Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, remains a global health concern despite vaccines, neutralizing antibodies, and antiviral drugs. The emergence of viral mutations that diminish the effectiveness of current interventions underscores the importance of alternative, host-directed strategies. Here, we show that pharmacological inhibition or knockdown of host N-myristoyltransferase 1 (NMT1), one of the two human enzymes that mediates protein N-myristoylation, significantly impairs SARS-CoV-2, Vesicular Stomatitis Virus (VSV) and Respiratory syncytial virus (RSV) infections. We demonstrate the antiviral efficacy and safety of this host-directed therapeutic strategy across multiple viral tropic sites, including human lung adenocarcinoma cell lines, primary nasal epithelial cells, and human choroid plexus-cortical brain organoids. NMT1 inhibition triggers a Golgi-bypassing pathway for SARS-CoV-2 progeny virion egress, through endoplasmic reticulum and lysosomal structures, which leads to perturbed progeny virion composition and spike maturation, impairing progeny virion infectivity.

Human N-myristoyltransferases (NMTs) catalyze N-terminal protein N-myristoylation and are promising targets in cancer, with an emerging mechanistic rationale for targeted therapy. Here, we screened 245 cancer cell lines against IMP-1320, a potent NMT inhibitor (NMTi), and conducted pathway-level analyses to identify that deregulated MYC increases cancer cell sensitivity to NMTis. Proteomics on detergent-enriched membrane fractions in MYC or MYCN-deregulated cancer cell models revealed that cell death is associated at least in part with loss of membrane association of mitochondrial respiratory complex I. This is concurrent with loss of myristoylation and degradation of the complex I assembly factor NDUFAF4, and induction of mitochondrial dysfunction, driven by MYC or MYCN-deregulation. NMTis eliminated or suppressed MYC- and MYCN-driven tumors in vivo without overt toxicity, suggesting that this constitutive co-translational protein modification can be targeted in MYC-driven cancers.

N-myristoyltransferases (NMTs) catalyze the protein N-terminal modification N-myristoylation, a lipidation event which affects >150 proteins and is involved in protein localization, stability, and function. NMT has been suggested as a target in cancers, but there has been a lack of rationale to identify patients who may respond to NMT inhibition. Additionally, the mechanism of action of NMT inhibitors (NMTi) is difficult to dissect, as NMT substrates feature in multiple biological pathways, and many studies have focused only on a single substrate. Here, a combination of bioinformatic, biochemical, proteomic, and cellular biology techniques have been combined to show that deregulation of MYC-family proteins sensitizes cells to NMT inhibition. Cell lines sensitive to NMTi were identified through screening of hundreds of cancer cell lines, and the transcriptome of sensitive and insensitive lines compared to obtain a “Sensitive to NMTi” gene set. This gene set correlated well with MYC-related gene sets and mutation, amplification, or chromosomal rearrangement in MYC(N) were predictive for NMTi sensitivity, suggesting that MYC deregulated cancer cells are sensitive to NMTi. This was verified by cytotoxicity assays in B-cell lymphoma and neuroblastoma lines, where highly MYC(N) expressing lines exhibited increased NMTi sensitivity, including in the SHEP21N and P493-6 lines, in which MYC(N) levels can be regulated. Furthermore, proteomic profiling identified multiple pathways which are affected by NMTi, and these were further validated. In particular, an impact on Complex I formation was seen, and mitochondrial dysfunction in high MYC(N) cells upon NMTi was seen through the loss of basal and maximal respiration, ATP production and spare respiratory capacity. Loss of Complex I formation was shown to be caused by the loss of the NMT substrate NDUFAF4 upon NMTi treatment. Furthermore, application of an orally bioavailable NMTi eliminated tumors in mouse models of both diffuse large B-cell lymphoma and neuroblastoma and were well tolerated with no changed in body weight, suggesting that NMT inhibitors are well tolerated and efficacious in vivo. Citation Format: James Zhang, Gregor A. Lueg, Monica Faronato, Evon Poon, Andrii Gorelik, Andrea G. Grocin, Eva Caamano-Gutierrez, Francesco Falciani, Roberto Solari, Robin Carr, Andrew S. Bell, Edward Bartlett, Jennie Hutton, Miriam Llorian-Sopena, Probir Chakravarty, Bernadette Brzezicha, Martin Janz, Matther J. Garnett, Louis Chesler, Dinis P. Calado, Edward W. Tate. Dysregulation of MYC-family proteins sensitizes cancers to NMT inhibition: identification of NMTi sensitivity and mechanism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4871.

Introduction: There is a need for ADC payloads with an improved therapeutic index, consequently we have coupled potent and selective inhibitors of NMT to trastuzumab to test their potential in both in vitro and in vivo models. Experimental Procedures: A highly potent NMT inhibitor was conjugated to trastuzumab using two different linkers to produce an ADCs with a DAR of 5. These ADCs were tested in vitro in cell killing studies using Her2+ and Her2- cell lines and subsequently tested in vivo in breast cancer and gastric cancer xenografts. Results: Our first Trastuzumab-NMT inhibitor ADC (MYX2449) caused cell death in Her2+ BT474 cells with an EC50 of 0.2nM and peaking after 8 days of incubation, but was inactive against Her2- cells MCF7 at concentrations up to 100nM. MYX2449 was tested in a BT474 xenograft implanted orthotopically in the mammary gland fat pad and compared to Trastuzumab and vehicle controls. Both antibodies were dosed IV once a week for four weeks at 2.5 and 5 mg/Kg. MYX2449 therapy at 2.5mg/Kg resulted in a partial response (TGI 55% on day 21) and 5mg/Kg resulted in a TGI of 108% on day 21 with 7/10 mice having undetectable tumor by day 33 with no significant body weight loss during the study. Trastuzumab resulted in no response at 2.5mg/Kg a partial response at 5mg/Kg. MYX2449 was subsequently tested in a gastric cancer xenograft, NCI-N87 inoculated subcutaneously and compared to an NMT inhibitor conjugated isotype control and to Trastuzumab and Trastuzumab deruxtecan. All antibodies were dosed IV at 2.5mg/Kg or 5mg/Kg on day 1 and day 8. Day 21 TGI for the 5mg/Kg Trastuzumab group was 193%, for the 5mg/Kg Trastuzumab-deruxtecan group TGI was 154% and for the 5mg/Kg MYX2449 group TGI was 280%. In the MYX2449 5mg/Kg group, 6/10 mice had undetectable tumors on day 21 and 2/10 of the MYX2449 2.5mg/Kg group. Mean body weights of all groups had increased by day 21. Conclusions: Potent small molecule inhibitors of NMT can be readily conjugated to therapeutic monoclonal antibodies to generate novel highly effective and well tolerated ADC payloads. NMT inhibitors represent a totally novel class of ADC payloads that exploit cancer cell dependency on myristloylated proteins. The potential of this novel mechanism to deliver high efficacy with better TI is currently being investigated in preclinical models. Examples of Myricx patented chemical structures will be disclosed. RS, ND, FF and RC are employees of Myricx Pharma.JW, FO and ET are funded by Myricx Pharma.ET is a board member and founder of Myricx Pharma. Citation Format: Robin A. Carr, Roberto Solari, Nikki D'Arcy, Ed Tate, Josephine Walton, Folake Orafidiya. N-Myristolytransferase (NMT) inhibitors as novel potent payloads for antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2635.

