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.

Summary Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-kB (NF-kB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-kB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IkB kinase ε (IKKε) to drive NF-kB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-kB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

Inflammasomes are multimeric protein complexes that induce the cleavage and release of bioactive IL-1 and cause a lytic form of cell death, termed pyroptosis. Due to its diverse triggers, ranging from infectious pathogens and host danger molecules to environmental irritants, the NOD-like receptor protein 3 (NLRP3) inflammasome remains the most widely studied inflammasome to date. Despite intense scrutiny, a universal mechanism for its activation remains elusive, although, recent research has focused on mitochondrial dysfunction or potassium (K + ) efflux as key events. In this review, we give a general overview of NLRP3 inflammasome activation and explore the recently emerging noncanonical and alternative pathways to NLRP3 activation. We highlight the role of the NLRP3 inflammasome in the pathogenesis of metabolic disease that is associated with mitochondrial and oxidative stress. Finally, we interrogate the mechanisms proposed to trigger NLRP3 inflammasome assembly and activation. A greater understanding of how NLRP3 inflammasome activation is triggered may reveal new therapeutic targets for the treatment of inflammatory disease. K E Y W O R D Scaspases, inflammasome, metabolism, mitochondria, reactive oxygen species Abbreviations: AIM2, absent in Melanoma; AMPK, AMP-activated protein kinase; APAF-1, apoptosis protease activating factor-1; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; BMDCs, bone marrow dendritic

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 emergence of SARS-CoV-2 causing the COVID-19 pandemic, has highlighted how a combination of urgency, collaboration and building on existing research can enable rapid vaccine development to fight disease outbreaks. However, even countries with high vaccination rates still see surges in case numbers and high numbers of hospitalized patients. The development of antiviral treatments hence remains a top priority in preventing hospitalization and death of COVID-19 patients, and eventually bringing an end to the SARS-CoV-2 pandemic. The SARS-CoV-2 proteome contains several essential enzymatic activities embedded within its non-structural proteins (nsps). We here focus on nsp3, that harbours an essential papain-like protease (PLpro) domain responsible for cleaving the viral polyprotein as part of viral processing. Moreover, nsp3/PLpro also cleaves ubiquitin and ISG15 modifications within the host cell, derailing innate immune responses. Small molecule inhibition of the PLpro protease domain significantly reduces viral loads in SARS-CoV-2 infection models, suggesting that PLpro is an excellent drug target for next generation antivirals. In this review we discuss the conserved structure and function of PLpro and the ongoing efforts to design small molecule PLpro inhibitors that exploit this knowledge. We first discuss the many drug repurposing attempts, concluding that it is unlikely that PLpro-targeting drugs already exist. We next discuss the wealth of structural information on SARS-CoV-2 PLpro inhibition, for which there are now ∼30 distinct crystal structures with small molecule inhibitors bound in a surprising number of distinct crystallographic settings. We focus on optimisation of an existing compound class, based on SARS-CoV PLpro inhibitor GRL-0617, and recapitulate how new GRL-0617 derivatives exploit different features of PLpro, to overcome some compound liabilities.

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Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling.