Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

Abstract

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

“…Our research group has developed an His 6 -MBP-FP-based, fluorescent protease assay for HTS-compatible substrate screening, the designed RFP substrate system has been successfully applied to study HIV-1 and TEV PRs [ 13 ], Ty1 retrotransposon PR [ 17 ], human paternally expressed gene 10 (PEG10) [ 16 ], the main protease of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [ 18 ] and the non-structural protein 2 protease of the alphavirus Venezuelan equine encephalitis virus (VEEV) [ 19 ]. The RFP substrates have versatile applications, can be digested in-solution, can be used in microbead-based assays and the product formation can be detected using either electrophoresis or fluorimetry.…”

“…Western-blot may be also used for fusion tag-specific detection (e.g., using anti-MBP antibody) [ 16 ]. The magnetic bead-based protease assay can be performed both in microcentrifuge-tubes [ 17 , 18 ] and in 96-well microtiter plates [ 15 , 19 ]. Purified enzymes are used in the conventional setup, but cleavage reactions may be performed in total cell lysates [ 16 ], as well.…”

Potential 3‐chymotrypsin‐like cysteine protease cleavage sites in the coronavirus polyproteins pp1a and pp1ab and their possible relevance to COVID‐19 vaccine and drug development

“…Meanwhile, a comparison based on the data 58,59 shows the absence of nsp11 and nsp16, and total 14 nsps in both SARS-CoV and SARS-CoV-2. 10 The absence of nsp16 in pp1ab draws our attention, leading us to explore nsp16 in the next section.…”

“…The 3‐chymotrypsin (C)‐like cysteine protease (3CL pro ) of coronaviruses (CoVs) is a target for developing antiviral drugs against SARS‐CoV, 1‐3 MERS‐CoV, 4‐6 and SARS‐CoV‐2 7‐10 . CoVs encode four structural and accessory proteins: spike protein (S‐protein), envelope protein (E‐protein), membrane protein (M‐protein), and nucleocapsid protein (N‐protein); and two replicase polyproteins (pp1a and pp1ab).…”

Molecular docking, DFT analysis, and dynamics simulation of natural bioactive compounds targeting ACE2 and TMPRSS2 dual binding sites of spike protein of SARS CoV-2

“…In recent times, in silico analysis is considered as one of the suitable and fastest approaches to screen the innumerable number of therapeutic molecules and/or drugs in order to discover the new drug for emerging diseases. Therefore, recently, numerous in-silico docking studies were carried out and published, primarily targeting the spike (S) protein [12] , [13] , main protease [14] , [15] , [16] , [17] , [18] , [19] , N protein [20] , [21] , [22] , [23] , and RNA-dependent RNA polymerase amongst others. Despite consistent scientific efforts around the world to contrive potential drug molecules, antibody cocktails, and vaccines against potential targets of this tiny virus, there have not been successful findings of the effective drug candidate for COVID-19 till date.…”