Tumor-associated macrophages (TAMs) play a central role in cancer by driving tumor growth, metastasis, therapy failure and cancer recurrence. Macrophage plasticity and diversity allows classification along a M1-M2 polarization axis, where TAMs have a M2-like polarization, associated with a pro-tumoral phenotype, whereas M1 macrophages exhibit anti-tumor functions. Reprogramming TAMs to a M1-like anti-cancer phenotype is an increasingly coveted therapeutic strategy in oncology. Here, we report TAMs can be favorably reprogrammed by modulating N-myristoyltransferase (NMT) activity using on-target, drug-like inhibitors (NMTi). We identified >100 N-myristoylated proteins differentially expressed by macrophages along the M1-M2 polarization axis. In TAM-like M2 macrophages, 42 N-myristoylated proteins exhibited higher NMTi sensitivity as compared to other polarizations, and these NMT substrates significantly enriched in anti-inflammatory, immunity- and metabolism-related pathways. Unique to TAM-like M2 macrophages, NMT modulation by NMTi induces significant transcriptomic and proteomic changes, effectively switching the polarization towards a M1-like anti-cancer phenotype that is characterized by a M1-like spindle morphology, a M1-like glycolytic state, and induction of a pro-inflammatory secretome exhibiting potent anti-tumoral activity towards ovarian cancer spheroids in vitro. In vivo proof-of-principle was established in the syngeneic, macrophage-driven ID8 mouse model for human ovarian cancer, where NMTi treatment significantly reprogrammed murine M2-like TAMs into a M1-like anti-cancer phenotype, concomitantly reducing tumor burden without observable side-effects, and significantly extending median survival by 16 days. We are currently further investigating the intricacies that N-myristoylation plays in macrophage polarization, as well as further establishing the scope of NMTi-driven in vivo reprogramming of TAMs beyond ovarian cancer. Citation Format: Wouter W. Kallemeijn, Sarah Spear, Josephine Walton, Claudio Bussi, Christelle Soudy, Helen R. Flynn, Mark Skehel, David Carling, Roberto Solari, Iain A. McNeish, Edward W. Tate. From foe to friend: In vivo reprogramming of tumor-associated macrophages to an anti-cancer phenotype by modulating N-myristoyltransferase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 439.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

Inhaled corticosteroids (ICS) are recommended treatments for all degrees of asthma severity and in combination with bronchodilators are indicated for COPD patients with a history of frequent exacerbations. However, the long-term side effects of glucocorticoids (GCs) may include increased risk of respiratory infections, including viral triggered exacerbations. Rhinovirus (RV) infection is the main trigger of asthma and COPD exacerbations. Thus, we sought to explore the influence of GCs on viral replication. We demonstrate the ICS fluticasone propionate (FP) and two selective non-steroidal (GRT7) and steroidal (GRT10) glucocorticoid receptor (GR) agonists significantly suppress pro-inflammatory (IL-6 and IL-8) and antiviral (IFN-λ1) cytokine production and the expression of the interferon-stimulated genes (ISGs) OAS and viperin in RV-infected bronchial epithelial cells, with a consequent increase of viral replication. We also show that FP, GRT7 and GRT10 inhibit STAT1 Y701 and/or STAT2 Y690 phosphorylation and ISG mRNA induction following cell stimulation with recombinant IFN-β. In addition, we investigated the effects of the ICS budesonide (BD) and the long-acting β2 agonist (LABA) formoterol, alone or as an ICS/LABA combination, on RV-induced ISG expression and viral replication. Combination of BD/formoterol increases the suppression of OAS and viperin mRNA observed with both BD and formoterol alone, but an increase in viral RNA was only observed with BD treatment and not with formoterol. Overall, we provide evidence of an impairment of the innate antiviral immune response by GC therapy and the potential for GCs to enhance viral replication. These findings could have important clinical implications.

Human N -myristoyltransferases (NMTs) catalyze N-terminal protein myristoylation, a modification regulating membrane trafficking and interactions of >100 proteins. NMT is a promising target in cancer, but a mechanistic rationale for targeted therapy remains poorly defined. Here, large-scale cancer cell line screens against a panel of NMT inhibitors (NMTi) were combined with systems-level analyses to reveal that NMTi is synthetic lethal with deregulated MYC. Synthetic lethality is mediated by post-transcriptional failure in mitochondrial respiratory complex I protein synthesis concurrent with loss of myristoylation and degradation of complex I assembly factor NDUFAF4, followed by mitochondrial dysfunction specifically in MYC-deregulated cancer cells. NMTi eliminated MYC-deregulated tumors in vivo without overt toxicity, providing a new paradigm in which targeting a constitutive co-translational protein modification is synthetically lethal in MYC-deregulated cancers. One-sentence summary N -myristoyltransferase inhibition leads to post-transcriptional complex I failure and cell death in MYC-deregulated cancers

Rationale: Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well characterized. Objectives: To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. Methods: Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16 and underwent bronchoscopy at baseline and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. Measurements and Main Results: At baseline, ILC2s were significantly higher in patients with asthma than in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with asthma than in healthy subjects (all comparisons P < 0.05). In healthy subjects, ILC1s increased from baseline at Day 3 (P = 0.001), while in patients with asthma, ILC1s increased from baseline at Day 8 (P = 0.042). Patients with asthma had significantly higher ILC2:ILC1 ratios at baseline (P = 0.024) and Day 8 (P = 0.005). Increased ILC2:ILC1 ratio in patients with asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid. Conclusions: An ILC2-predominant inflammatory profile in patients with asthma was associated with increased severity and duration of rhinovirus infection compared with healthy subjects, supporting the potential role of ILC2s in the pathogenesis of virus-induced asthma exacerbations.

