Boceprevir, Calpain Inhibitors II and XII, and GC-376 Have Broad-Spectrum Antiviral Activity against Coronaviruses.

As the COVID-19 pandemic continues to unfold, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII, and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. In this study, we further characterized the mechanism of action of these four compounds using the SARS-CoV-2 pseudovirus neutralization assay. It was found that GC-376 and calpain inhibitors II and XII have a dual mechanism of action by inhibiting both viral Mpro and host cathepsin L in Vero cells. To rule out the cell-type dependent effect, the antiviral activity of these four compounds against SARS-CoV-2 was also confirmed in type 2 transmembrane serine protease-expressing Caco-2 cells using the viral yield reduction assay. In addition, we found that these four compounds have broad-spectrum antiviral activity in inhibiting not only SARS-CoV-2 but also SARS-CoV, and MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift-binding assay and enzymatic fluorescence resonance energy transfer assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 might be promising starting points for further development against existing human coronaviruses as well as future emerging CoVs.

Vitamin D, zinc and glutamine: Synergistic action with OncoTherad immunomodulator in interferon signaling and COVID­19 (Review).

Coronavirus disease 2019 (COVID­19), caused by severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2), was identified in December, 2019 in Wuhan, China. Since then, it has continued to spread rapidly in numerous countries, while the search for effective therapeutic options persists. Coronaviruses, including SARS­CoV­2, are known to suppress and evade the antiviral responses of the host organism mediated by interferon (IFN), a family of cytokines that plays an important role in antiviral defenses associated with innate immunity, and has been used therapeutically for chronic viral diseases and cancer. On the other hand, OncoTherad, a safe and effective immunotherapeutic agent in the treatment of non­muscle invasive bladder cancer (NMIBC), increases IFN signaling and has been shown to be a promising therapeutic approach for COVID­19 in a case report that described the rapid recovery of a 78­year­old patient with NMIBC with comorbidities. The present review discusses the possible synergistic action of OncoTherad with vitamin D, zinc and glutamine, nutrients that have been shown to facilitate immune responses mediated by IFN signaling, as well as the potential of this combination as a therapeutic option for COVID­19.

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development.

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 µM), calpain inhibitors II and XII (IC50 ~20-25 µM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human ß-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.

Degradation of bacterial permeability family member A1 (BPIFA1) by house dust mite (HDM) cysteine protease Der p 1 abrogates immune modulator function.

Bacterial permeability family member A1 (BPIFA1) is one of the most abundant proteins present in normal airway surface liquid (ASL). It is known to be diminished in asthmatic patients' sputum, which causes airway hyperresponsiveness (AHR). What is currently unclear is how environmental factors, such as allergens' impact on BPIFA1's abundance and functions in the context of allergic asthma. House dust mite (HDM) is a predominant domestic source of aeroallergens. The group of proteases found in HDM is thought to cleave multiple cellular protective mechanisms, and therefore foster the development of allergic asthma. Here, we show that BPIFA1 is cleaved by HDM proteases in a time-, dose-, and temperature-dependent manner. We have also shown the main component in HDM that is responsible for BPIFA1's degradation is Der p1. Fragmented BPIFA1 failed to bind E. coli lipopolysaccharide (LPS), and hence elevated TNFα and IL-6 secretion in human whole blood. BPIFA1 degradation is also observed in vivo in bronchoalveolar fluid (BALF) of mice which are intranasally instilled with HDM. These data suggest that proteases associated with environmental allergens such as HDM cleave BPIFA1 and therefore impair its immune modulator function.

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease.

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.