Functional Protease Enzymes to Expedite SARS-CoV-2 Drug Discovery

SARS-CoV-2, commonly known as COVID19, is a newly discovered novel coronavirus and infectious disease for which no approved vaccines or antiviral drugs exist.

Since the initial outbreak and subsequent COVID19 pandemic, an international community of medical researchers have accelerated their efforts on an unprecedented scale in an attempt to uncover and identify small molecule inhibitors of SARS-CoV-2. The aim is to ultimately prevent viral entry and replication by targeting key enzymes crucial for the viral replication cycle and any virus-host cell interaction. Currently, some of the leading antiviral drugs in the market target two significant viral protease enzymes among the coronavirus family: papain-like protease (PLpro) and 3C-like protease (3CLpro).

Since the beginning of the SARS-CoV-2 outbreak, SignalChem, a worldwide leader in drug discovery, cell signaling, and protein engineering, has engaged in heavy research that focuses on new and novel coronavirus strains. So as to successfully engineer active recombinant SARS-CoV-2 and COVID-19 related human proteins, scientists at SignalChem have used their resources to better comprehend the protein interaction map of SARS-CoV-2.

The goal is to offer researchers across the globe vital biochemical tools essential for their ongoing research programs and potential drug discovery. Using highly-developed protein engineering technology and advanced AI drug development software, SignalChem has initiated projects advancing the expression systems of key SARS-CoV-2 related proteins, including sought-after drug targets like protease enzymes PLpro and 3CLpro. These active proteins are fully functional and ready to be included in current research projects focused on drug discovery directed against SARS-CoV-2 and other coronavirus infections.

SARS-CoV-2 3CLpro, active (C19CL-G241H)

SignalChem’s Recombinant full-length 2019-nCoV 3CLpro, part of a large replicase polyprotein 1ab (S3264-Q3569), was expressed in E. coli cells with an N-terminal His tag.

The main proteinase (Mpro) found in SARS-CoV-2 is known as 3-Chymotrypsin like protease (3CLpro), a cysteine protease enzyme crucial in proteolytic processing of viral polyproteins, proteins essential for viral replication and function. 3CLpro cleaves the C-terminus of the large replicase polyprotein 1ab (790 kDa) at more than 11 different sites. Therefore, it can be assumed that the inhibitors that obstruct the cleavage function of 3CLpro will impede virus replication, making this protease enzyme one of the major targets advised for drug discovery development and the potent treatment of COVID19.

The sequences of 3CLpro found in both SARS-CoV-2 and the already known SARS-CoV share 96% identity, and the great structural parallel between the two protease enzymes was also verified by the recently resolved crystal structure of SARS-CoV-2 3CLpro. The amalgamation of protease enzyme inhibitors lopinavir/ritonavir (LPV/r), which have exhibited activity against retroviral infections including SARS-CoV-2, have recently been put forward as potential effective treatments for COVID19.

Regarding the therapeutic treatment of a COVID19 patient in South Korea, a case report emphasized that administration of LPV/r to the patient notably diminished the viral loads with little to no coronavirus titers observed upon further treatment.

Protease enzyme activity of 3CLpro (SignalChem Catalog No. C19CL-G241H), as detected in a FRET-based assay.

Figure 1. Protease enzyme activity of 3CLpro (SignalChem Catalog No. C19CL-G241H), as detected in a FRET-based assay. Image Credit: SignalChem Biotech Inc.

SARS-CoV-2 PLpro, active (C19PL-G241H)

SignalChem’s Recombinant 2019-nCoV PLpro/papain-like protease, part of a large replicase polyprotein 1ab (E1564-Y1882), was expressed in E. coli cells with an N-terminal His tag.

SARS-CoV-2 contains a multifunctional cysteine protease enzyme, the papain-like protease (PLpro), that processes both host cell proteins and viral polyprotein by hydrolyzing the peptide and isopeptide bonds in cellular and viral substrates inducing the virus replication. Thus, PLpro identifies the tetrapeptide LXGG motif established at three sites of the viral polyprotein and unleashes its cleavage activity to produce mature non-structural proteins (nsp) 1, 2 and 3, all of which play specific roles in the life cycle of the virus.

Moreover, PLpro has the ability to suppress the host immune response against viral infection, either by affecting the ubiquitin and/or ISG15-dependent pathways through its deubiquitinating and deISGylating activities, or by interfering with the production of cytokines and chemokines that control the activation of an innate immune response.

Previous attempts to design inhibitors against SARS-CoV PLpro produced encouraging results, thus, new efforts in drug discovery projects seeking SARS-CoV-2 PLpro-targeted therapeutics for Covid19 are currently gathering a lot of speed. Several research groups have announced the effectiveness of drugs already approved and in circulation as potential inhibitors against the novel coronavirus, SARS-CoV-2.

Included in this list are anti-SARS drugs (GRL-0667, GRL-0617, and Mycophenolic acid), anti-HCV drugs (Grazoprevir, Telaprevir, and Boceprevir) and those used for a wide range of therapeutic treatments (e.g. Formoterol, Chloroquine). One particular drug, isotretinoin, a medication predominantly used to treat severe acne, is currently being tested in four clinical trials for its effectiveness to treat COVID-19.

Activity profiles of PLpro (SignalChem Catalog No. C19PL-G241H). A. Deubiquitinase activity of PLpro detected in a bioluminescence assay. B. PLpro inhibition profile of PR-619, a broad-spectrum DUB inhibitor. C. Protease activity of PLpro detected in a FRET-based assay.

Figure 2. Activity profiles of PLpro (SignalChem Catalog No. C19PL-G241H). A. Deubiquitinase activity of PLpro detected in a bioluminescence assay. B. PLpro inhibition profile of PR-619, a broad-spectrum DUB inhibitor. C. Protease activity of PLpro detected in a FRET-based assay. Image Credit: SignalChem Biotech Inc.

About SignalChem Biotech Inc.

SignalChem is a biotech company focused on the research, development, and production of innovative and high-quality human recombinant cell signaling products.

Throughout the years, Signalchem has capitalized on its core expertise in cellular signaling, molecular biology, and protein biochemistry to generate more than 2,000 functional protein products and has established itself as the leader in the industry to produce highly active human recombinant signaling enzymes, especially protein kinases, disease-related mutant kinases, lipid kinases, epigenetic enzymes, ubiquitination-related enzymes, and neurodegenerative disease-related enzymes and proteins.

SignalChem strives to support scientists in academia, pharma and biotech companies around the world by creating effective research tools to advance the basic research in life sciences and to facilitate the efforts in drug discovery and development.


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Last updated: Oct 9, 2020 at 10:52 AM

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