BackgroundAsthma is a chronic airway disease driven by complex genetic–environmental interactions. The role of epigenetic modifications in bronchial epithelial cells (BECs) in asthma is poorly understood.MethodsWe piloted genome-wide profiling of the enhancer-associated histone modification H3K27ac in BECs from people with asthma (n = 4) and healthy controls (n = 3).ResultsWe identified n = 4,321 (FDR < 0.05) regions exhibiting differential H3K27ac enrichment between asthma and health, clustering at genes associated predominately with epithelial processes (EMT). We identified initial evidence of asthma-associated Super-Enhancers encompassing genes encoding transcription factors (TP63) and enzymes regulating lipid metabolism (PTGS1). We integrated published datasets to identify epithelium-specific transcription factors associated with H3K27ac in asthma (TP73) and identify initial relationships between asthma-associated changes in H3K27ac and transcriptional profiles. Finally, we investigated the potential of CRISPR-based approaches to functionally evaluate H3K27ac-asthma landscape in vitro by identifying guide-RNAs capable of targeting acetylation to asthma DERs and inducing gene expression (TLR3).ConclusionOur small pilot study validates genome-wide approaches for deciphering epigenetic mechanisms underlying asthma pathogenesis in the airways.

CCR4 is the sole receptor for the chemokines CCL22 and CCL17. Clinical studies of asthmatic airways have shown levels of both ligands and CCR4(+) Th2 cells to be elevated, suggestive of a role in disease. Consequently, CCR4 has aroused much interest as a potential therapeutic target and an understanding of how its cell surface expression is regulated is highly desirable. To this end, receptor expression, receptor endocytosis, and chemotaxis were assessed using transfectants expressing CCR4, CCR4(+) human T cell lines, and human Th2 cells polarized in vitro. CCL17 and CCL22 drove rapid endocytosis of CCR4 in a dose-dependent manner. Replenishment at the cell surface was slow and sensitive to cycloheximide, suggestive of de novo synthesis of CCR4. Constitutive CCR4 endocytosis was also observed, with the internalized CCR4 found to be significantly degraded over a 6-h incubation. Truncation of the CCR4 C-terminus by 40 amino acids had no effect on cell surface expression, but resulted in significant impairment of ligand-induced endocytosis. Consequently, migration to both CCL17 and CCL22 was significantly enhanced. In contrast, truncation of CCR4 did not impair constitutive endocytosis or degradation, suggesting the use of alternative receptor motifs in these processes. We conclude that CCR4 cell surface levels are tightly regulated, with a degradative fate for endocytosed receptor. We postulate that this strict control is desirable, given that Th2 cells recruited by CCR4 can induce the further expression of CCR4 ligands in a positive feedback loop, thereby enhancing allergic inflammation.

Since 1996, when the number of FDA drug approvals reached a peak of 53 NMEs, the rate of success declined to a low of only 17 in 2002. However, there is now strong evidence of a revival in the rate of approval of new drugs which first appeared in 2011 (30 NMEs) and is now gathering pace with the endorsement of 59 NMEs in 2018 [1]. On the basis of this data alone, previous reports of the demise of the Pharma industry appear premature. However, how much of this apparent resurgence is down to Pharma and Biotech’s home-grown projects and how much is down to accessing innovation from academia and new initiatives and partnerships with academia and not-for-profit organizations?

