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KINOMEscan selectivity profiling of JBJ-04-125-02 and IC50 of EAI045, JBJ-02-112-05 and JBJ-04-125-02 in BaF3 cells.

JBJ-04-125-02 is more potent than EAI045 and JBJ-02-112-05 in the presence of cetuximab and is effective in inhibiting EGFR-dependent proliferation and phosphorylation in L858R/ C797S Ba/F3 cells.

Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant–selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer.Significance:The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib.This article is highlighted in the In This Issue feature, p. 813

Supplemental methods for structural modeling of inhibitor binding, pharmacokinetic assays, in vivo studies, crosslinking and biotinylated drug pull-down assays.

Specificity for a desired enzyme target is an essential property of small-molecule inhibitors. Molecules targeting oncogenic driver mutations in the epidermal growth factor receptor (EGFR) kinase domain have had a considerable clinical impact due to their selective binding to cancer-causing mutants compared to wild type. Despite the availability of clinically approved drugs for cancers driven by EGFR mutants, persistent challenges in drug resistance in the past decades have led to newer generations of drugs with divergent chemical structures. The present clinical complications are mainly due to acquired resistance to third-generation inhibitors by the acquisition of the C797S mutation. Diverse fourth-generation candidates and tool compounds with C797S selectivity have emerged and their structural characterization has allowed for understanding of the molecular factors that allow for EGFR mutant selective binding. Here, we have reviewed all known structurally-characterized EGFR TKIs targeting clinically-relevant mutations to identify consistent binding mode features that enable C797S inhibition. Newer generation EGFR inhibitors exhibit consistent and previously underutilized hydrogen bonding interactions with the conserved K745 and D855 residue side chains. We also consider binding modes and hydrogen bonding interactions of inhibitors targeting the classical ATP and the more unique allosteric sites.

The optimization of linkers that connect fragments within drug binding sites represents an impediment in fragment-based drug discovery (FBDD). To improve our understand of the molecular factors that enable effective fragment linking, we have produced a series of compounds that bind to the ATP and allosteric sites of the EGFR kinase domain connected by two distinct linker structures. We find the linker is responsible for opposing impacts on potency against EGFR mutants, the most potent of which are active in human cancer cells. Comparison of X-ray cocrystal structures of active versus inactive molecules provide unique experimentally derived insights into linker design criteria such as how fragment flexibility and intermolecular interactions can serve compound design broadly in drug optimization.

Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 μM) and L858R/T790M (4.4 μM) variants.

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation–dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA– or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation–based activation of Src and EGFR and was attenuated in Duox1−/− and Src+/− mice, indicating that IL-33–induced epithelial signaling and subsequent airway responses involve DUOX1/Src–dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. Structures show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure-inspired approaches to developing targeted therapies in medicinal oncology.

Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

The C797S mutation confers resistance to covalent EGFR inhibitors used in the treatment of lung tumors with the activating L858R mutation. Isoindolinones such as JBJ-4-125-02 bind in an allosteric pocket and are active against this mutation, with high selectivity over wild-type EGFR. The most potent examples we developed from that series have a potential chemical instability risk from the combination of the amide and phenol groups. We explored a scaffold hopping approach to identify new series of allosteric EGFR inhibitors that retained good potency in the absence of the phenol group. The 5-F quinazolinone 34 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg/kg.

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

Protein kinases are essential mediators of cellular signal transduction and are often dysregulated in disease. Among these, protein tyrosine kinases (PTKs) have received specific interest due to their common roles in various diseases including cancer, and emerging observations indicating that PTK signalling pathways are susceptible to regulation by reactive oxygen species (ROS), which are also frequently implicated in disease pathology. While it is well recognized that ROS can impact on tyrosine kinase signalling by inhibiting tyrosine phosphatases, more recent studies highlight additional modes of redox-based regulation of tyrosine kinase signalling by direct redox modification of non-catalytic cysteines within tyrosine kinases or other protein components of this signalling pathway. In this review, we will present recent advancements with respect to redox-based mechanisms in regulating PTK signalling, with a specific focus on recent studies demonstrating direct redox regulation of Src-family kinases and epidermal growth factor receptor kinases. Importantly, redox-based modulation of tyrosine kinases may be relevant for many other kinases and has implications for current approaches to develop pharmacological inhibitors for these proteins.

