References for host proteases in SARS.

J Proteome Res. 2020 Nov 6; 19(11): 4316–4326.

Targeting Proteases for Treating COVID-19

Binquan Luan,† Tien Huynh,† Xuemei Cheng,‡ Ganhui Lan,§⊥ and Hao-Ran Wangcorresponding author*∥

Abstract

The unprecedented pandemic of coronavirus disease 2019 (COVID-19) demands effective treatment for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The infection of SARS-CoV-2 critically depends on diverse viral or host proteases, which mediate viral entry, viral protein maturation, as well as the pathogenesis of the viral infection. Endogenous and exogenous agents targeting for proteases have been proved to be effective toward a variety of viral infections ranging from HIV to influenza virus, suggesting protease inhibitors as a promising antiviral treatment for COVID-19. In this Review, we discuss how host and viral proteases participated in the pathogenesis of COVID-19 as well as the prospects and ongoing clinical trials of protease inhibitors as treatments.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640965/

Front Mol Biosci. 2020; 7: 215.Published online 2020 Oct 9.

COVID-19: Targeting Proteases in Viral Invasion and Host Immune Response

Sanchit Seth, Jyotsna Batra, and Srilakshmi Srinivasan*

Abstract

An acute respiratory disorder (COVID-19) that accelerated across the globe has been found to be caused by a novel strain of coronaviruses (SARS-CoV-2). The absence of a specific antiviral drug or vaccination has promoted the development of immediate therapeutic responses against SARS-CoV-2. As increased levels of plasma chemokines and, cytokines and an uncontrolled influx of inflammatory cells were observed in lethal cases, it was concluded that the severity of the infection corresponded with the imbalanced host immunity against the virus. Tracing back the knowledge acquired from SERS and MERS infections, clinical evidence suggested similar host immune reactions and host ACE2 receptor-derived invasion by SARS-CoV-2. Further studies revealed the integral role of proteases (TMPRSS2, cathepsins, plasmin, etc.) in viral entry and the immune system. This review aims to provide a brief review on the latest research progress in identifying the potential role of proteases in SARS-CoV-2 viral spread and infection and combines it with already known information on the role of different proteases in providing an immune response. It further proposes a multidisciplinary clinical approach to target proteases specifically, through a combinatorial administration of protease inhibitors. This predictive review may help in providing a perspective to gain deeper insights of the proteolytic web involved in SARS-CoV-2 viral invasion and host immune response.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581869/

Biochem Pharmacol. 2020 Dec; 182: 114225. Published online 2020 Sep 19.

Role of proteolytic enzymes in the COVID-19 infection and promising therapeutic approaches

Magda Gioia,a,⁎ Chiara Ciaccio,a,⁎ Paolo Calligari,b Giovanna De Simone,c Diego Sbardella,d Grazia Tundo,d Giovanni Francesco Fasciglione,a Alessandra Di Masi,c Donato Di Pierro,a Alessio Bocedi,b Paolo Ascenzi,c,e and Massimo Colettaa,⁎

Abstract

In the Fall of 2019 a sudden and dramatic outbreak of a pulmonary disease (Coronavirus Disease COVID-19), due to a new Coronavirus strain (i.e., SARS-CoV-2), emerged in the continental Chinese area of Wuhan and quickly diffused throughout the world, causing up to now several hundreds of thousand deaths.

As for common viral infections, the crucial event for the viral life cycle is the entry of genetic material inside the host cell, realized by the spike protein of the virus through its binding to host receptors and its activation by host proteases; this is followed by translation of the viral RNA into a polyprotein, exploiting the host cell machinery. The production of individual mature viral proteins is pivotal for replication and release of new virions.

Several proteolytic enzymes either of the host and of the virus act in a concerted fashion to regulate and coordinate specific steps of the viral replication and assembly, such as (i) the entry of the virus, (ii) the maturation of the polyprotein and (iii) the assembly of the secreted virions for further diffusion. Therefore, proteases involved in these three steps are important targets, envisaging that molecules which interfere with their activity are promising therapeutic compounds.

In this review, we will survey what is known up to now on the role of specific proteolytic enzymes in these three steps and of most promising compounds designed to impair this vicious cycle. FDA, Food and Drug Agency

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501082/

Life Sci Alliance. 2020 Sep; 3(9): e202000786.

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

Dorothea Bestle,1,* Miriam Ruth Heindl,1,* Hannah Limburg,1,* Thuy Van Lam van,2 Oliver Pilgram,2 Hong Moulton,3 David A Stein,3 Kornelia Hardes,2,4 Markus Eickmann,1,5 Olga Dolnik,1,5 Cornelius Rohde,1,5 Hans-Dieter Klenk,1 Wolfgang Garten,1 Torsten Steinmetzer,2 and Eva Böttcher-Friebertshäuser1

Abstract

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.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383062/

Virology. Volume 554, February 2021, Pages 48-54

Repurposing of renin inhibitors as SARS-COV-2 main protease inhibitors: A computational study

H. Refaey, Mohamed K El-Ashrey, Y. M. Nissan

Highlights

For rapid management of COVID-19, drug repurposing was widely carried out.
Renin inhibitors downregulate ACE2, decreasing the risk of SARS-COV-2 infection.
SARS-COV-2 main protease is a potential target for renin inhibitors.
Pharmacophore model was generated and virtually screened on renin inhibitors.
Remikiren was a hit, furtherly studied through molecular docking and dynamics.

Abstract

The COVID-19 pandemic has urged for the repurposing of existing drugs for rapid management and treatment. Renin inhibitors down regulation of ACE2, which is an essential receptor for SARS-CoV-2 infection that is responsible for COVID-19, in addition to their ability to act as protease inhibitors were encouraging aspects for their investigation as possible inhibitors of main protease of SARS-CoV-2 via computational studies. A Pharmacophore model was generated using the newly released SARS-COV-2 main protease inhibitors. Virtual screening was performed on renin inhibitors, and Drug likeness filter identified remikiren and 0IU as hits. Molecular docking for both compounds showed that the orally active renin inhibitor remikiren (Ro 42–5892) of Hoffmann–La Roche exhibited good molecular interaction with Cys145 and His41 in the catalytic site of SARS-CoV-2 main protease. Molecular dynamics simulation suggested that the drug is stable in the active site of the enzyme.

https://www.sciencedirect.com/science/article/pii/S0042682220302476?sid=SCITRUS

Study shows SARS-CoV-2 cell receptor is a novel target of the gamma-secretase enzyme complex

By Dr. Tomislav Meštrović, MD, Ph.D.Sep 6 2020

Based on structural similarities with other targets of the gamma-secretase system, researchers from Columbia University Irving Medical Center in the United States show that the SARS-CoV-2 receptor for cell entry may be affected by its proteolytic activity. Their results are currently freely available in a bioRxiv* preprint paper.

The coronavirus disease (COVID-19) health crisis, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused massive disruption worldwide with no drug or vaccine yet on sight. However, some primary research endeavors show promising results in the design of an effective and targeted therapeutic.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-linked ectoenzyme that processes Angiotensin II but also mediates the entry of SARS-CoV-2 into the human cell by means of binding the viral spike glycoprotein.

More specifically, spike glycoprotein binding to ACE2 sets off membrane fusion and viral entry, but only after spike priming by transmembrane protease serine 2 (TMPRSS2), which also cleaves the ectodomain of ACE2.

ACE2 cleavage (or shedding) can additionally be fostered by the disintegrin and metallopeptidase domain 17 (ADAM17), found to compete with TMPRSS2. Accordingly, there are conflicting reports of ADAM17-mediated shedding that affect coronavirus cell entry.

Human ACE2 receptor, 3D illustration. Image Credit: Kateryna Kon / Shutterstock

The role of gamma-secretase

The gamma-secretase (γS) protein complex is comprised of a Presenilin 1/2 aspartyl protease catalytic core with regulatory (Aph-1a or Aph-1b), enhancer (PEN2), and targeting (Nicastrin) subunits. In short, this is a prototype intramembrane-cleaving protease (I-CLiP).

Dozens of putative γS targets have been identified, determined by a rather specific transmembrane conformational structure and accessibility. Nonetheless, the validation of novel targets is hampered by the lack of definite common features and ectodomain shedding demands.

Based on the structural similarity of ACE2 to known γS targets, the researchers from the Columbia University Irving Medical Center in the United States (led by Dr. Alberto Bartolomé) hypothesized that γS can regulate intramembrane cleavage of ACE2 and may impact the biology of SARS-CoV-2.

From cell cultures to advanced methods

A myriad of different methods was used to study the aforementioned hypothesis. For cell culture purposes, the researchers have used Presenilin-deficient (Psen1/2 double knockout) and control mouse embryonic fibroblasts (MEFs), as well as Nicastrin knockout and control MEFs.

Furthermore, to test the hypothesis whether ACE2ΔE and γS physically interact, the researchers performed co-immunoprecipitation of endogenous γS with C-terminally tagged ACE2 and detected the association with both Nicastrin and Presenilin1 with ACE2ΔE, but not full-length ACE2.

Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus

Eric Bergeron 1, Martin J Vincent, Louise Wickham, Josée Hamelin, Ajoy Basak, Stuart T Nichol, Michel Chrétien, Nabil G Seidah

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS. Analysis of SARS-CoV spike glycoprotein (S) using recombinant plasmid and virus infections demonstrated that the S-precursor (proS) exists as a approximately 190 kDa endoplasmic reticulum form and a approximately 210 kDa Golgi-modified form. ProS is subsequently processed into two C-terminal proteins of approximately 110 and approximately 80 kDa. The membrane-bound proprotein convertases (PCs) furin, PC7 or PC5B enhanced the production of the approximately 80 kDa protein. In agreement, proS processing, cytopathic effects, and viral titers were enhanced in recombinant Vero E6 cells overexpressing furin, PC7 or PC5B. The convertase inhibitor dec-RVKR-cmk significantly reduced proS cleavage and viral titers of SARS-CoV infected cells. In addition, inhibition of processing by dec-RVKR-cmk completely abrogated the virus-induced cellular cytopathicity. A fluorogenically quenched synthetic peptide encompassing Arg(761) of the spike glycoprotein was efficiently cleaved by furin and the cleavage was inhibited by EDTA and dec-RVKR-cmk. Taken together, our data indicate that furin or PC-mediated processing plays a critical role in SARS-CoV spread and cytopathicity, and inhibitors of the PCs represent potential therapeutic anti-SARS-CoV agents.

https://pubmed.ncbi.nlm.nih.gov/15596135/

Int J Mol Sci. 2020 May; 21(10): 3622.

Proteasome Inhibitors as a Possible Therapy for SARS-CoV-2

Lucia Longhitano,1,† Daniele Tibullo,1,† Cesarina Giallongo,2,* Giacomo Lazzarino,3 Nicola Tartaglia,4 Sara Galimberti,5 Giovanni Li Volti,1,* Giuseppe Alberto Palumbo,2,‡ and Arcangelo Liso4,‡

Abstract

The COVID-19 global pandemic is caused by SARS-CoV-2, and represents an urgent medical and social issue. Unfortunately, there is still not a single proven effective drug available, and therefore, current therapeutic guidelines recommend supportive care including oxygen administration and treatment with antibiotics. Recently, patients have been also treated with off-label therapies which comprise antiretrovirals, anti-inflammatory compounds, antiparasitic agents and plasma from convalescent patients, all with controversial results. The ubiquitin–proteasome system (UPS) is important for the maintenance of cellular homeostasis, and plays a pivotal role in viral replication processes. In this review, we discuss several aspects of the UPS and the effects of its inhibition with particular regard to the life cycle of the coronaviruses (CoVs). In fact, proteasome inhibition by various chemical compounds, such as MG132, epoxomycin and bortezomib, may reduce the virus entry into the eucariotic cell, the synthesis of RNA, and the subsequent protein expression necessary for CoVs. Importantly, since UPS inhibitors reduce the cytokine storm associated with various inflammatory conditions, it is reasonable to assume that they might be repurposed for SARS-CoV-2, thus providing an additional tool to counteract both virus replication as well as its most deleterious consequences triggered by abnormal immunological response.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279248/

Life Sciences. Volume 258, 1 October 2020, 118170

Molecular profiling of immune cell-enriched Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interacting protein USP13

BurcuBiterge Süt

Abstract

Aims

Coronavirus disease 2019 (COVID-19), which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a major health concern worldwide. Due to the lack of specific medication and vaccination, drug-repurposing attempts has emerged as a promising approach and identified several human proteins interacting with the virus. This study aims to provide a comprehensive molecular profiling of the immune cell-enriched SARS-CoV-2 interacting protein USP13.

