Accelerating antiviral drug discovery: lessons from COVID-19

Abstract

During the coronavirus disease 2019 (COVID-19) pandemic, a wave of rapid and collaborative drug discovery efforts took place in academia and industry, culminating in several therapeutics being discovered, approved and deployed in a 2-year time frame. This article summarizes the collective experience of several pharmaceutical companies and academic collaborations that were active in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral discovery. We outline our opinions and experiences on key stages in the small-molecule drug discovery process: target selection, medicinal chemistry, antiviral assays, animal efficacy and attempts to pre-empt resistance. We propose strategies that could accelerate future efforts and argue that a key bottleneck is the lack of quality chemical probes around understudied viral targets, which would serve as a starting point for drug discovery. Considering the small size of the viral proteome, comprehensively building an arsenal of probes for proteins in viruses of pandemic concern is a worthwhile and tractable challenge for the community.

Fig. 1. Key targets in the SARS-CoV-2 replication cycle.

a, Stage 1: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell upon binding to the extracellular receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). Stage 2: following viral uncoating, the viral RNA is released and two large open reading frames (ORF1a and ORF1b) are translated into polyproteins. Stage 3: these polyproteins are co- and post-translationally processed by viral proteases into non-structural proteins (NSPs) that form the viral replication complex. Continuous cleavage of the polyprotein is required for sustained RNA synthesis, suggesting that formation of the replication complex is dynamic and occurs continually. Stage 4: the central enzyme of the replication complex is the RNA-dependent RNA polymerase (RdRp), which synthesizes viral RNA. Other enzymes such as the NSP13 helicase and the NSP14 N-methyltransferase contribute to initiation of replication, RNA unwinding, proofreading and sustaining RNA synthesis. Stage 5: genomic viral RNA is encapsulated by nucleocapsid protein (N), and viral structural proteins translocate to the endoplasmic reticulum. Stage 6: structural proteins transit through the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) to the Golgi for glycosylation and progression into exocytic vesicles. Encapsidated genomic RNA buds into the final virion, acquiring a lipid bilayer that contains structural proteins spike (S), membrane (M) and envelope (E). Stage 7: the virion is released from the infected cell by exocytosis. Key viral targets are listed in the boxes. b, As part of the innate immune response towards SARS-CoV-2 infection, the host’s pattern recognition receptors such as proteins retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) recognize viral RNA and trigger downstream signalling cascades involving the mitochondrial antiviral signalling protein (MAVS), leading to activation of the IRF3 and nuclear factor-κB (NF-κB) transcription factors that induce interferon-β (IFNβ) transcription. Viral proteins such as those listed in the box interfere with components of the pathway. M^pro, main protease; P, phosphorylation; PL^pro, papain-like protease.

Fig. 2. Co-crystal structures of the SARS-CoV-2 main protease active site.

a, The inhibitor ensitrelvir (PDB: 7VU6) is bound. b, The inhibitor nirmatrelvir (PDB: 7RFS) is bound. The colours indicate the various binding pockets following the standard Schechter and Berger nomenclature for proteases: P1′ (orange), P1 (yellow), P2 (blue) and P3–5 (cyan). SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Fig. 3. The pharmacokinetic profile of a hypothetical antiviral compound administered twice daily.

The free (unbound) drug plasma concentration (blue) is depicted, including the maximum drug concentration (C_max) and minimum drug concentration (C_min) at steady state. The 90% effective concentration (EC₉₀; red dotted line), the concentration at which 90% inhibition of viral replication is observed in cellular antiviral assays, is corrected for plasma protein binding. BID, twice daily.

Fig. 4. A typical cascade approach for antiviral screening.

After the initial primary enzymatic assays, a high-throughput tier 1 cellular assay is used to drive medicinal chemistry, and a lower-throughput tier 2 assay is used to predict human dose. A549-hACE2, A549 cell line overexpressing human angiotensin-converting enzyme 2; HAEC, human airway epithelial cell; iPSC, induced pluripotent stem cell; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Fig. 5. Common variables in cellular antiviral assays that impact the final read-out.

a, The cell lines used. b, The choice of infecting virus strain. c, The various experimental parameters. ACE2, angiotensin-converting enzyme 2; GFP, green fluorescent protein; iPSC, induced pluripotent stem cell; Luc, firefly luciferase; MHV, mouse hepatitis virus; qPCR, quantitative polymerase chain reaction; TMPRSS2, transmembrane protease serine 2.