Lung cancers are frequently characterized by inappropriate activation of epidermal growth factor receptor (EGFR)-dependent signaling and epigenetic silencing of the NADPH oxidase (NOX) enzyme DUOX1, both potentially contributing to worse prognosis. Based on previous findings linking DUOX1 with redox-dependent EGFR activation, the present studies were designed to evaluate whether DUOX1 silencing in lung cancers may be responsible for altered EGFR regulation. In contrast to normal epithelial cells, EGF stimulation of lung cancer cell lines that lack DUOX1 promotes EGF-induced EGFR internalization and nuclear localization, associated with induction of EGFR-regulated genes and related tumorigenic outcomes. Each of these outcomes could be reversed by overexpression of DUOX1 or enhanced by shRNA-dependent DUOX1 silencing. EGF-induced nuclear EGFR localization in DUOX1-deficient lung cancer cells was associated with altered dynamics of cysteine oxidation of EGFR, and an overall reduction of EGFR cysteines. These various outcomes could also be attenuated by silencing of glutathione S-transferase P1 (GSTP1), a mediator of metabolic alterations and drug resistance in various cancers, and a regulator of cysteine oxidation. Collectively, our findings indicate DUOX1 deficiency in lung cancers promotes dysregulated EGFR signaling and enhanced GSTP1-mediated turnover of EGFR cysteine oxidation, which result in enhanced nuclear EGFR localization and tumorigenic properties.

Precision oncology is premised on identifying and drugging proteins and pathways that drive tumorigenesis or are required for survival of tumor cells. Across diverse cancer types, the signaling pathway emanating from receptor tyrosine kinases on the cell surface to RAS and the MAP kinase pathway is the most frequent target of oncogenic mutations, and key proteins in this signaling axis including EGFR, SHP2, RAS, BRAF, and MEK have long been a focus in cancer drug discovery. In this review, we provide an overview of historical and recent efforts to develop inhibitors targeting these nodes with an emphasis on the role that an understanding of protein structure and regulation has played in inhibitor discovery and characterization. Beyond its well-established role in structure-based drug design, structural biology has revealed mechanisms of allosteric regulation, distinct effects of activating oncogenic mutations, and other vulnerabilities that have opened new avenues in precision cancer drug discovery.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

Inhibitors developed to target the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harbouring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations within the receptor itself has motivated development of successive generations of inhibitors that bind in the ATP-site, and third-generation agent osimertinib is now a first-line treatment for this disease. More recently, allosteric inhibitors have been developed to overcome the C797S mutation that confers resistance to osimertinib. In this study, we present the rational structure-guided design and synthesis of a mutant-selective EGFR inhibitor that spans the ATPand allosteric sites. The lead compound consists of a pyridinyl imidazole scaffold that binds irreversibly in the orthosteric site fused with a benzylisoindolinedione occupying the allosteric site. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with relative sparing of wild-type EGFR (47 nM). Additionally, this compound achieves cetuximab-independent, mutant-selective cellular efficacy on the L858R and L858R/T790M variants

The multicopper oxidases (MCOs) couple four 1e(−) oxidations of substrate to the 4e(−) reduction of O(2) to H(2)O. These divide into two groups: those that oxidize organic substrates with high turnover frequencies (TOFs) up to 560 s(−1) and those that oxidize metal ions with low TOFs, ~1 s(−1) or less. The catalytic mechanism of the organic oxidases has been elucidated, and the high TOF is achieved through rapid intramolecular electron transfer (IET) to the Native Intermediate (NI), which only slowly decays to the resting form. Here we uncover the factors that govern the low TOF in Fet3p, a prototypical metallooxidase, in the context of the MCO mechanism. We determine that the NI decays rapidly under optimal turnover conditions, and the mechanism thereby becomes rate-limited by slow IET to the resting enzyme. Development of a catalytic model leads to the important conclusions that proton delivery to the NI controls the mechanism and enables the slow turnover in Fet3p that is functionally significant in Fe metabolism enabling efficient ferroxidase activity while avoiding ROS generation.