Inhaled corticosteroids (ICS) are the mainstay of asthma management. ICS/long-acting β2 agonists (LABA) combinations are widely used as maintenance treatment to prevent exacerbations in asthma and COPD. Viral infections are considered the main trigger of exacerbations in both obstructive lung diseases. However, there is concern on ICS suppression of the antiviral innate immune response. Here, we evaluated by qRT-PCR the modulatory effect of the ICS Budesonide (Bud), alone or in combination with the LABA Formoterol (Form), on interferon stimulated gene (ISG) expression and viral genome quantification in bronchial epithelial cells. BEAS2B cells were pre-treated for 1h with scalar concentrations of tested compounds before 1h rhinovirus 1B (HRV-1B) infection. Treatment was continued with tested compounds for 24h. Bud and to a lesser extent Form reduced in a dose-dependent manner mRNA expression of two antiviral ISGs, viperin and OAS. At a concentration of 1nM, Bud suppressed viperin and OAS gene expression of 92±6% and 82±9% respectively over control vehicle. At a concentration of 10nM, Form suppressed viperin and OAS of 60±36% and 45±42% respectively over control vehicle. The combination Bud0.1nM/Form10nM potentiated viperin and OAS gene suppression compared to the individual effects of Bud and Form at the same concentrations. Furthermore, we observed an increase of viral RNA after exposure to 10nM Bud (1.5-fold) but not Form. Thus, Bud and to a lesser extent Form impaired the early innate immune response in bronchial epithelial cells by suppression of antiviral ISG expression, an effect potentiated by the combination Bud/Form.

There are currently no approved drugs for the treatment of rhinovirus (RV) or indeed of any picornavirus infection, despite decades of drug discovery efforts. Likewise there are no licensed vaccines for RV even though trials began in the late 1960s. However, a large number of experimental approaches that target RV itself, the RV-induced inflammatory response, or promote broad RV-specific immunity have been through preclinical development and clinical trials with mixed success. This chapter will amalgamate these studies and highlight the most promising and applicable therapeutic approaches.

RATIONALE:Immunophenotypes of antiviral responses, and their relationship with asthma, allergy, and lower respiratory tract infections, are poorly understood. OBJECTIVES:We characterized multiple cytokine responses of peripheral blood mononuclear cells to rhinovirus stimulation, and their relationship with clinical outcomes. METHODS:In a population-based birth cohort, we measured 28 cytokines after stimulation with rhinovirus-16 in 307 children aged 11 years. We used machine learning to identify patterns of cytokine responses, and related these patterns to clinical outcomes, using longitudinal models. We also ascertained phytohemagglutinin-induced T-helper cell type 2 (Th2)-cytokine responses (PHA-Th2). MEASUREMENTS AND MAIN RESULTS:We identified six clusters of children based on their rhinovirus-16 responses, which were differentiated by the expression of four cytokine/chemokine groups: interferon-related (IFN), proinflammatory (Inflam), Th2-chemokine (Th2-chem), and regulatory (Reg). Clusters differed in their clinical characteristics. Children with an IFNmodInflamhighestTh2-chemhighestReghighest rhinovirus-16-induced pattern had a PHA-Th2low response, and a very low asthma risk (odds ratio [OR], 0.08; 95% confidence interval [CI], 0.01-0.81; P = 0.03). Two clusters had a high risk of asthma and allergic sensitization, but with different trajectories from infancy to adolescence. The IFNlowestInflamhighTh2-chemlowRegmod cluster exhibited a PHA-Th2lowest response and was associated with early-onset asthma and sensitization, and the highest risk of asthma exacerbations (OR, 1.37; 95% CI, 1.07-1.76; P = 0.014) and lower respiratory tract infection hospitalizations (OR, 2.40; 95% CI, 1.26-4.58; P = 0.008) throughout childhood. In contrast, the IFNhighestInflammodTh2-chemmodReghigh cluster with a rhinovirus-16-cytokine pattern was characterized by a PHA-Th2highest response, and a low prevalence of asthma/sensitization in infancy that increased sharply to become the highest among all clusters by adolescence (but with a low risk of asthma exacerbations). CONCLUSIONS:Early-onset troublesome asthma with early-life sensitization, later-onset milder allergic asthma, and disease protection are each associated with different patterns of rhinovirus-induced immune responses.

