Activities within the SCORE consortium
1: Drug discovery in phenotypic assays
The SCORE project focuses on the development of Coronavirus-specific therapeutics and will do so
- short-term, by developing combinations of repurposed drugs;
- mid-term, by expediting studies on compounds already in development;
- longer-term, by pursuing de novo discovery and development of inhibitors.
This multi-pace approach is warranted because it is unclear at the moment whether the current virus will spread to cause a full-blown pandemic and/or continue to circulate for a prolonged time. Moreover, its animal reservoirs remain to be identified and resolved (if at all possible) and the current virus may evolve to further adapt to the human host, with uncertain consequences for its epidemiological profile and pathogenic potential. As exemplified by the prior SARS-CoV and MERS-CoV outbreaks, and the current SARS-CoV-2 epidemic, it is clearly only a matter of time until the next zoonotic coronavirus will appear on the world stage. Therefore, it is key to also invest in a longer-term effort with the purpose of developing novel, specific, and potent broad-spectrum inhibitors of coronavirus infection or replication.
2: Nucleoside/tide inhibitors
The spectrum of Nucleoside Analogue Inhibitors (NAIs) active against human pathogenic CoVs has been recently reviewed (27). There are five NAIs (Remdesivir, B-D-N4 hydroxycytidine, Gemcitabine, 6-Azauridine, Mizoribine, Acyclovir-Fleximer) showing moderate-to-potent activity against 3 human pathogenic CoVs (SARS-CoV, MERS-CoV, and HCoV-NL63). One striking feature of this activity spectrum is the structural peculiarity of analogues relative to their potency towards CoVs as well as other RNA viruses. The activity spectrum is linked to both the structure of the NAI and several yet-to-be determined MoAs of the nucleoside 5'-triphosphate analogues. Hence, a wide variety of different chemical modifications of NAIs (e.g., on the ribose, on the base, on both) lead to the observed antiviral effects for CoVs.
The NAI "classical" first inhibition mechanism is through termination of RNA synthesis, either obligate (immediate, e.g. the analogue lacks a 3'-OH), or non-obligate (the analogue is incorporated few times in a row and perturbates enough RNA synthesis to stop it). These analogues will be called Nucleotide Inhibitors (NIs). For HCV, the sofosbuvir-derived nucleotide acts through this mechanism.
There is a second mechanism by which nucleotide analogues can act: they do not terminate RNA synthesis, but get incorporated into the nascent RNA and thereby alter the genetic make-up of the virus, ultimately causing ‘lethal mutagenesis’. These analogues, which are not inhibitors of RNA synthesis, will be termed Mutagenic Nucleoside Analogues (MNAs). The viral RdRp’s (in-)fidelity is directly responsible for the antiviral effect of such analogues.
3: Inhibitors of viral proteases
The 5' two-thirds of the RNA genome of coronaviruses encode two large viral replicase polyproteins, pp1a and ppa1ab, which harbor the majority of protein components of the RNA-synthesizing machinery. These crucial polyproteins are processed by two viral proteases, the papain-like protease (PLpro in nsp3) and the main protease (Mpro, also called 3C-like protease, 3CLpro; in nsp5), to yield a total of 16 nonstructural proteins (nsps). Many of these proteins are crucial for viral RNA synthesis and genome expression, and their release from the polyprotein by proteolytic cleavage is critical for their maturation/activation (30). Therefore, inhibition of the function of PLpro and Mpro will block replication of the virus.
4: Entry inhibitors
To successfully initiate infection the membrane-enveloped coronaviruses need to deliver their genome into the host cell through a two-step process: receptor binding followed by fusion of the viral envelope and the cellular membrane (35). The spikes on the surface of coronavirus particles mediate this first and critical step in infection. Each spike consists of three spike (S) glycoprotein molecules, each containing the receptor-binding subunit S1 and the membrane-anchored subunit S2. The S1 subunits are used to select and bind the host cell by engaging the host receptor ACE2 for SARS-CoV-2 (36, 37). Upon receptor binding, the three S2 subunits - that form the stem of the spike - mediate membrane fusion. Two processes are key in the CoV membrane fusion event. (i) Cleavage of the Spike protein by cellular proteases is essential for activation (priming) of the fusion function. The Coronavirus spike (S) is unusual in that a range of different proteases (i.e. TMPRSS2, cathepsins and/or furin,(38) can cleave and activate it. These host proteases can cleave the Spike protein at or in the target cell and induce a so-called ‘metastable energy state’ that confers fusion capacity. A key feature of Coronavirus S is that the proteolytic cleavage events required for membrane fusion occur both at the interface of the receptor binding (S1) and fusion (S2) domains (S1/S2), as well as immediately upstream of a fusion peptide within S2 (S2′). (ii) Upon cleavage, the spikes can undergo conformational changes leading to virus-cell fusion. Rearrangement of the spike structure includes exposure of the hydrophobic fusion peptides that insert into the target membrane, followed by an S2 hairpinning event that merges the viral and cellular membrane. Hairpinning of S2 occurs through interaction of two ‘heptad repeat’ regions HR1 and HR2 into a highly stable HR1-HR2 six-helical bundle. Both the process of priming activation by proteolytic cleavage and that of stable bundle formation are required for membrane fusion (i.e. virus entry) and hence key targets for drug development.
5: nCoV toolbox and assay development
For SCORE partners (and external stakeholders), it is crucial that standardized tools, materials, assays and basic information on the SARS-CoV-2 replication cycle will become available, obtained using standardized protocols. For example, there is the need for a well-characterized standard strain/isolate, a panel of different clinical isolates and reporter-gene expressing viruses, cDNA clones for reverse genetics studies, protein expression constructs, purified proteins, antibodies, RNA standards, enzymatic and cell-based assays. WP5 aims to develop this 'nCoV toolbox' of SOPs, assays, protocols, and materials for studying virus replication, identifying inhibitors, and quantifying inhibitor activity and mode-of-action. These tools will be shared with other WPs (and the scientific community at large /stakeholders outside SCORE). WP5 will benefit from and build on the vast available Coronavirus expertise of our partners, e.g., on reverse genetics systems, on purification and characterization of viral proteins, and development of biochemical assays to study viral enzyme activities and develop assays suitable for use in high-throughput (HTP) screening. This is crucial for identifying inhibitors and to perform SAR (structure-activity relationship) analysis on compounds arising from the medicinal chemistry efforts that follow initial hit identification. The expert knowledge, abundant experience, state-of-the-art tools, and infrastructure already available should ensure a smooth start and high success rate in the development of crucial assays and materials within SCORE.
6: From lead to pre-clinical candidate and proof-of-concept in small-animal models
The most promising inhibitors of coronavirus replication emerging from WP1-4 will be evaluatedin cell culture and in vitro models to assess the many properties that determine whether a compound can be developed into an actual drug. In a next step, the compounds will be evaluated ex vivo in primary human airway epithelial cultures, which represent the human lung epithelium. Finally, we will assess the in vivo efficacy of the most promising compounds in two animal models (Syrian golden hamsters and humanized ACE2 mice) to obtain final proof-of-concept.