SARS-CoV-2: An Update on Key Targets, Trends and Discoveries

Since the emergence of SARS-CoV-2 the effort among researchers finding ways to stop the virus has been unprecedented. Huge advances have been made in our knowledge of both the virus (SARS-CoV-2) and associated disease (COVID-19). These advances highlight that infectious disease research must receive more attention to further develop the resources available in this field.

This blog draws on recent trends in the published literature, as well as discussion forums, to highlight important known and emerging targets for SARS-CoV-2 research. For an introduction to SARS-CoV-2 and coronaviruses see our previous blog post on the subject.

SARS-CoV-2 Viral Replication

SARS-CoV-2 relies on viral replication machinery to proliferate and transcribe the 30kb positive sense RNA genome. This is performed by the replication-transcription complex (RTC), comprised of non-structural proteins (NSP)7-16. Each NSP has a defined function within the complex and as such may constitute a possible target for inhibiting viral replication. The most well understood NSP in the RTC is NSP12, or RNA-dependent RNA polymerase (RdRp). This target has received the most attention, as among many RNA viruses it is seen as the engine of the RTC. A range of nucleosides have been developed against viral RdRps including favipiravir (Cat. No. 7225), galidesivir, EIDD-2801 (the prodrug of EIDD 1931, Cat. No. 7231) and remdesivir (Cat. No. 7226). These compounds are incorporated into viral RNA, mimicking the natural substrate of RdRp, where they cause lethal mutation or termination of the RNA chain. Remdesivir is the first antiviral to receive FDA approval for the treatment of COVID-19.

Other components of the RTC are also potential targets for SARS-CoV-2 antivirals. NSP14, a viral exonuclease (proofreading enzyme), ensures low error rates and high replicative fidelity of the viral genome. This target is particularly relevant since effective incorporation of nucleoside analogues into the viral genome requires evasion of this proofreading mechanism. Another essential component in the RTC is NSP13, the viral helicase. Structural insights into how this protein interacts with RdRp has been achieved with cryo-EM and in turn could help guide development of new antivirals.

SARS-CoV-2 Viral Proteases

Inhibitors of viral proteases have emerged as important therapeutic targets against a number of different viruses. The first demonstration of this was saquinavir (Cat. No. 4418), approved for the treatment of HIV in 1995. More recently, structure-based design has been used to develop inhibitors for coronavirus proteases such as Mpro N3 (Cat. No. 7230) and Mpro 13b (Cat. No. 7228).

Figure 1: The SARS-CoV-2 3CL protease in complex with inhibitor Mpro 13b (Cat. No. 7228). Adapted from Zhang et aI (2020) Science 368 409. PDB ID: 6Y2F.

There are two essential proteases encoded in the SARS-CoV-2 genome, the 3CL protease (also known as M^pro) encoded by NSP5, and papain-like protease (PLpro) encoded within NSP3. These enzymes process the polyproteins pp1a and pp1ab into their functional components. Currently there is only one active clinical trial targeting the SARS-CoV-2 viral proteases, which is using an M^pro inhibitor, PF-007304814, a prodrug of a compound originally developed during the SARS-CoV outbreak of 2002/2003.

Aside from its role in polyprotein processing, PLpro possesses deISGylase and deubiquitinase activity. This enables the viral protease to suppress the type I interferon response by cleaving ISG15 (a ubiquitin-like modification) from IRF3; a signal that would otherwise instigate an immune response within the cell. Owing to these multiple roles within the viral lifecycle, PLpro could potentially be an Achilles heels of SAR-CoV-2. Several potent inhibitors have demonstrated efficacy against PLpro in vitro, including GRL 0617 (Cat. No. 7280) and PLpro inhibitor 6 (Cat. No. 7357).

To facilitate the efforts of researchers studying the SARS-CoV-2 proteases, recombinant versions of these proteins are now also commercially available from our sister brand, R&D Systems.

Viral Cell Entry

The primary host cell receptor for binding of the SARS-CoV and SARS-CoV-2 spike (S) protein is the angiotensin-converting enzyme 2 (ACE2) receptor.

For several coronaviruses transmembrane protease serine 2 (TMPRSS2) plays an important role in processing the S2 domain of the S protein, required for fusion to the host cell. Camostat (Cat. No. 3193), an orally active serine protease inhibitor approved in Japan for the treatment of chronic pancreatitis and postoperative reflux esophagitis, has been shown to inhibit viral replication in vitro and in animal studies. Several clinical trials have been initiated to determine whether camostat can be repurposed as an antiviral against SARS-CoV-2.

Understanding differences between viral entry mechanisms could identify new targets against SARS-CoV-2. A key difference between the S protein of SARS-CoV and SARS-CoV-2, is the presence of a furin cleavage site at the S1-S2  boundary in SARS-CoV-2. This is thought to be an important factor in the enhanced mechanism for infectivity. It has recently been shown that the furin cleavage sequence complies to the "C-end rule" (CendR), enabling binding of the processed S protein to neuropilin-1 (NRP1), a host cell membrane receptor. EG 00229 (Cat. No. 6986), a small molecule antagonist, has been shown to inhibit NRP1-S protein binding and reduce infectivity in vitro.

 Emerging Targets

Several exciting technologies targeting the viral lifecycle have also been applied to SARS-CoV-2 research. Professor Matthew Disney’s group at Scripps Research Institute have turned their RIBOTAC method for achieving targeted RNA degradation towards the SARS-CoV-2 genome. Through binding the attenuator hairpin (an internal loop of RNA) within the frameshifting element, the group was able to recruit a cellular nuclease to degrade the viral RNA.

Another new modality with potential in SARS-CoV-2 research and therapy is de novo protein design. Professor David Baker’s group have used their in silico design software to create miniproteins that tightly bind to the receptor binding domain (RBD) of the S protein and prevent binding to the ACE2 receptor. They are now moving towards clinical trials where they envisage the protein being used in an intranasal spray that would act as a prophylactic against viral infection.

If you would like to learn more about the latest thinking around SARS-CoV-2, watch the NIH Virtual SARS-CoV-2 Antiviral Therapeutics Summit on demand.

References

Yang, H et al (2005) Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biology 3 e324.

Zhou, Y et al (2015) Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research 116 76–84. 

Cao, L et al (2020) De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370 426–431. 

Daly, J et al (2020) Neuropilin-1 is a host factor for SARS-CoV-2 infection. Science 370 861–865. 

Haniff, HS et al (2020) Targeting the SARS-COV-2 RNA genome with small molecule binders and ribonuclease targeting chimera (RiboTAC) degraders. ACS Central Science 6 1713–1721. 

Freitas, BT et al (2020) Characterization and noncovalent inhibition of the deubiquitinase and deISGylase activity of SARS-CoV-2 papain-like protease. ACS Infectious Diseases 6 2099–2109. 

Chen, J et al (2020) Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex. Cell 182, 1560. 

Hoffmann, M et al (2020) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181 271. 

Shannon, J et al (2020) Remdesivir and SARS-CoV-2: structural requirements at both nsp12 RdRp and nsp14 exonuclease active-sites. Antiviral Research 178 104793.

Source: Tocris