WEHI-P8 for COVID-19

<title>Abstract</title> <p>The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has highlighted the vulnerability of a globally connected population to zoonotic viruses. The only FDA-approved coronavirus antiviral, Paxlovid, targets the essential SARS-CoV-2 main protease, Mpro. Whilst effective in the acute phase of an infection, Paxlovid cannot be used by all patients, can lead to viral recurrence, and does not protect against post-acute sequelae of COVID-19 (PASC), commonly known as Long COVID, an emerging significant health burden that remains poorly understood and untreated. Alternative antivirals that are addressing broader patient needs are urgently required. We here report our drug discovery efforts to target PLpro, a further essential coronaviral protease, for which we report a novel chemical scaffold that targets SARS-CoV-2 PLpro with low nanomolar activity, and which exhibits activity against PLpro of other pathogenic coronaviruses. Our lead compound shows excellent in vivo efficacy in a mouse model of severe acute disease. Importantly, our mouse model recapitulates long-term pathologies matching closely those seen in PASC patients, including lung, heart, gut and brain dysfunction. Our lead compound offers protection against PASC in this model, prevents lung pathology and reduces brain dysfunction, providing a potential treatment option for PASC sufferers going forward.</p>

Abstract The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has highlighted the vulnerability of a globally connected population to zoonotic viruses. The FDA-approved coronavirus antiviral Paxlovid targets the essential SARS-CoV-2 main protease, Mpro. Whilst effective in the acute phase of a COVID infection, Paxlovid cannot be used by all patients, can lead to viral recurrence, and does not protect against post-acute sequelae of COVID-19 (PASC), commonly known as long COVID, an emerging significant health burden that remains poorly understood and untreated. Alternative antivirals that are addressing broader patient needs are urgently required. We here report our drug discovery efforts to target PLpro, a further essential coronaviral protease, for which we report a novel chemical scaffold that targets SARS-CoV-2 PLpro with low nanomolar activity, and which exhibits activity against PLpro of other pathogenic coronaviruses. Our lead compound shows excellent in vivo efficacy in a mouse model of severe acute disease. Importantly, our mouse model recapitulates long-term pathologies matching closely those seen in PASC patients. Our lead compound offers protection against a range of PASC symptoms in this model, prevents lung pathology and reduces brain dysfunction. This provides proof-of-principle that PLpro inhibition may have clinical relevance for PASC prevention and treatment going forward.

ABSTRACT Antiviral drugs have been approved for the treatment of COVID‐19. However, they present pharmacological limitations, a mixed efficacy profile and target just two coronavirus proteins. To extend the range of druggable coronavirus proteins, researchers explored small molecule N‐glycan binders as inhibitors of SARS‐CoV‐2 spike protein interaction with the cell receptor. Other groups investigated lipopeptides as inhibitors of cell fusion by viral spikes. High throughput screening of chemical libraries yielded viral maturation inhibitors that targeted the viral M protein. Massive screening led to inhibitors of the non‐structural coronavirus protein NSP14, a methyltransferase involved in viral mRNA cap synthesis. Machine learning–driven scans of chemical space revealed inhibitors of non‐structural coronavirus protein NSP3, a papain‐like protease subverting innate immune response to viral infection. A chimera of a nucleotide analogue coupled to an RNase L attractor bound the RNA‐dependent RNA polymerase NSP12 and mediated degradation of the viral RNA. Several of these compounds showed comparable or even superior antiviral efficacy as approved COVID‐19 drugs in preclinical animal tests. Parallel efforts were made to develop chemical compounds targeting host proteins needed for viral multiplication. Peptidomimetic tetrapeptides acted as inhibitors of the host protease TMPRSS2 involved in cell fusion by the viral spike protein. A repurposed TMPRSS2 inhibitor was tested in COVID‐19 patients without demonstrating efficacy. A genetic screen demonstrated an enzyme involved in sphingomyelin synthesis and its inhibitor which impaired SARS‐CoV‐2 replication. A viral‐cell protein interactome study showed 332 cellular proteins interacting with 26 coronaviral proteins. A chemoinformatic search found inhibitors for the interaction of NSP9 with host elongation factor eIF4A and for NSP13 with elongation factor eEF1A. Plitidepsin, a clinically used eEF1A inhibitor, was tested in human clinical trials with COVID‐19 patients demonstrating in vivo antiviral activity and a trend for clinical amelioration in an underpowered phase 3 clinical trial.

The SARS-CoV-2 non-structural proteome remains the most clinically validated and strategically important landscape for direct-acting small-molecule antiviral drug discovery. The success of inhibitors targeting the main protease (Mpro, Nsp5) and RNA-dependent RNA polymerase (RdRp, Nsp12) has firmly established viral replication enzymes as tractable, druggable, and therapeutically relevant targets, while setting clear benchmarks for translational antiviral development. Building on this foundation, a second wave of non-structural protein (Nsp) targets has emerged with increasing translational promise, including the papain-like protease (PLpro), the bifunctional Nsp14 proofreading and capping machinery, Nsp16 2′-O-methyltransferase, Nsp13 helicase, and Nsp15 endoribonuclease. In parallel, additional components such as Nsp1 and the Mac1 domain of Nsp3 continue to expand the antiviral design space, although they remain at earlier stages of chemical validation. In this review, we comprehensively assess SARS-CoV-2 non-structural proteins through a medicinal chemistry and translational lens, with an emphasis on structural tractability, mechanism of action, quality of chemical matter, cellular and in vivo antiviral evidence, evolutionary conservation, resistance liabilities, and developability. Particular attention is given to the features that distinguish tool compounds from genuinely actionable leads and to the opportunities for rational combination regimens that extend beyond first-generation protease- and polymerase-centred therapy. Collectively, the non-structural proteome offers the strongest foundation for next-generation and potentially broader-spectrum coronavirus antivirals with improved resilience to viral evolution.

Drug-escape, where a target evolves to escape inhibition from a drug, has the potential to lead to cross-resistance where drugs that are structurally related or share similar binding mechanisms all become less effective. PLpro inhibitors are currently under development and many emerging PLpro inhibitors are derived from GRL0617, a repurposed SARS-CoV PLpro inhibitor with moderate activity against SARS-CoV-2. Two leading derivatives, PF-07957472 and Jun12682, demonstrate low nanomolar activity and display activity in mice. WEHI-P8 is structurally distinct but binds to a similar pocket adjacent to the active site as GRL0617-like compounds. Using deep mutational scanning, we assessed the potential for PLpro to develop resistance to PF-07957472, Jun12682, and WEHI-P8. PF-07957472 and Jun12682 exhibited largely overlapping escape mutations due to their shared scaffold and binding modes, whereas WEHI-P8 resistance mutations were distinct. These findings underscore the importance of developing structurally diverse inhibitors to minimize resistance risks and ensure that viral mutations against one compound do not compromise the efficacy of others.