Materials and methods

The list of immune cell-enriched proteins interacting with SARS-CoV-2 was retrieved from The Human Protein Atlas. Genomic alterations were identified using cBioPortal. Survival analysis was performed via Kaplan-Meier Plotter. Analyses of protein expression and tumor infiltration levels were carried out by TIMER.

Key findings

14 human proteins that interact with SARS-CoV-2 were enriched in immune cells. Among these proteins, USP13 had the highest frequency of genomic alterations. Higher USP13 levels were correlated with improved survival in breast and lung cancers, while resulting in poor prognosis in ovarian and gastric cancers. Furthermore, copy number variations of USP13 significantly affected the infiltration levels of distinct subtypes of immune cells in head & neck, lung, ovarian and stomach cancers. Although our results suggested a tumor suppressor role for USP13 in lung cancer, in other cancers, its role seemed to be context-dependent.

Significance

It is critical to identify and characterize human proteins that interact with SARS-CoV-2 in order to have a better understanding of the disease and to develop better therapies/vaccines. Here, we provided a comprehensive molecular profiling the immune cell-enriched SARS-CoV-2 interacting protein USP13, which will be useful for future studies.

https://www.sciencedirect.com/science/article/abs/pii/S002432052030922X

Nat Commun. 2017 May 23;8:15534. doi: 10.1038/ncomms15534.

USP13 negatively regulates antiviral responses by deubiquitinating STING

He Sun 1, Qiang Zhang 1, Ying-Ying Jing 1, Man Zhang 1, Hai-Ying Wang 1, Zeng Cai 1, Tianzi Liuyu 1, Zhi-Dong Zhang 2, Tian-Chen Xiong 2, Yan Wu 3, Qi-Yun Zhu 4, Jing Yao 1, Hong-Bing Shu 2, Dandan Lin 5, Bo Zhong 1 2

Abstract

STING (also known as MITA) is critical for host defence against viruses and the activity of STING is regulated by ubiquitination. However, the deubiquitination of STING is not fully understood. Here, we show that ubiquitin-specific protease 13 (USP13) is a STING-interacting protein that catalyses deubiquitination of STING. Knockdown or knockout of USP13 potentiates activation of IRF3 and NF-κB and expression of downstream genes after HSV-1 infection or transfection of DNA ligands. USP13 deficiency results in impaired replication of HSV-1. Consistently, USP13 deficient mice are more resistant than wild-type littermates to lethal HSV-1 infection. Mechanistically, USP13 deconjugates polyubiquitin chains from STING and prevents the recruitment of TBK1 to the signalling complex, thereby negatively regulating cellular antiviral responses. Our study thus uncovers a function of USP13 in innate antiviral immunity and provides insight into the regulation of innate immunity.

https://pubmed.ncbi.nlm.nih.gov/28534493/

doi: https://doi.org/10.1101/2020.03.01.971499

Kallikrein 13: a new player in coronaviral infections

Aleksandra Milewska, Katherine Falkowski, Magdalena Kalinska, Ewa Bielecka, Antonina Naskalska, Pawel Mak, Adam Lesner, Marek Ochman, Maciej Urlik, Jan Potempa, Tomasz Kantyka, View ORCID ProfileKrzysztof Pyrc

This article is a preprint and has not been certified by peer review [what does this mean?].

ABSTRACT

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host specificity, but virus entry is a complex process requiring a concerted action of multiple cellular elements. Here, we show that KLK13 is required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that protease distribution and specificity predetermines the tissue and cell specificity of the virus and may also regulate interspecies transmission. It is also of importance that presented data may be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand the differences in their zoonotic potential.

https://www.biorxiv.org/content/10.1101/2020.03.01.971499v1.full

Sci Signal. 2020 Nov 24;13(659):eaba9902. doi: 10.1126/scisignal.aba9902.

Kallikrein 13 serves as a priming protease during infection by the human coronavirus HKU1

Aleksandra Milewska 1 2, Katherine Falkowski 2, Magdalena Kulczycka 3, Ewa Bielecka 3, Antonina Naskalska 1, Pawel Mak 4, Adam Lesner 5, Marek Ochman 6, Maciej Urlik 6, Elftherios Diamandis 7 8 9 10, Ioannis Prassas 8 10, Jan Potempa 2 11, Tomasz Kantyka 3 12, Krzysztof Pyrc 13

Abstract

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.

https://pubmed.ncbi.nlm.nih.gov/33234691/

Advances in Traditional Medicine (2020). Published: 06 October 2020

Matrix metalloproteinase inhibitors identified from Camellia sinensis for COVID-19 prophylaxis: an in silico approach

Nikita Kanbarkar & Sanjay Mishra

Abstract

To respond to the public panic, government and private research organizations of every country keep working on the COVID-19 pandemic, even though still there is a lack of more efficacious medicine for the choice of Coronavirus disease treatment. To counteract on this situation several approved drugs including anti-malarial (hydroxychloroquine and chloroquine), and few anti-viral (remdesvir) agents are choice of treatment for COVID-19. However, these agents suffer from certain limitation in their uses and pointed that there is no specific treatment or vaccine available to counter this contagious disease. Hence, there is urgent requirement to find a specific cure for the disease. In this view, there are several ongoing clinical trials of both western and traditional medicines. In present study, phytochemicals from Camellia sinensis were retrieved from the database and identified based on their ability to inhibit matrix metalloproteinase (MMPs) against SARS-CoV-2 main protease. Camellia sinensis entails of a massive number of phytochemicals with a good source of polyphenols such as Catechin, Epicatechin, Epigallocatechin and (–)-Epigallocatechin gallate. Molecular docking was performed using the GLIDE docking module of Schrodinger Suite software. The analysis displayed docking score for the five polyphenols i.e. theaflavin (− 8.701), 1-O-caffeoylquinic acid (− 7.795), Genistein (− 7.168), Epigallocatechin 3-gallate (− 6.282) and Ethyl trans-caffeate (− 5.356). Interestingly, theaflavin and Epigallocatechin 3-gallate have not revealed any side effects. These polyphenolic compounds had a strong binding affinity with hydrogen bonds and a good drug-likeness score. Therefore, Camellia sinensis could be the beneficial option in the prophylaxis of the COVID-19 outbreak.

https://www.google.com/search?rlz=1C1CHBF_esES924ES925&sxsrf=ALeKk00ltaAFZkDYtaLdfHOj4mNaf8QWZw%3A1610730912446&ei=oM0BYPbXGpGgUKGmr4AB&q=metalloproteinase+sars&oq=metalloproteinase+sars&gs_lcp=CgZwc3ktYWIQAzoECCMQJ1DWYVjWYWCdZWgAcAB4AIABlgGIAeEBkgEDMS4xmAEAoAEBqgEHZ3dzLXdpesABAQ&sclient=psy-ab&ved=0ahUKEwj2p_WeuJ7uAhUREBQKHSHTCxAQ4dUDCA0&uact=5

Antiviral Res. 2020 Nov;183:104867. doi: 10.1016/j.antiviral.2020.104867. Epub 2020 Aug 2.

Gamma secretase inhibition impairs HCMV replication by reduction of immediate early gene expression at the transcriptional level

Sun Min Lee 1, Dasol Han 1, Mookwang Kwon 1, Hogyun Noh 1, Ju Hyun Lee 1, Youngik Yoon 1, Jae Youl Cho 1, Jin-Hyun Ahn 2, Keejung Yoon 3

Abstract

Due to diverse pathogenic potentials, there is a growing need for anti-HCMV agents. In this study, we show that treatment with DAPT, a γ-secretase inhibitor (GSI), impairs HCMV replication as assessed by a progeny assay based on immunostaining. This effect is not limited to DAPT because other GSIs with different structures and distinct mechanisms of action also exhibit a similar level of inhibitory effects on HCMV viral production, indicating that γ-secretase activity is required for efficient HCMV replication. Western blot and qPCR analyses reveal that DAPT does not interfere with the viral entry process, but reduces expression of the immediate early protein IE1 at the transcriptional level. Furthermore, we exclude the possible involvement of Notch signaling pathway during HCMV replication by showing that expression of the dominant-negative form of MAML1, which disrupts the transactivational ability of Notch intracellular domain (NICD), does not reduce viral particle formation, and that NICD cannot rescue the DAPT-treated outcomes. Taken together, these findings indicate that γ-secretase activity plays an important role in a key step of the HCMV life cycle and γ-secretase inhibition could potentially be used as a novel preventive and therapeutic strategy against HCMV infection and HCMV-related diseases.

