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, non‐covalent 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.
Synopsis
Crystal structures explain the specificity of SARS‐CoV‐2 papain‐like protease, PLpro, for ISG15 and Lys48‐linked diubiquitin, and specific inhibition of PLpro by small molecules, shows strong antiviral effects.
SARS‐CoV-2 PLpro preferentially cleaves ISG15 and also targets longer Lys48‐linked ubiquitin chains.
Preference for ISG15 is provided by the S1 ubiquitin binding site in PLpro, while Lys48‐polyubiquitin specificity is provided by the S2 ubiquitin binding site.
In a high‐throughput screen, FDA approved drugs and late stage clinical compounds are unable to inhibit PLpro in vitro.
Known SARS PLpro inhibitors also target SARS2 PLpro and show anti‐viral efficacy.
Crystal structure and biochemical analysis explains the specificity of SARS‐CoV‐2 PLpro for ISG15 and longer Lys48‐linked ubiquitin chains leading to the identification of inhibitors that show promising antiviral activity in a SARS‐CoV‐2 infection model.
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-κB (NF-κB)-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-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB 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.
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•TBK1 is dispensable for NF-κB activation downstream of STING in vitro and in vivo•TBK1 and IKKε act redundantly to elicit STING-induced NF-κB activation•STING-NF-κB is less sensitive to TBK1/IKKε kinase inhibition than type I IFN•TAK1 and IKK complexes are required for STING-mediated NF-κB responses
Activation of NF-κB via STING is considered to be exclusively dependent on TBK1. Balka et al. now show that, although TBK1 and its kinase activity are critical for IRF3 activation and type I IFNs, it is dispensable for NF-κB. Instead, TBK1 and IKKε act redundantly to mediate STING-induced NF-κB responses.
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.
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•TDP-43 enters mitochondria, triggers mtDNA release via the mPTP•TDP-43-induced cytosolic mtDNA accumulation activates the cGAS/STING pathway•Evidence of cytoplasmic mtDNA was found in ALS patient cells and disease models•Blocking STING prevents inflammation and neurodegeneration in vitro and in vivo
TDP-43 causes inflammation in ALS by stimulating mitochondrial DNA release, which is subsequently sensed by the cytosolic cGAS/STING pathway, suggesting that inhibition of cGAS/STING could help alleviate inflammation-related damage in ALS.
Inhibitors of SARS-CoV-2 PLpro Calleja, Dale J; Lessene, Guillaume; Komander, David
Frontiers in chemistry,
04/2022, Letnik:
10
Journal Article
Recenzirano
Odprti dostop
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.
Inflammasomes are multimeric protein complexes that induce the cleavage and release of bioactive IL‐1β 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.
Review of the ambiguities surrounding mitochondrial damage‐mediated activation of NLRP3, including new models for how cell death may activate NLRP3 to influence inflammation.
Abstract Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation ...and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.
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.
Understanding SOCS protein specificity Linossi, Edmond M.; Calleja, Dale J.; Nicholson, Sandra E.
Growth factors (Chur, Switzerland),
07/2018, Letnik:
36, Številka:
3-4
Journal Article
Recenzirano
The development and activity of our immune system are largely controlled by the action of pleiotropic cytokines and growth factors, small secreted proteins, which bind to receptors on the surface of ...immune cells to initiate an appropriate physiological response. Cytokine signalling is predominantly executed by intracellular proteins known as the Janus kinases (JAKs) and the signal transducers and activators of transcriptions (STATs). Although the 'nuts and bolts' of cytokine-activated pathways have been well established, the nuanced way in which distinct cellular outcomes are achieved and the precise molecular details of the proteins that regulate these pathways are still being elucidated. This is highlighted by the intricate role of the suppressor of cytokine signalling (SOCS) proteins. The SOCS proteins act as negative feedback inhibitors, dampening specific cytokine signals to prevent excessive cellular responses and returning the cell to a homeostatic state. A great deal of study has demonstrated their ability to inhibit these pathways at the receptor complex, either through direct inhibition of JAK activity or by targeting the receptor complex for proteasomal degradation. Detailed analysis of individual SOCS proteins is slowly revealing the complex and highly controlled manner by which they can achieve specificity for distinct substrates. However, for many of the SOCS, a level of detail is still lacking, including confident identification of the full suite of tyrosine phosphorylated targets of their SH2 domain. This review will highlight the general mechanisms which govern SOCS specificity of action and discuss the similarities and differences between selected SOCS proteins, focusing on CIS, SOCS1 and SOCS3. Because of the functional and sequence similarities within the SOCS family, we will also discuss the evidence for functional redundancy.
NLRP1 is an innate immune sensor that can form cytoplasmic inflammasome complexes. Polymorphisms in NLRP1 are linked to asthma; however, there is currently no functional or mechanistic explanation ...for this.
We sought to clarify the role of NLRP1 in asthma pathogenesis.
Results from the GALA II cohort study were used to identify a link between NLRP1 and asthma in Mexican Americans. In vitro and in vivo models for NLRP1 activation were applied to investigate the role of this inflammasome in asthma at the molecular level.
We document the association of an NLRP1 haplotype with asthma for which the single nucleotide polymorphism rs11651270 (M1184V) individually is the most significant. Surprisingly, M1184V increases NLRP1 activation in the context of N-terminal destabilization, but decreases NLRP1 activation on dipeptidyl peptidase 9 inhibition. In vitro studies demonstrate that M1184V increases binding to dipeptidyl peptidase 9, which can account for its inhibitory role in this context. In addition, in vivo data from a mouse model of airway inflammation reveal a protective role for NLRP1 inflammasome activation reducing eosinophilia in this setting.
Linking our in vitro and in vivo results, we found that the NLRP1 variant M1184V reduces inflammasome activation in the context of dipeptidyl peptidase 9 inhibition and could thereby increase asthma severity. Our studies may have implications for the treatment of asthma in patients carrying this variant of NLRP1.
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