Acquired drug resistance in epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer is a persistent challenge in cancer therapy. Previous studies of trisubstituted imidazole inhibitors led to the serendipitous discovery of inhibitors that target the drug resistant EGFR(L858R/T790M/C797S) mutant with nanomolar potencies in a reversible binding mechanism. To dissect the molecular basis for their activity, we determined the binding modes of several trisubstituted imidazole inhibitors in complex with the EGFR kinase domain with X-ray crystallography. These structures reveal that the imidazole core acts as an H-bond acceptor for the catalytic lysine (K745) in the "αC-helix out" inactive state. Selective N-methylation of the H-bond accepting nitrogen ablates inhibitor potency, confirming the role of the K745 H-bond in potent, noncovalent inhibition of the C797S variant. Insights from these studies offer new strategies for developing next generation inhibitors targeting EGFR in non-small-cell lung cancer.

Reversible protein phosphorylation, mediated by reciprocal actions of protein kinases and phosphatases, represents a major mode of posttranslational regulation of protein function in all organisms. During the past 20 years, it has become clear that protein phosphorylation is subject to oxidative mechanisms, which not only can inhibit major classes of protein phosphatase, particularly tyrosine phosphatases, but also can directly regulate various protein kinases as well as recognition of phosphorylated proteins, both positively and negatively. Such redox-based regulation typically involves reversible oxidation of conserved cysteine residues, although other oxidative modifications are also involved. The diverse modes of orthogonal regulation of protein phosphorylation by such reversible oxidative events indicate an intricate relationship between redox-dependent signaling mechanisms and protein kinase signaling pathways, especially in aerobic life. Dysregulation of such redox-dependent regulatory mechanisms during conditions of oxidative stress may contribute to abnormal protein phosphorylation and development of disease.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated., The activity of several protein kinases is increased upon cellular production of reactive oxygen species, which can cause cysteine oxidation. Here the authors show that sulfenylation of specific cysteine residues within Src induce local structural changes that directly impact its activation.

NADPH oxidases (NOXs) represent a family of enzymes that mediate regulated cellular production of reactive oxygen species (ROS) and play various functional roles in physiology. Among the NOX family, the dual oxidases DUOX1 and DUOX2 are prominently expressed in epithelial cell types at mucosal surfaces and have therefore been considered to have important roles in innate host defence pathways. Recent studies have revealed important insights into the host defence mechanisms of DUOX enzymes, which control innate immune response pathways in response to either microbial or allergic triggers. In this review, we discuss the current level of understanding with respect to the biological role(s) of DUOX enzymes and the unique role of DUOX1 in mediating innate immune responses to epithelial injury and allergens and in the development of allergic disease. These novel findings highlight DUOX1 as an attractive therapeutic target, and opportunities for the development of selective inhibitor strategies will be discussed.

The reversible oxidation of protein cysteine residues (Cys-SH) is a key reaction in cellular redox signaling involving initial formation of sulfenic acids (Cys-SOH), which are commonly detected using selective dimedone-based probes. Here, we report that significant portions of dimedone-tagged proteins are susceptible to cleavage by DTT reflecting the presence of perthiosulfenic acid species (Cys-SSOH) due to similar oxidation of hydropersulfides (Cys-SSH), since Cys-S-dimedone adducts are stable toward DTT. Combined studies using molecular modeling, mass spectrometry, and cell-based experiments indicate that Cys-SSH are readily oxidized to Cys-SSOH, which forms stable adducts with dimedone-based probes. We additionally confirm the presence of Cys-SSH within protein tyrosine kinases such as EGFR, and their apparent oxidation to Cys-SSOH in response NADPH oxidase activation, suggesting that such Cys-SSH oxidation may represent a novel, as yet uncharacterized, event in redox-based signaling., Graphical abstract fx1

