A Network Pharmacology Approach for the Identification of Common Mechanisms of Drug-Induced Peripheral Neuropathy

Drug-induced peripheral neuropathy is a side effect of a variety of therapeutic agents that can affect therapeutic adherence and lead to regimen modifications, impacting patient quality of life. The molecular mechanisms involved in the development of this condition have yet to be completely described in the literature. We used a computational network pharmacology ap-proach to explore the Connectivity Map, a large collection of transcriptional profiles from drug perturbation experiments to identify common genes affected by peripheral neuropathy-inducing drugs. Consensus profiles for 98 of these drugs were used to construct a drug–gene perturbation network. We identified 27 genes significantly associated with neuropathy- inducing drugs. These genes may have a potential role in the action of neuropathy-inducing drugs. Our results suggest that molecular mechanisms, including alterations in mitochondrial function, microtubule and cytoskeleton function, ion chan-nels, transcriptional regulation including epigenetic mechanisms, signal transduction, and wound healing, may play a critical role in drug-induced peripheral neuropathy

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis

<div><p>Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. <i>MYC</i> and <i>TAF4</i> regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. <i>CTCF</i>, <i>RHOA</i>, and <i>CDKN1B</i>) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.</p></div

Caracterización proteómica de la interacción y la inserción de proteínas de membrana

Las proteínas de membrana tienen funciones muy diversas en la célula, desde controlar el tráfico molecular hasta facilitar la transducción de señales. Sin embargo, el estudio de estas proteínas supone todo un reto por sus particulares características fisico-químicas. Su naturaleza hidrofóbica requiere un elevado control durante su síntesis. Uno de los pasos críticos en esta síntesis es la inserción en la bicapa lipídica. Generalmente, este proceso se produce a través de un mecanismo (co-traduccional) acoplado al proceso de traducción. Esta vía co-traduccional ha sido ampliamente estudiada y la maquinaria implicada en el proceso se conoce con bastante profundidad. Sin embargo, algunas proteínas (aquellas que presentan el primer segmento transmembrana lejos del inicio de la proteína) siguen una ruta para su inserción diferente. Esta ruta alternativa, es conocida como post-traduccional, debido a que la inserción en la bicapa se produce tras la traducción de la proteína. Los detalles moleculares de esta vía alternativa están pobremente caracterizados. En la primera parte de esta tesis hemos descrito un procedimiento experimental para identificar proteínas involucradas en la inserción de proteínas de membrana a través de la ruta post-traduccional. Para ello hemos creado una quimera que contiene la nucleasa de Staphylococcus aureus seguida del fragmento transmembrana de Glicoforina A (proteína modelo de membrana) en el extremo carboxilo terminal. Una proteína quimérica idéntica, pero carente del segmento transmembrana fue utilizada como control. A continuación, hemos analizado el interactoma de ambas quimeras utilizando diferentes estrategias experimentales como espectrometría de masas, entrecruzamiento químico, geles de dos dimensiones no reductores/reductores y fraccionamiento celular con la esperanza de encontrar proteínas que se asocien de manera específica a la quimera con segmento transmembrana y que participen en el proceso de inserción de la misma. A continuación, validamos la interacción de dos de las proteínas identificadas (HslU, una proteína con función chaperona y MetH, una proteína implicada en el metabolismo de la metionina) mediante técnicas de cromatografía de exclusión molecular, ensayos de luz dispersada e identificación de los fragmentos entrecruzados. Los datos obtenidos nos han permitido identificar nuevos componentes de la ruta post-traduccional así como analizar la multifuncionalidad del proteoma de E.coli, . Comprender las interacciones entre proteínas facilita información sobre el funcionamiento de las mismas y, entre otras aplicaciones, puede resultar de utilidad para el diseño de fármacos. De hecho, el estudio de las interacciones proteína-proteína entre la célula hospedadora y los virus, ha permitido la identificación de componentes celulares implicados en la infección viral, que pueden ser potenciales dianas para el desarrollo de vacunas o medicamentos con acción antiviral. En esta segunda parte de las tesis, hemos analizado mediante técnicas de espectrometría de masas las proteínas celulares presentes en partículas virales (VLPs) del virus Nipah. Este virus pertenece a la familia Paramyxoviridae, la cual está compuesta por virus con genomas de ssRNA de cadena negativa y envoltura lipídica. Actualmente carecemos de tratamientos específicos contra el virus Nipah, siendo estos una prioridad en la investigación biomédica debido a la alta tasa de mortalidad asociada a la infección y el amplio rango de hospedadores a los que puede afectar. Para identificar las proteínas celulares incorporadas en el virión de Nipah, se generaron VLPs (Virus Like Particles) mediante la transfección de las proteínas virales F, G y M en cultivos celulares humanos. Posteriormente las VLPs producidas fueron purificadas y los componentes celulares asociados a las mismas analizados mediante espectrometría de masas. Nuestros resultados mostraron la presencia de proteínas celulares en las VLPs de Nipah que participaban principalmente en el transporte de proteínas y/o vesículas desde los compartimentos internos hasta la superficie celular. Estas proteínas celulares podrían tener una implicación en el ciclo viral y son por lo tanto potenciales dianas terapéuticas contra el virus Nipah.Membrane proteins perform different functions in the cell, controlling molecular trafficking and facilitating signal transduction. However, the study of these proteins is challenging due to their particular physicochemical characteristics. One critical step in the synthesis of membrane proteins is their insertion into the lipid bilayer. Generally, this process occurs through a co-translational mechanism, where insertion is coupled to the translation process. This co-translational pathway has been widely studied and the machinery involved in the process is well known. However, some proteins follow a different route for insertion which molecular details are poorly characterized. This alternative route is known as post-translational, because the insertion in the bilayer occurs once the protein translation is complete. In the first part of this thesis, we have described an experimental procedure to identify proteins involved in membrane protein insertion through the post-translational route. For this purpose we desinged a chimera containing the Staphylococcus aureus nuclease followed by the transmembrane fragment of Glycoforin A, located at the carboxyl terminal end of the chimera. An identical chimeric protein lacking the transmembrane segment was used as a control. Next, we analyzed the interactome associated with both chimeras using different strategies (mass spectrometry, chemical cross-linking, non-reducing / reducing two-dimensional gels and cell fractionation) with the aim of finding proteins that associate specifically with the chimera containing the transmembrane domain. We identified XXXX proteins interacting with our membrane chimera. Next, we validate the interaction of two of these proteins (HslU, with chaperone function and MetH, involved in methionine metabolism) using molecular exclusion chromatography techniques, light scattering assays and identification of cross-linked peptides within mass spectra. In this work we have identify new components of the post-translational route. However, their precise role on the insertion of Ct anchoring proteins remains elusive. Understanding protein interactions can provide a strong foundation for the complete understanding of cell mechanisms, information that is valuable for drug discovery and development. Identification of the protein-protein interactions between host cells and viruses has allowed the identification of many components involved in viral infection, which may be potential targets for vaccine or drug development. In the second part of the thesis, we analyzed the cellular proteins present in viral particles of the Nipah virus using mass spectrometry techniques. Nipah virus belongs to the family Paramyxoviridae, composed of single-stranded RNA viruses with negative genome polarity and lipid envelope. We currently lack specific treatments against Nipah virus. This virus infects a wide range of animals and causes severe disease and death in people, making it a public health concern. To identify the cellular proteins incorporated in the Nipah virion, VLPs (Virus Like Particles) were generated by transfection of human cells with the viral proteins F, G and M. Next, VLPs produced were purified and the cellular components analyzed by mass spectrometry. We identified 67 host protein in the viral particles. These proteins i participate mainly in the transport of membrane vesicles from internal membranous compartments to the plasma membrane or viceversa. Collectively, our data might contribute to decipher viral budding necessities and particularly to a more profound knowledge of NiV life cycle

