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*∥
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.
J Genet Genomics. 2020 Feb 20; 47(2): 119–121.
Potential inhibitors against 2019-nCoV coronavirus M protease from clinically approved medicines
Xin Liu, Xiu-Jie Wang.
Starting from December 2019, a novel coronavirus, named 2019-nCoV, was found to cause Severe Acute Respiratory (SARI) symptoms and rapid pandemic in China. With the hope to identify candidate drugs for 2019-nCoV, we adopted a computational approach to screen for available commercial medicines which may function as inhibitors for the Mpro of 2019-nCoV. Up to 10 commercial medicines that may form hydrogen bounds to key residues within the binding pocket of 2019-nCoV Mpro were identified, which may have higher mutation tolerance than lopinavir/ritonavir and may also function as inhibitors for other coronaviruses with similar Mpro binding sites and pocket structures.
J Med Chem. 2016 Jul 28; 59(14): 6595–6628.
An Overview of Severe Acute Respiratory Syndrome–Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy
Thanigaimalai Pillaiyar,corresponding author*† Manoj Manickam,∥ Vigneshwaran Namasivayam,† Yoshio Hayashi,§ and Sang-Hun Jung∥
Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 (10–15%) fatalities in 2003. The causative agent of SARS has been identified as a novel human coronavirus (SARS-CoV), and its viral protease, SARS-CoV 3CLpro, has been shown to be essential for replication and has hence been recognized as a potent drug target for SARS infection. Currently, there is no effective treatment for this epidemic despite the intensive research that has been undertaken since 2003 (over 3500 publications). This perspective focuses on the status of various efficacious anti-SARS-CoV 3CLpro chemotherapies discovered during the last 12 years (2003–2015) from all sources, including laboratory synthetic methods, natural products, and virtual screening. We describe here mainly peptidomimetic and small molecule inhibitors of SARS-CoV 3CLpro. Attempts have been made to provide a complete description of the structural features and binding modes of these inhibitors under many conditions.
ACS Pharmacol Transl Sci. 2020 Aug 4;3(5):1017-1019. doi: 10.1021/acsptsci.0c00093. eCollection 2020 Oct 9.
Can Papain-like Protease Inhibitors Halt SARS-CoV-2 Replication?
Biplab K Maiti 1
SARS-CoV-2 encoded papain-like protease (PLpro) harbors a labile Zn site (Cys189-X-X-Cys192-X n -Cys224-X-Cys226) and a classic catalytic site (Cys111-His272-Asp286), which play key roles for viral replication and hence represent promising drug targets. In this Viewpoint, both sulfur-based drugs and peptides-based inhibitors may block Cys residues in the catalytic and/or Zn site of CoV-2-PLpro, leading to dysfunction of CoV-2-PLpro and thereby halting viral replication.
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,*
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.
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
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.
Virol Sin. 2020 Sep 10 : 1–9.
Comparative Antiviral Efficacy of Viral Protease Inhibitors against the Novel SARS-CoV-2 In Vitro
Leike Zhang,#1 Jia Liu,#1 Ruiyuan Cao,#2 Mingyue Xu,1 Yan Wu,1 Weijuan Shang,1 Xi Wang,1 Huanyu Zhang,1 Xiaming Jiang,1 Yuan Sun,1 Hengrui Hu,1 Yufeng Li,1 Gang Zou,3 Min Zhang,2 Lei Zhao,2 Wei Li,2 Xiaojia Guo,2 Xiaomei Zhuang,2 Xing-Lou Yang,1 Zheng-Li Shi,1 Fei Deng,1 Zhihong Hu,1 Gengfu Xiao,corresponding author1 Manli Wang,corresponding author1 and Wu Zhongcorresponding author2
The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is of urgent demand to cure the huge number of patients. Virus-encoded proteases are considered potential drug targets. The human immunodeficiency virus protease inhibitors (lopinavir/ritonavir) has been recommended in the global Solidarity Trial in March launched by World Health Organization. However, there is currently no experimental evidence to support or against its clinical use. We evaluated the antiviral efficacy of lopinavir/ritonavir along with other two viral protease inhibitors in vitro, and discussed the possible inhibitory mechanism in silico. The in vitro to in vivo extrapolation was carried out to assess whether lopinavir/ritonavir could be effective in clinical. Among the four tested compounds, lopinavir showed the best inhibitory effect against the novel coronavirus infection. However, further in vitro to in vivo extrapolation of pharmacokinetics suggested that lopinavir/ritonavir could not reach effective concentration under standard dosing regimen [marketed as Kaletra®, contained lopinavir/ritonavir (200 mg/50 mg) tablets, recommended dosage is 400 mg/10 mg (2 tablets) twice daily]. This research concluded that lopinavir/ritonavir should be stopped for clinical use due to the huge gap between in vitro IC50 and free plasma concentration. Nevertheless, the structure–activity relationship analysis of the four inhibitors provided further information for de novel design of future viral protease inhibitors of SARS-CoV-2.
Bioorg Med Chem Lett. 2020 Sep 1; 30(17): 127377.
The SARS-CoV-2 main protease as drug target
Sven Ullrich and Christoph Nitsche⁎
The unprecedented pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is threatening global health. The virus emerged in late 2019 and can cause a severe disease associated with significant mortality. Several vaccine development and drug discovery campaigns are underway. The SARS-CoV-2 main protease is considered a promising drug target, as it is dissimilar to human proteases. Sequence and structure of the main protease are closely related to those from other betacoronaviruses, facilitating drug discovery attempts based on previous lead compounds. Covalently binding peptidomimetics and small molecules are investigated. Various compounds show antiviral activity in infected human cells.
Virol J. 2020 Nov 26;17(1):190. doi: 10.1186/s12985-020-01457-0.
Analysis of the efficacy of HIV protease inhibitors against SARS-CoV-2’s main protease
Mohamed Mahdi 1, János András Mótyán 2, Zsófia Ilona Szojka 2 3, Mária Golda 2 3, Márió Miczi 2 3, József Tőzsér 2
Background: The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of infections worldwide. While the search for an effective antiviral is still ongoing, experimental therapies based on repurposing of available antivirals is being attempted, of which HIV protease inhibitors (PIs) have gained considerable interest. Inhibition profiling of the PIs directly against the viral protease has never been attempted in vitro, and while few studies reported an efficacy of lopinavir and ritonavir in SARS-CoV-2 context, the mechanism of action of the drugs remains to be validated.
Methods: We carried out an in-depth analysis of the efficacy of HIV PIs against the main protease of SARS-CoV-2 (Mpro) in cell culture and in vitro enzymatic assays, using a methodology that enabled us to focus solely on any potential inhibitory effects of the inhibitors against the viral protease. For cell culture experiments a dark-to-bright GFP reporter substrate system was designed.
Results: Lopinavir, ritonavir, darunavir, saquinavir, and atazanavir were able to inhibit the viral protease in cell culture, albeit in concentrations much higher than their achievable plasma levels, given their current drug formulations. While inhibition by lopinavir was attributed to its cytotoxicity, ritonavir was the most effective of the panel, with IC50 of 13.7 µM. None of the inhibitors showed significant inhibition of SARS-CoV-2 Mpro in our in vitro enzymatic assays up to 100 µM concentration.
Conclusion: Targeting of SARS-CoV-2 Mpro by some of the HIV PIs might be of limited clinical potential, given the high concentration of the drugs required to achieve significant inhibition. Therefore, given their weak inhibition of the viral protease, any potential beneficial effect of the PIs in COVID-19 context might perhaps be attributed to acting on other molecular target(s), rather than SARS-CoV-2 Mpro.