The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, noncovalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

32 Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that 33 are essential for viral replication and evasion of host innate immune 34 mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and 35 reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein 36 modifications 1,2 . Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 37PLpro) may hence be effective as treatments or prophylaxis for COVID-19, 38 reducing viral load and reinstating innate immune responses 3 . 39 We here characterise SARS2 PLpro in molecular and biochemical detail. 40 SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with 41 high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the 42 S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 43 binding site provides Lys48 chain specificity and cleavage efficiency.44 We further exploit two strategies to target PLpro. A repurposing approach, 45 screening 3727 unique approved drugs and clinical compounds against 46 SARS2 PLpro, identified no compounds that inhibited PLpro consistently or 47 that could be validated in counterscreens. More promisingly, non-covalent 48 small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with 49 high potency and excellent antiviral activity in SARS-CoV-2 infection models. 50 51The COVID-19 pandemic unfolding globally in the first half of 2020 is caused by the 52 novel Coronavirus SARS-CoV-2, and has highlighted, amongst many things, the 53 general lack of antiviral small molecule drugs to fight a global coronavirus pandemic. 54Proteolytic enzymes are critical for viruses expressing their protein machinery as a 55 polyprotein that requires cleavage into functional units. As a result, viruses with 56 blocked protease activity do not replicate efficiently in cells; this concept extends to 57 coronaviruses 4 . Drugging the proteases in SARS-CoV-2 is therefore a current focus 58 of concerted global academic and pharma efforts 3 . 59 60 SARS-CoV-2 encodes two proteases, the papain-like protease (PLpro, encoded 61 within non-structural protein (nsp) 3), and 3-chymotrypsin-like 'main' protease 62 (3CLpro or Mpro, encoded within nsp5). PLpro generates nsp1, nsp2, and nsp3 63 (Figure 1a) and 3CLpro generates the remaining 13 non-structural proteins. After 64 3 their generation, the nsps assemble the viral replicase complex on host membranes, 65 initiating replication and transcription of the viral genome 1,5 . 66 67Viral proteases can have additional functions, and can for example act to inhibit host 68 innate immune responses that are mounted initially as an inflammatory response, 69 and subsequently as an interferon response. The interferon system generates an 70 antiviral state in host cells through transcriptional upregulation of more than 300 71 interferon-stimulated genes (ISGs), to efficiently detect and respond to viral threats 6 . 72Dysregulated inflammatory responses are a hallmark of COVID-19, and substantial 73 morb...

The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.