Pharmacology of novel approaches designed to target picornaviral infections

Many Picornaviridae family members infect humans. Enterovirus (EV), the largest genus, causes diseases ranging from the common cold to fatal heart disease and paralysis. Human parechoviruses (HPeV) are at least as prevalent as EVs. Currently, no antipicornavirus drugs are used clinically and the large number of viruses precludes vaccination. New drugs are therefore required. Picornavirus infection exploits viral and host proteins, lipids and cellular systems, all potentially targetable by inhibitors. In addition to compounds with a known target, natural products are potential sources of new drugs. Plant material such as berries are cheap, and some extracts have proven antiviral activity. The aim of this project was to repurpose known drugs, approved and used for several years for various medical indications, as well as identify new compounds from natural sources. Coxsackievirus A9 (CAV9) was used as a typical enterovirus and a plaque reduction assay was used to assess antivirus activity. Both approaches gave promising agents. Fluoxetine and dibucaine caused a complete CAV9 inhibition at low concentrations. All drug resistant mutants (DRM) against these compounds had a protein 2C I227V mutation, suggesting this protein is targeted, in accord with previous work on coxsackievirus B3. The identification of DRMs with a single mutation suggests that drug resistance could be problematical if dibucaine and fluoxetine are used clinically. A previously observed interaction between lipid droplets (LDs) and CAV9 and HPeV1 led to studies of LD targeting agents. DGAT inhibitors A922500 and betulinic acid were the most active compounds, in addition to promising results from aspirin and metformin. Redcurrant extract showed antiviral activity, in addition to antioxidant and photodynamic activity. Some microalgae extracts also showed antiviral effects. Although much works remains to be done to fully develop these novel approaches, they have great potential to combat the serious effect of picornavirus infections