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-β. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

In patients with asthma or chronic obstructive pulmonary disease, rhinovirus (RV) infections can provoke acute worsening of disease, and limited treatment options exist. Viral replication in the host cell induces significant remodeling of intracellular membranes, but few studies have explored this mechanistically or as a therapeutic opportunity. We performed unbiased lipidomic analysis on human bronchial epithelial cells infected over a 6 h period with the RV-A1b strain of RV to determine changes in 493 distinct lipid species. Through pathway and network analysis, we identified temporal changes in the apparent activities of a number of lipid metabolizing and signaling enzymes. In particular, analysis highlighted FA synthesis and ceramide metabolism as potential anti-rhinoviral targets. To validate the importance of these enzymes in viral replication, we explored the effects of commercially available enzyme inhibitors upon RV-A1b infection and replication. Ceranib-1, D609, and C75 were the most potent inhibitors, which confirmed that FAS and ceramidase are potential inhibitory targets in rhinoviral infections. More broadly, this study demonstrates the potential of lipidomics and pathway analysis to identify novel targets to treat human disorders.

Inhaled corticosteroids (ICS) have limited efficacy in reducing chronic obstructive pulmonary disease (COPD) exacerbations and increase pneumonia risk, through unknown mechanisms. Rhinoviruses precipitate most exacerbations and increase susceptibility to secondary bacterial infections. Here, we show that the ICS fluticasone propionate (FP) impairs innate and acquired antiviral immune responses leading to delayed virus clearance and previously unrecognised adverse effects of enhanced mucus, impaired antimicrobial peptide secretion and increased pulmonary bacterial load during virus-induced exacerbations. Exogenous interferon-β reverses these effects. FP suppression of interferon may occur through inhibition of TLR3- and RIG-I virus-sensing pathways. Mice deficient in the type I interferon-α/β receptor (IFNAR1−/−) have suppressed antimicrobial peptide and enhanced mucin responses to rhinovirus infection. This study identifies type I interferon as a central regulator of antibacterial immunity and mucus production. Suppression of interferon by ICS during virus-induced COPD exacerbations likely mediates pneumonia risk and raises suggestion that inhaled interferon-β therapy may protect. Corticosteroid therapy is frequently used for chronic obstructive pulmonary disease (COPD) but its use is associated with increased risk of pneumonia. Here the authors show that corticosteroid use impairs innate and adaptive immunity to rhinovirus infection, which is restored by exogenous IFNβ.

Background Asthma is a chronic airway disease driven by complex genetic-environmental interactions. The role of epigenetic modifications in bronchial epithelial cells (BECs) in asthma is poorly understood. We undertook genome-wide profiling of the enhancer-associated histone modification H3K27ac in BECs from people with asthma (n=4) and healthy controls (n=3).Results We identified n=4,321 (FDR <0.05) regions exhibiting differential H3K27ac enrichment between asthma and health clustering at genes associated predominately with epithelial processes (EMT). Asthma dramatically influenced the BEC enhancer landscape and we identified asthma-associated Super-Enhancers encompassing genes encoding transcription factors ( TP63 ) and enzymes regulating lipid metabolism ( PTGS1 ). We integrated published datasets to identify epithelium-specific transcription factors associated with H3K27ac in asthma ( TP73 ) and identify initial relationships between asthma-associated changes in H3K27ac, DNA methylation, genetic susceptibility and transcriptional profiles. Finally, we used a CRISPR-based approach to functionally evaluate components of the H3K27ac-asthma landscape in vitro and provide proof of principal that asthma-associated gene expression ( TLR3 ) is driven in part by aberrant histone acetylation.Conclusion Our small study validates the combination of genome-wide and epigenome-editing approaches in deciphering the molecular mechanisms underlying asthma pathogenesis.* 3D : three dimensions BECs : bronchial epithelial cells CGI’s – CpG Islands ChIP-Seq : Chromatin-immunoprecipitation coupled with high-through-put sequencing. CRISPR-Cas9 : Clustered, regularly interspaced, short palindromic repeats. DERs : differentially enriched regions DEX : dexamethasone ENCODE : Encyclopedia of DNA Elements consortia gRNA : guide RNA H3K27ac : Histone H3 Lys27 acetylation H3K4me2 : Histone H3 Lys4 dimethylation kb : kilobase RPKM : reads per kilobase per million mapped reads SEs : super enhancers SNPs : single nucleotide polymorphisms T2 : Type 2 airway inflammation TFs : transcription factors TSSs : transcriptional start sites