Chandra Mohan, Ph.D. Biology.Technical Marketing MilliporeSigma, Temecula, CA, A business of Merck KGaA, Darmstadt, Germany
Wayne K. Way, Ph.D. Head of Protein Preparation and Reagents, MilliporeSigma, Bellefonte, PA A business of Merck KGaA, Darmstadt, Germany
SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, has caused millions of infections and more than five million deaths worldwide to date. Although the speed with which COVID-19 vaccines have been developed is remarkable, the long-term protection and effectiveness they provide against emerging and potential future variants of SARS-CoV-2 and other coronaviruses remains to be determined. Considering this, directly acting anti-viral therapeutics are being explored as drug candidates. Viral protease inhibitors have enabled therapies for viral infections such as HIV, so the essential role of cysteine proteases in the maturation and replication of SARS-CoV-2 makes them an attractive target for an anti-viral therapeutic against COVID-19.
The role and mechanism of cysteine protease activation
Cysteine proteases are involved in several cellular functions, including extracellular matrix turnover, antigen presentation, digestion, immune invasion, and the processing of surface proteins. These protein-degrading hydrolase enzymes are divided into at least 21 families on the basis of their sequences or tertiary structures. Nearly half of these families occur in known viruses.
Cysteine proteases are synthesized as zymogens containing a regulatory pro-domain region that serves as an endogenous inhibitor of the mature enzyme and blocks the access of substrate to the active site. Enzyme activation occurs following the removal of the pro-domain within a subcellular compartment or the extracellular environment. Within the acidic environment of lysosomes, cysteine proteases are activated by controlled proteolysis that involves either autocatalysis or transactivation. Autocatalysis involves the cleavage of the pro-domain by a catalytic site present inside the catalytic cleft of the enzyme under the influence of pH change. Transactivation involves cleavage by another molecule of the same enzyme or by the action of other proteases that cleave within the residues lying at the junction of the pro-domain and the mature domain. The active site of cysteine proteases contains a Cys-His-Asn triad where the histidine residue serves as a proton donor and enhances the nucleophilicity of the cysteine residue.
The cysteine proteases of SARS-CoV-2
The COVID-19 pandemic has generated a lot of interest in the role of cysteine proteases in processing SARS-CoV-2 proteins. To reach the replication stage, the SARS-CoV-2 genome produces 15 non-structural proteins (NSPs) from two large polyproteins, pp1a and pp1ab, which are processed by two main cysteine proteases that are reported to be essential for the maturation and the proliferation cycles of the coronavirus: chymotrypsin-like main protease (3CLpro or Mpro) and papain-like protease (PLpro). 3CLpro cuts at eleven sites in the nonstructural polyprotein pp1a. Proper polyprotein processing is reported to be essential for the release and maturation of the 15 NSPs and assembly into multicomponent replicase-transcriptase complexes (RTCs) that are responsible for guiding the replication, transcription, and maturation of the viral genome.
Although 3CLpro and PLpro process the viral polyprotein in a coordinated manner, PLpro performs several additional functions. In addition to processing pp1a/pp1ab, it also has deubiquitinating activity and disassembles mono-, di-, and polyubiquitin chains. Ubiquitin contains the PLpro recognition motif at its C-terminal region, and deubiquitination in host cells interferes with host response to viral infection. PLpro is also reported to inactivate TANK-binding kinase 1 (TBK1), to block NF-κB signaling, to prevent translocation of interferon regulatory factor 3 (IRF3), and to inhibit TLR7 signaling. Hence, in addition to its role in processing viral polyproteins, PLpro also acts on the host cell proteins to disrupt the host’s viral response machinery and thus to facilitate viral proliferation and replication. Due to its key role in viral replication, PLpro has emerged as an excellent candidate for a therapeutic against SARS-CoV-2. Studies are also underway to repurpose existing drug substances to block the catalytic dyad (His41 and Cys145) of 3CLpro as well as the catalytic triad (Cys111, His272, and Asp286) of PLpro. If successful, these efforts will pave the way for rapid in-vitro and in-vivo studies to battle the SARS-CoV-2 or future coronavirus related infections.
Reagents for protease research
As with the development of vaccines against SARS-CoV-2, time is of the essence. To support and speed up the drug discovery and screening process, several specific products are now available on the market. These include research grade proteases such as Mpro, 3CL (SAE0172, Sigma-Aldrich), protease inhibitors such as SARS-CoV-2 main protease inhibitor (SML2877, Sigma-Aldrich) as well as protease detection kits. One kit, the SARS-CoV-2 (COVID-19) Inhibitors Screening Kit (CS2000, Sigma-Aldrich), is designed to simplify the identification and characterization of SARS-CoV-2 inhibitors.
References
Cho, C-C., et al. (2022). ChemMed Chem. 17; e202100455.
Mellott, DM., et al. (2021). ACS Chem. Biol. 16; 642-650.
Mengist, HM., et al. (2021). Front. Chem. 9; 622898.
Osipiuk, J., et al. (2021). Nat. Commun. 12; Article number 743.
Rut, W., et al. (2020). Sci. Adv. 6(42); abd4596.
Verma, S., et al. (2016). Front. Pharmacol. 7; 107.
Baez-Santos, YM., et al. (2015). Antiviral Res. 115; 21-38
Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered right to your inbox!
Sign up now!