Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

Correction: Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy.

[This corrects the article DOI: 10.1371/journal.ppat.1009340.].

Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy.

Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.

Influenza A virus causes considerable morbidity and mortality largely because of a lack of effective antiviral drugs. Viral neuraminidase inhibitors, which inhibit viral release from the infected cell, are currently the only approved drugs for influenza, but have recently been shown to be less effective than previously thought. Growing resistance to therapies that target viral proteins has led to increased urgency in the search for novel anti-influenza compounds. However, discovery and development of new drugs have been restricted because of differences in susceptibility to influenza between animal models and humans and a lack of translation between cell culture and in vivo measures of efficacy. To circumvent these limitations, we developed an experimental approach based on ex vivo infection of human bronchial tissue explants and optimized a method of flow cytometric analysis to directly quantify infection rates in bronchial epithelial tissues. This allowed testing of the effectiveness of TVB024, a vATPase inhibitor that inhibits viral replication rather than virus release, and to compare efficacy with the current frontline neuraminidase inhibitor, oseltamivir. The study showed that the vATPase inhibitor completely abrogated epithelial cell infection, virus shedding, and the associated induction of proinflammatory mediators, whereas oseltamivir was only partially effective at reducing these mediators and ineffective against innate responses. We propose, therefore, that this explant model could be used to predict the efficacy of novel anti-influenza compounds targeting diverse stages of the viral replication cycle, thereby complementing animal models and facilitating progression of new drugs into clinical trials.

Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

Anti-citrullinated histone monoclonal antibody CIT-013, a dual action therapeutic for neutrophil extracellular trap-associated autoimmune diseases.

Neutrophil extracellular traps (NETs) contribute to the pathophysiology of multiple inflammatory and autoimmune diseases. Targeting the NETosis pathway has demonstrated significant therapeutic potency in various disease models. Here, we describe a first-in-class monoclonal antibody (CIT-013) with high affinity for citrullinated histones H2A and H4, which inhibits NETosis and reduces tissue NET burden in vivo with significant anti-inflammatory consequences. We provide a detailed understanding of the epitope selectivity of CIT-013. Detection of CIT-013 epitopes in rheumatoid arthritis (RA) synovium provides evidence that RA is an autoimmune disease with excessive citrullinated NETs that can be targeted by CIT-013. We show that CIT-013 acts upon the final stage of NETosis, binding to its chromatin epitopes when plasma membrane integrity is compromised to prevent NET release. Bivalency of CIT-013 is necessary for NETosis inhibition. In addition, we show that CIT-013 binding to NETs and netting neutrophils enhance their phagocytosis by macrophages in an Fc-dependent manner. This is confirmed using a murine neutrophilic airway inflammation model where a mouse variant of CIT-013 reduced tissue NET burden with significant anti-inflammatory consequences. CIT-013's therapeutic activity provides new insights for the development of NET antagonists and indicates the importance of a new emerging therapy for NET-driven diseases with unmet therapeutic needs.

An anti-diabetic drug targets NEET (CISD) proteins through destabilization of their [2Fe-2S] clusters.

Elevated levels of mitochondrial iron and reactive oxygen species (ROS) accompany the progression of diabetes, negatively impacting insulin production and secretion from pancreatic cells. In search for a tool to reduce mitochondrial iron and ROS levels, we arrived at a molecule that destabilizes the [2Fe-2S] clusters of NEET proteins (M1). Treatment of db/db diabetic mice with M1 improved hyperglycemia, without the weight gain observed with alternative treatments such as rosiglitazone. The molecular interactions of M1 with the NEET proteins mNT and NAF-1 were determined by X-crystallography. The possibility of controlling diabetes by molecules that destabilize the [2Fe-2S] clusters of NEET proteins, thereby reducing iron-mediated oxidative stress, opens a new route for managing metabolic aberration such as in diabetes.

A GAL4-based yeast three-hybrid system for the identification of small molecule-target protein interactions.

We report the development of a yeast strain designed for assaying compound-protein interactions through activation of reporter gene expression. Activation of lacZ expression, driven by the GAL4 promoter, has been demonstrated for precedented compound-protein interactions between FK506 and FK506 binding protein 12 (FKBP12) and also between methotrexate (MTX) and dihydrofolate reductase (DHFR). Reporter gene expression was completely abrogated in a competitive manner by the presence of excess FK506 or MTX, respectively. In addition, a strain expressing a mutated DHFR clone with decreased binding affinity for MTX was not capable of activating reporter gene expression. While strain sensitivity is compound-dependent, the minimum compound concentration necessary to drive reporter gene expression was 20 nM for the FK506-FKBP12 interaction. The utility of this strain as a tool for identifying unknown compound-binding proteins has been demonstrated by screening a mouse cDNA library for clones that encode proteins capable of binding MTX. Four library clones of mouse DHFR were identified after screening 5 x 10(6) clones. The screen background was low and false positives were easily identified, making this yeast system particularly amenable for use in a screening context for novel compound-protein interactions.

Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance.

The incidence and prevalence of pathological fibrosis increase with advancing age, although mechanisms for this association are unclear. We assessed the capacity for repair of lung injury in young (2 months) and aged (18 months) mice. Whereas the severity of fibrosis was not different between these groups, aged mice demonstrated an impaired capacity for fibrosis resolution. Persistent fibrosis in lungs of aged mice was characterized by the accumulation of senescent and apoptosis-resistant myofibroblasts. These cellular phenotypes were sustained by alterations in cellular redox homeostasis resulting from elevated expression of the reactive oxygen species-generating enzyme Nox4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-4] and an impaired capacity to induce the Nrf2 (NFE2-related factor 2) antioxidant response. Lung tissues from human subjects with idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, also demonstrated this Nox4-Nrf2 imbalance. Nox4 mediated senescence and apoptosis resistance in IPF fibroblasts. Genetic and pharmacological targeting of Nox4 in aged mice with established fibrosis attenuated the senescent, antiapoptotic myofibroblast phenotype and led to a reversal of persistent fibrosis. These studies suggest that loss of cellular redox homeostasis promotes profibrotic myofibroblast phenotypes that result in persistent fibrosis associated with aging. Our studies suggest that restoration of Nox4-Nrf2 redox balance in myofibroblasts may be a therapeutic strategy in age-associated fibrotic disorders, potentially able to resolve persistent fibrosis or even reverse its progression.

hCLCA1 and mCLCA3 are secreted non-integral membrane proteins and therefore are not ion channels.

Proteins of the CLCA gene family have been proposed to mediate calcium-activated chloride currents. In this study, we used detailed bioinformatics analysis and found that no transmembrane domains are predicted in hCLCA1 or mCLCA3 (Gob-5). Further analysis suggested that they are globular proteins containing domains that are likely to be involved in protein-protein interactions. In support of the bioinformatics analysis, biochemical studies showed that hCLCA1 and mCLCA3, when expressed in HEK293 cells, could be removed from the cell surface and could be detected in the extracellular medium, even after short incubation times. The accumulation in the medium was shown to be brefeldin A-sensitive, demonstrating that hCLCA1 is constitutively secreted. The N-terminal cleavage products of hCLCA1 and mCLCA3 could be detected in bronchoalveolar lavage fluid taken from asthmatic subjects and ovalbumin-challenged mice, demonstrating release from cells in a physiological setting. We conclude that hCLCA1 and mCLCA3 are non-integral membrane proteins and therefore cannot be chloride channels in their own right.