivatives, macrocyclic inhibitors addressing nonprimed binding regions, and bicyclic 14-mer SFTI-1 analogues blocking both, primed and nonprimed binding sites of plasmin. Furthermore, several allosteric plasmin inhibitors based on heparin mimetics have been developed.

Albumin is an exquisite tool of nature used in biomedicine to achieve long blood residence time for drugs, but the payload it can carry is typically limited to one molecule per protein. In contrast, synthetic macromolecular prodrugs contain multiple copies of drugs per polymer chain but offer only a marginal increase in the circulation lifetime of the drugs. We combine the benefits of the two platforms and at the same time overcome their respective limitations. Specifically, we develop the synthesis of albumin–polymer–drug conjugates to obtain long circulating, high payload drug delivery vehicles. In vivo data validate that albumin endows the conjugate with a blood residence time similar to that of the protein and well exceeding that of the polymer. Therapeutic activity of the conjugates is validated using prodrugs of panobinostat, an HIV latency reversal agent, in which case the conjugates matched the drug in terms of efficacy of treatment.

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 lM in HIEC-6. These inhibitors did not cause elevations in extracellular H 2 O 2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 lM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 lM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.