The reversible oxidation of protein cysteine residues is well recognized as an important regulatory mechanism in redox-dependent cell signaling. Cysteine oxidation is diverse in nature and involves various post-translational modifications (sulfenic acids, disulfides, etc.) and the specific functional or structural impact of these specific oxidative events is still poorly understood. The proximal product of protein cysteine oxidation by biological reactive oxygen species (ROS) is sulfenic acid (Cys-SOH), and experimental evidence is accruing for the formation of Cys-SOH as intermediate in protein cysteine oxidation in various biological settings. However, the plausibility of protein Cys-SH oxidation by ROS has often been put in question because of slow reaction kinetics compared to more favorable reactions with abundant thiol-based reductants such as peroxiredoxins (Prx) or glutathione (GSH). This commentary aims to address this controversy by highlighting the unique physical properties in cells that may restrict ROS diffusion and allow otherwise less favorable cysteine oxidation of proteins. Some limitations of analytical tools to assess Cys-SOH are also discussed. We conclude that formation of Cys-SOH in biological systems cannot always be predicted based on kinetic analyses in homogenous solution, and may be facilitated by unique structural and physical properties of Cys-containing proteins within e.g. signaling complexes.

Summary Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

[Image: see text] Allosteric kinase inhibitors represent a promising new therapeutic strategy for targeting kinases harboring oncogenic driver mutations in cancers. Here, we report the discovery, optimization, and structural characterization of allosteric mutant-selective EGFR inhibitors comprising a 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one scaffold. Our structure-based medicinal chemistry effort yielded an inhibitor (3) of the EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S) mutants with an IC(50) of ∼10 nM and high selectivity, as assessed by kinome profiling. Further efforts to develop allosteric dibenzodiazepinone inhibitors may serve as the basis for new therapeutic options for targeting drug-resistant EGFR mutations.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and that this interaction is required for efficient oxidative folding of HA in vitro. However it is unknown whether these host-viral protein interactions occur during active infection and whether this interaction represent a putative target for the treatment of influenza infection. Current therapies targeting viral proteins are often limited in their effectiveness due to rapid development of viral resistance; targeting host proteins may avoid these limitations. Here we show that PDIA3 is specifically upregulated in IAV infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Among PDI inhibitors tested, we found LOC14 inhibits PDIA3 at an IC50 of 5μM. Treatment with LOC14 inhibits PDIA3 activity in lung epithelial cells and subsequently decreases disulfide bonds, and oligomerization (maturation) of HA in both pandemic H1N1 and seasonal H3N2 infected lung epithelial cells. Disulfide labeling analysis indicated that inhibition of PDIA3 activity also decreased disulfide bonds in IAV-neuraminidase (NA) and these differences in oxidative folding correspond to a subsequent decrease in NA activity. Furthermore, these decreases in disulfide bonds significantly decrease viral load, and pro-inflammatory response from primary lung epithelial cells. Lung epithelial specific deletion of PDIA3 in mice results in a significant decreases in viral burden and levels of inflammatory-immune markers in mouse lung, as well as significantly improved airway mechanics (function). Taken together, these results suggest PDIA3 is required for effective influenza pathogenesis in vivo, and inhibition of PDIA3 may aid future development anti-influenza therapies based on inhibition of host PDIs to circumvent antigenic drifts and shifts of influenza and may lend critically needed help during pandemic and severe influenza seasons.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons., Graphical abstract fx1

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H2O2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H2O2-induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.

Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant–selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer. Significance: The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib. This article is highlighted in the In This Issue feature, p. 813

Activating mutations in protein kinases are a frequent cause of cancer, and selecting drugs that act on these oncogenic kinases can lead to effective therapies. Targeted or whole-genome sequencing of tumor samples can readily reveal the presence of mutations, but discerning previously uncharacterized activating “driver” mutations that will respond to drug treatment from much more abundant but inconsequential “passenger” mutations is problematic. Chakroborty et al. apply a screening approach that leverages error-prone PCR and a proliferating cell model to identify such gain-of-function mutants in the epidermal growth factor receptor (EGFR) kinase. The screen is validated by the identification of known cancer-promoting mutations and reveals a previously unappreciated oncogenic EGFR mutation, A702V, demonstrating its power for discovery of driver mutations.

