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The murine caspase-11 non-canonical inflammasome responds to various bacterial infections. Caspase-11 activation-induced pyroptosis, in response to cytoplasmic lipopolysaccharide (LPS), is critical for endotoxic shock in mice. The mechanism underlying cytosolic LPS sensing and the responsible pattern recognition receptor are unknown. Here we show that human monocytes, epithelial cells and keratinocytes undergo necrosis upon cytoplasmic delivery of LPS. LPS-induced cytotoxicity was mediated by human caspase-4 that could functionally complement murine caspase-11. Human caspase-4 and the mouse homologue caspase-11 (hereafter referred to as caspase-4/11) and also human caspase-5, directly bound to LPS and lipid A with high specificity and affinity. LPS associated with endogenous caspase-11 in pyroptotic cells. Insect-cell purified caspase-4/11 underwent oligomerization upon LPS binding, resulting in activation of the caspases. Underacylated lipid IVa and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) could bind to caspase-4/11 but failed to induce their oligomerization and activation. LPS binding was mediated by the CARD domain of the caspase. Binding-deficient CARD-domain point mutants did not respond to LPS with oligomerization or activation and failed to induce pyroptosis upon LPS electroporation or bacterial infections. The function of caspase-4/5/11 represents a new mode of pattern recognition in immunity and also an unprecedented means of caspase activation. Caspase-4 and caspase-11 are shown to be the direct sensors for cytoplasmic lipopolysaccharide in humans and mice, respectively, mediating inflammatory cell death in intracellular bacterial infection. Sensing role for caspases in innate immunity A 'non-canonical' innate immune pathway, independent of Toll-like receptor 4 but involving caspase-11, was recently discovered in mice, where it acts to recognize lipopolysaccharide (LPS) from pathogenic bacteria. Here Feng Shao and colleagues investigate this pathway and a similar one in humans. They find that caspase-11 and caspase-4 are the direct sensors for cytoplasmic LPS in mice and humans, respectively, mediating inflammatory cell death in intracellular bacterial infection.

An allosteric inhibitor, EAI045, is reported that is selective for certain drug-resistant EGFR mutants, but spares the wild-type receptor; combination therapy of EAI045 with EGFR-dimerization-blocking antibodies is effective in mouse models of lung cancer driven by mutant versions of EGFR that are resistant to all previously developed inhibitors. Novel EGFR-directed therapeutics Currently available small-molecule inhibitors targeting epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases bind the ATP site of the kinase, and therefore typically inhibit a number of 'off-target' kinases owing to the high conservation of this site. In addition, the common binding site of these drugs leads to shared susceptibility to resistance-conferring mutations in EGFR. Here, Michael Eck and colleagues describe an allosteric inhibitor, EAI045, that is selective for certain drug-resistant EGFR mutants but spares the wild-type receptor. Although EAI045 is not effective in blocking EGFR-driven cell proliferation as a single agent, it has synergistic inhibitory activity when combined with an antibody that blocks EGFR dimerization. This combination therapy is effective in mouse models of lung cancer driven by mutant versions of EGFR that are resistant to all previously developed inhibitors. The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase 1 , 2 , but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor 3 , 4 . Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant 5 , 6 , but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond 7 . All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state 8 . We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization 9 , 10 , rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

Although clinical trials have shown that RAF inhibitors prolong the survival of patients with BRAF-mutant melanoma, resistance inevitably develops; resistance is shown here to be frequently mediated by the expression of splicing variants of mutant BRAF. Mechanism of RAF inhibitor resistance Although recent clinical trials have shown the efficacy of B-RAF inhibitors in the treatment of melanomas with activating B-RAF mutations, the patients inevitably develop resistance. David Solit and colleagues now identify a mechanism of acquired resistance conferred by a structural change in B-RAF itself. The expression of a 61-kilodalton splice variant of mutant B-RAF leads to enhanced B-RAF dimerization, rendering it resistant to kinase inhibitors. This variant was found to be expressed in 6 of 19 patients who had developed resistance to the B-RAF inhibitor PLX4032. Activated RAS promotes dimerization of members of the RAF kinase family 1 , 2 , 3 . ATP-competitive RAF inhibitors activate ERK signalling 4 , 5 , 6 , 7 by transactivating RAF dimers 4 . In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E) 8 . However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4–8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS–GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type I interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.

G-protein-coupled receptors (GPCRs) are key cell-surface proteins that transduce external environmental cues into biochemical signals across the membrane. GPCRs are intrinsically allosteric proteins; they interact via spatially distinct yet conformationally linked domains with both endogenous and exogenous proteins, nutrients, metabolites, hormones, small molecules and biological agents. Here we explore recent high-resolution structural studies, which are beginning to unravel the atomic details of allosteric transitions that govern GPCR biology, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics. High-resolution structural studies of GPCRs have led to insights into the role of allostery in GPCR-mediated signal transduction.

The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca 2+ influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca 2+ influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis. Liu and colleagues find that MLKL translocates to the plasma membrane to induce TNF-induced necroptosis, possibly through an effect on calcium influx and the action of the cation channel TRPM7.

Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.

Upon sensing cytosolic DNA, the enzyme cGAS induces innate immune responses that underpin anti-microbial defenses and certain autoimmune diseases. Missense mutations of PRKDC encoding the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) are associated with autoimmune diseases, yet how DNA-PK deficiency leads to increased immune responses remains poorly understood. In this study, we report that DNA-PK phosphorylates cGAS and suppresses its enzymatic activity. DNA-PK deficiency reduces cGAS phosphorylation and promotes antiviral innate immune responses, thereby potently restricting viral replication. Moreover, cells isolated from DNA-PKcs-deficient mice or patients carrying PRKDC missense mutations exhibit an inflammatory gene expression signature. This study provides a rational explanation for the autoimmunity of patients with missense mutations of PRKDC , and suggests that cGAS-mediated immune signaling is a potential target for therapeutic interventions. The enzyme cGAS induces innate immune responses upon recognition of cytosolic DNA. Here, using in vitro and in vivo models, the authors identify DNA-PK as a negative regulator of cGAS signalling.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K . BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.