A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells (original) (raw)
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A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells
ACS Chemical Biology
Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC 50 < 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC 90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC 50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC 50 was >10 μM. There was no toxicity to any of the host cell lines at 10−100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.
Biochemical and Biophysical Research Communications, 2021
There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.
Dual Inhibitors of Main Protease (M Pro ) and Cathepsin L as Potent Antivirals against SARS-CoV2
Journal of American Chemical Society, 2022
Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (M Pro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, M Pro is a novel therapeutic target. We identified two novel M Pro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the M Pro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PL Pro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC 50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of M Pro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.
Cysteine focused covalent inhibitors against the main protease of SARS-CoV-2
Journal of Biomolecular Structure and Dynamics, 2020
In viral replication and transcription, the main protease (Mpro) of SARS-CoV-2 plays an important role and appears to be a vital target for drug design. In Mpro, there is a Cys-His catalytic dyad, and ligands that interact with the Cys145 assumed to be an effective approach to inhibit the Mpro. In this study, approximately 1400 cysteine-focused ligands were screened to identify the best candidates that can act as potent inhibitors against Mpro. Our results show that the selected ligands strongly interact with the key Cys145 and His41 residues. Covalent docking was performed for the selected candidates containing the acrylonitrile group, which can form a covalent bond with Cys145. All atoms molecular dynamics (MD) simulation was performed on the selected four inhibitors including L1, L2, L3 and L4 to validate the docking interactions. Our results were also compared with a control ligand, a-ketoamide (11r). Principal component analysis on structural and energy data obtained from the MD trajectories shows that L1, L3, L4 and a-ketoamide (11r) have structural similarity with the apo-form of the Mpro. Quantitative structure-activity relationship method was employed for pattern recognition of the best ligands, which discloses that ligands containing acrylonitrile and amide warheads can show better performance. ADMET analysis displays that our selected candidates appear to be safer inhibitors. Our combined studies suggest that the best cysteine focused ligands can help to design an effective lead drug for COVID-19 treatment.
2022
Recurrent waves of SARS CoV2 infections remain a major global health concern. Emergence of highly infectious variants with reduced sensitivity to neutralization by vaccines and monoclonal antibodies (mAb) necessitates a deeper understanding of factors involved in SARS CoV2 infections and identification of drug candidates to halt infection. Here, we determined the primacy of endosomal protease cathepsin-L in mediating SARS CoV2 entry and screened a library of well-annotated bioactive compounds for potent cathepsin-L inhibitory activity. Whilst the potent cathepsin-L inhibitors were capable of inhibiting SARS CoV2 entry and cytopathic effect (CPE) in less susceptible cell lines such as human ACE2 expressing 293T cells, these drugs failed to inhibit SARS CoV2 in highly susceptible cell lines such as human TMPRSS2 or human-ACE2-TMPRSS2 overexpressing Vero E6 cells. Only drugs with dual inhibitory effect on both host cathepsin-L and virus 3CL-Protease enzymes such as Z-FA-FMK and GC-376 ...
Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19
ACS Infectious Diseases, 2021
Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both M pro and PL pro proteases. These efforts identified a small number of hits for the M pro protease and no viable hits for the PL pro protease. Of the M pro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead M pro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of M pro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting M pro and PL pro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.
Prospective Role of Peptide-Based Antiviral Therapy Against the Main Protease of SARS-CoV-2
Frontiers in Molecular Biosciences, 2021
The recently emerged coronavirus (SARS-CoV-2) has created a crisis in world health, and economic sectors as an effective treatment or vaccine candidates are still developing. Besides, negative results in clinical trials and effective cheap solution against this deadly virus have brought new challenges. The viral protein, the main protease from SARS-CoV-2, can be effectively targeted due to its viral replication and pathogenesis role. In this study, we have enlisted 88 peptides from the AVPdb database. The peptide molecules were modeled to carry out the docking interactions. The four peptides molecules, P14, P39, P41, and P74, had more binding energy than the rest of the peptides in multiple docking programs. Interestingly, the active points of the main protease from SARS-CoV-2, Cys145, Leu141, Ser139, Phe140, Leu167, and Gln189, showed nonbonded interaction with the peptide molecules. The molecular dynamics simulation study was carried out for 200 ns to find out the docked complex’s...
Infection and Drug Resistance
The current global pandemic of a novel severe acute respiratory syndrome coronavirus-2 continues with its public health disaster beginning from late December 2019 in Wuhan, Hubei province, China. The scientific community has tried to fight against this novel coronavirus through vaccine development and designing different candidate drugs. However, there is no well-defined therapy to prevent 2019-nCov infection, thus complete prevention of the virus remains difficult. Therefore, it is a critical factor for death of millions worldwide. Many clinical trials and insights are ongoing in the struggle with this pandemic of SARS-CoV-2. SARS-CoV-2 entry into the host cell requires host cell angiotensinconverting enzyme-2 (ACE2) and glucose regulated protein 78 (GRP78). On the other hand, proteolytic activation of the viral spike protein (S protein) needs the host cell serine proteases, including transmembrane serine protease 2 (TMPRSS2), cathepsins, trypsin and furin. This review focuses on the protein involved in the mechanism of entry, and proteolytic activation. In addition, it looks at current therapeutic options for SARS-CoV-2.
Validation and invalidation of SARS-CoV-2 papain-like protease inhibitors
2021
SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. The PLpro is involved in the cleavage of viral polyproteins as well as immune modulation through removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screening. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and the cell based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of the P...
Microorganisms, 2021
The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is considered the greatest challenge to the global health community of the century as it continues to expand. This has prompted immediate urgency to discover promising drug targets for the treatment of COVID-19. The SARS-CoV-2 viral proteases, 3-chymotrypsin-like protease (3CLpro) and papain-like cysteine protease (PLpro), have become the promising target to study due to their essential functions in spreading the virus by RNA transcription, translation, protein synthesis, processing and modification, virus replication, and infection of the host. As such, understanding of the structure and function of these two proteases is unavoidable as platforms for the development of inhibitors targeting this protein which further arrest the infection and spread of the virus. While the abundance of reports on the screening of natural compounds such as SARS-CoV-2 proteases inhibitors are available, the m...