https://pubmed.ncbi.nlm.nih.gov/32755660/

Version 2. bioRxiv. Preprint. 2020 Jan 6.

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

Chunlong Ma,1 Michael D. Sacco,2 Brett Hurst,3,4 Julia A. Townsend,5 Yanmei Hu,1 Tommy Szeto,1 Xiujun Zhang,2 Bart Tarbet,3,4 Michael T. Marty,5 Yu Chen,2,* and Jun Wang1,*

Abstract

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), 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 6.96% as of May 4, 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 Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three monomers 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.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263507/

Sci Adv. 2020 Dec 9;6(50):eabe0751. doi: 10.1126/sciadv.abe0751. Print 2020 Dec.

Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M pro and cathepsin L

Michael Dominic Sacco 1, Chunlong Ma 2, Panagiotis Lagarias 3, Ang Gao 2, Julia Alma Townsend 4, Xiangzhi Meng 5, Peter Dube 5, Xiujun Zhang 1, Yanmei Hu 2, Naoya Kitamura 2, Brett Hurst 6 7, Bart Tarbet 6 7, Michael Thomas Marty 4, Antonios Kolocouris 3, Yan Xiang 5, Yu Chen 8, Jun Wang 9

Abstract

The main protease (Mpro) of SARS-CoV-2 is a key antiviral drug target. While most Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of Mpro in complex with calpain inhibitors II and XII and three analogs of GC-376 The structure of Mpro with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725459/

Biochem J. 2015 Jun 1; 468(2): 215–226.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

Miklós Békés,* Wioletta Rut,† Paulina Kasperkiewicz,† Monique P. C. Mulder,‡ Huib Ovaa,‡ Marcin Drag,† Christopher D. Lima,§ and Tony T. Huang*,1

Abstract

Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447217/

Comput Biol Chem. 2020 Dec; 89: 107376.

Identification of novel human USP2 inhibitor and its putative role in treatment of COVID-19 by inhibiting SARS-CoV-2 papain-like (PLpro) protease

Muhammad Usman Mirza,a Sarfraz Ahmad,b Iskandar Abdullah,b and Matheus Froeyena,*

Abstract

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb–palm–fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 μM) and MOTL-4 cells (11.8 μM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487165/

Br J Pharmacol. 2020 Nov; 177(21): 4971–4974.

Proteinase‐activated receptor 1: A target for repurposing in the treatment of COVID‐19?

Krishna Sriram 1 and Paul A. Inselcorresponding author 1 , 2

Abstract

In the search to rapidly identify effective therapies that will mitigate the morbidity and mortality of COVID‐19, attention has been directed towards the repurposing of existing drugs. Candidates for repurposing include drugs that target COVID‐19 pathobiology, including agents that alter angiotensin signalling. Recent data indicate that key findings in COVID‐19 patients include thrombosis and endotheliitis. Activation of proteinase‐activated receptor 1 (PAR1), in particular by the serine protease thrombin, is a critical element in platelet aggregation and coagulation. PAR1 activation also impacts on the actions of other cell types involved in COVID‐19 pathobiology, including endothelial cells, fibroblasts and pulmonary alveolar epithelial cells. Vorapaxar is an approved inhibitor of PAR1, used for treatment of patients with myocardial infarction or peripheral arterial disease. We discuss evidence for a possible beneficial role for vorapaxar in the treatment of COVID‐19 patients and other as‐yet non‐approved antagonists of PAR1 and proteinase‐activated receptor 4 (PAR4).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361899/

Int J Mol Sci. 2020 May; 21(10): 3622.

Proteasome Inhibitors as a Possible Therapy for SARS-CoV-2

Abstract

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279248/