NADPH oxidases (NOXs) represent a family of enzymes that mediate regulated cellular production of reactive oxygen species (ROS) and play various functional roles in physiology. Among the NOX family, the dual oxidases DUOX1 and DUOX2 are prominently expressed in epithelial cell types at mucosal surfaces and have therefore been considered to have important roles in innate host defence pathways. Recent studies have revealed important insights into the host defence mechanisms of DUOX enzymes, which control innate immune response pathways in response to either microbial or allergic triggers. In this review, we discuss the current level of understanding with respect to the biological role(s) of DUOX enzymes and the unique role of DUOX1 in mediating innate immune responses to epithelial injury and allergens and in the development of allergic disease. These novel findings highlight DUOX1 as an attractive therapeutic target, and opportunities for the development of selective inhibitor strategies will be discussed.

Dysregulated oxidative metabolism is a well-recognized aspect of cancer biology, and many therapeutic strategies are based on targeting cancers by altering cellular redox pathways. The NADPH oxidases (NOXes) present an important enzymatic source of biological oxidants, and the expression and activation of several NOX isoforms are frequently dysregulated in many cancers. Cell-based studies have demonstrated a role for several NOX isozymes in controlling cell proliferation and/or cell migration, further supporting a potential contributing role for NOX in promoting cancer. While various NOX isoforms are often upregulated in cancers, paradoxical recent findings indicate that dual oxidases (DUOXes), normally prominently expressed in epithelial lineages, are frequently suppressed in epithelial-derived cancers by epigenetic mechanisms, although the functional relevance of such DUOX silencing has remained unclear. This review will briefly summarize our current understanding regarding the importance of reactive oxygen species (ROS) and NOXes in cancer biology, and focus on recent observations indicating the unique and seemingly opposing roles of DUOX enzymes in cancer biology. We will discuss current knowledge regarding the functional properties of DUOX, and recent studies highlighting mechanistic consequences of DUOX1 loss in lung cancer, and its consequences for tumor invasiveness and current anticancer therapy. Finally, we will also discuss potentially unique roles for the DUOX maturation factors. Overall, a better understanding of mechanisms that regulate DUOX and the functional consequences of DUOX silencing in cancer may offer valuable new diagnostic insights and novel therapeutic opportunities.

Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite-induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.

The airway epithelium forms the primary defense against inhaled allergens and environmental insults to the respiratory system. Aside from acting as a physical barrier, the epithelium also signals for reparative and inflammatory responses by generating various epithelial-derived cytokines and mediators. Chief among these is the epithelial secretion of the alarmin cytokine IL-33, which in turn induces type 2 immune responses. Although these processes serve a protective mechanism by promoting epithelial regenerative pathways, chronic activation of such type 2 responses contribute to chronic respiratory diseases such as asthma and COPD. We recently demonstrated that epithelial IL-33 secretion in response to various injurious triggers is dependent on H2O2 production by the NADPH oxidase dual oxidase 1 (DUOX1), and that this was associated with activation and oxidation of the non-receptor tyrosine kinase Src. However, the precise role of Src in these responses is still unclear, and we hypothesized that redox-dependent activation of Src is critical for increased IL-33 secretion and downstream type 2 responses to allergens. Pretreatment of mice with the Src family kinase inhibitor AZD0530 was found to significantly attenuate epithelial Src activation, as well as expression and secretion of IL-33 and the downstream type 2 cytokines IL-5 and IL-13, in response to acute challenge with house dust mite (HDM). Similar findings were obtained using isolated mouse tracheal epithelial cells (MTEC), in which siRNA-dependent silencing of Src resulted in diminished HDM-induced production of IL-33, as well as IL-5 and IL-13. Interestingly, we noted that HDM-induced IL-33 secretion is diminished upon blockade of the IL-33 receptor ST2, suggesting that IL-33 regulates its own secretion through a positive feedback mechanism. Indeed, induction of type 2 cytokine secretion (IL-13) in airway epithelial cells by direct stimulation with IL-33 was found to be DUOX1-dependent, and associated with increased H2O2 production and activation/oxidation of Src. Building on our recent findings that DUOX1-dependent activation of Src involves oxidation of its C185 and C277 residues, preliminary studies using H292 lung epithelial cells transfected with C185A or C277A Src variants indicate that expression of these variants leads to suppressed HDM-induced IL-33 secretion. Overall, our studies identify Src as a critical mediator of allergen-induced IL-33 secretion and innate type 2 immune responses, and highlight the importance of various redox-sensitive Cys residues in these responses.

The airway epithelium forms a first line of defense against environmental triggers, and through secretion of epithelial cytokines, such as IL-33, presents a critical innate response mechanism to injurious insults by initiating type 2 cytokine responses. We recently demonstrated that airway epithelial IL-33 secretion depends on activation of the NADPH oxidase dual oxidase 1 (DUOX1) and redox-dependent activation of the epidermal growth factor receptor (EGFR) and Src kinases. Curiously, activation of type 2 responses by direct airway administration of IL-33 was markedly diminished in DUOX1-deficient mice, suggesting DUOX1 is activated by IL-33 and participates in its downstream signaling. Furthermore, activation of epithelial IL-33 secretion in response to injury or allergens was dramatically attenuated by blockade or siRNA-mediated deletion of the IL-33 receptor (IL-33R/ST2), suggesting that epithelial IL-33 evokes a positive autocrine or paracrine feedback mechanism in which it regulates its own secretion through IL-33R/ST2-mediated signaling. Studies with human bronchial epithelial cells (H292) and primary mouse tracheal epithelial cells (MTEC) showed that IL-33 can directly trigger EGFR and Src activation and type 2 cytokine secretion, via intermediate activation of DUOX1. The activation of these kinases was shown to be associated with increased levels of DUOX1-dependent sulfenylation. Additionally, IL-33 stimulation resulted in ST2-mediated calcium-dependent extracellular secretion of ATP, a known damage signal capable of activating DUOX1. Consistent with its activation by IL-33, inhibition of Src with AZD0530 or siRNA-mediated deletion of Src led to decreased secretion of type 2 cytokines, confirming that Src plays a role in these downstream processes. Overall, our findings suggest that the role of DUOX1 in allergen-induced IL-33 secretion, and activation of type 2 responses, is largely mediated by its involvement in IL-33-dependent signaling and subsequent oxidation and activation of Src and EGFR.

The mu-eta(2):eta(2)-peroxodicopper(II) complex synthesized by reacting the Cu(I) complex of the bis-diamine ligand N,N'-di-tert-butyl-ethylenediamine (DBED) with O(2) is a functional and spectroscopic model of the coupled binuclear copper protein tyrosinase. This complex reacts with 2,4-di-tert-butylphenolate at low temperature to produce a mixture of the catechol and quinone products, which proceeds through three intermediates (A-C) that have been characterized. A, stabilized at 153 K, is characterized as a phenolate-bonded bis-mu-oxo dicopper(III) species, which proceeds at 193 K to B, presumably a catecholate-bridged coupled bis-copper(II) species via an electrophilic aromatic substitution mechanism wherein aromatic ring distortion is the rate-limiting step. Isotopic labeling shows that the oxygen inserted into the aromatic substrate during hydroxylation derives from dioxygen, and a late-stage ortho-H(+) transfer to an exogenous base is associated with C-O bond formation. Addition of a proton to B produces C, determined from resonance Raman spectra to be a Cu(II)-semiquinone complex. The formation of C (the oxidation of catecholate and reduction to Cu(I)) is governed by the protonation state of the distal bridging oxygen ligand of B. Parallels and contrasts are drawn between the spectroscopically and computationally supported mechanism of the DBED system, presented here, and the experimentally derived mechanism of the coupled binuclear copper protein tyrosinase.