Influenza virus-host interactions and their modulation by small molecules

Influenza viruses cause pandemics and annual epidemics which have serious consequences for public health and global economy. The severity of infections with influenza viruses can vary from asymptomatic to life-threatening viral pneumonias. Currently, four licensed anti-influenza drugs are available for the prevention and treatment of influenza virus infections. However, resistance to the licensed antivirals develops rapidly. Therefore, there is a need for next-generation antiviral agents to combat influenza virus infections. Recent advances in understanding influenza virus-host interactions have revealed a number of host targets for potential antiviral interventions. In particular, basic cellular functions, metabolic and biosynthesis pathways, as well as the signaling cascades could be modulated by small-molecule inhibitors to block virus infection. Moreover, temporal inhibition of these host functions will be less likely to induce viral drug resistance. In addition, many of the inhibitors of cellular functions are already approved or in clinical development for other diseases. Drug repurposing will facilitate their introduction for treatment of viral infections, since the pharmacokinetics and toxicity profile of these drugs are already known. In this work, a library of small-molecule inhibitors targeting host factors and potentially interfering with influenza virus infection was built and screened. Inhibitors of vacuolar proton-ATPase (v-ATPase), Akt kinase, ribonucleotide reductase and the anti-apoptotic B-cell lymphoma-2 family proteins showed antiviral activity in vitro. Saliphenylhalamide, an inhibitor of v-ATPase, was the most potent antiviral agent and it was effective against a broad range of influenza viruses and some other RNA viruses in vitro, and against a mouse adapted influenza strain in vivo. In order to overcome the low water solubility and high toxicity of saliphenylhalamide, bioavailability was optimized using a porous silicon particle-based delivery system for the putative clinical trials. The results presented in this study expand the understanding of influenza virus-host interactions, and provide a novel perspective for ways to adopt a rational approach in the discovery of new antiviral agents.Influenssavirukset aiheuttavat maailmanlaajuisia pandemioita ja jokavuotisia epidemioita joilla on mittavia vaikutuksia kansanterveyteen ja maailmantalouteen. Influenssavirusinfektioiden vakavuus vaihtelee oireettomasta infektiosta hengenvaaralliseen virusperäiseen keuhkokuumeeseen. Influenssavirusinfektioiden ehkäisyyn ja hoitoon on tällä hetkellä olemassa neljä hyväksyttyä lääkettä, mutta virusten vastustuskyky näille lääkeaineille kehittyy nopeasti. Tästä johtuen tarvitaan uusia, seuraavan sukupolven lääkeaineita torjumaan influenssavirusinfektioita. Viimeaikaiset edistysaskeleet tietämyksessä influenssaviruksen ja isännän vuorovaikutuksista ovat paljastaneet useita mahdollisia isäntäsolukohteita uusille virusinfektioita ehkäiseville lääkeaineille. Erityisesti solujen perustoimintoja, aineenvaihdunta-ja biosynteesireittejä, sekä signalointireittejä voitaisiin moduloida pienmolekyylisalpaajien avulla ja siten estää virusinfektio. Lisäksi väliaikainen isäntäsolun toiminnan esto ei todennäköisesti aiheuttaisi virusten lääkeresistenssin kehittymistä. Monet pienimolekyyliset solun toimintojen estäjät on jo hyväksytty muiden sairauksien hoitoon tai ne ovat parhaillaan kliinisessä lääkekehityksessä. Lääkeaineiden uudelleenkohdennus edesauttaisi niiden käyttöönottoa virusinfektioiden hoitoon, koska farmakokineettiset ominaisuudet ja toksisuusprofiilit näille lääkeaineille ovat jo tiedossa. Tässä väitöskirjatyössä rakennettiin ja seulottiin molekyylikirjasto joka koostui isäntäsolun toimintoihin kohdistuvista ja siten mahdollisesti influenssavirusinfektiota estävistä pienmolekyylisalpaajista. Vakuolaarisen protonipumpun (v-ATPaasi ), Akt -kinaasin, ribonukleotidireduktaasin ja anti-apoptoottisen B-solulymfooma-2 perheen proteiinin salpaajat estivät influenssavirusinfektiota in vitro olosuhteissa. V-ATPaasin estäjä, saliphenylhalamide, oli näistä lupaavin, estäen tehokkaasti monia erilaisia influenssaviruksia ja muita RNA-viruksia in vitro olosuhteissa, sekä hiiriadaptoidun influenssaviruksen infektion in vivo olosuhteissa. Saliphenylhalamiden käyttöä lääkeaineena haittaa sen alhainen vesiliukoisuus ja korkea toksisuus, joten saliphenylhalamiden biosaatavuutta optimoitiin onnistuneesti käyttämällä huokoisiin piipartikkeleihin pohjautuvaa lääkeaineen annostelusysteemiä mahdollisia tulevia kliinisiä tutkimuksia varten. Tässä työssä esitetyt tulokset laajentavat ymmärtämystä influenssaviruksen ja isännän vuorovaikutuksista ja tarjoavat uusia näkökulmia rationaaliseen lähestymistapaan uusien viruslääkeaineiden löytämisessä

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.Peer reviewe

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.Peer reviewe

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.Peer reviewe

Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14–15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome‐wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late‐stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy‐lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.</p

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma

Contains fulltext : 226259.pdf (publisher's version ) (Open Access

The Effect of Mechanical Activation and Lignin Impurities on the Hydrolysis-Dehydration of Cellulose in the Presence of Sibunit-4 Solid Acidic Carbon Catalysts

Исследовано влияние механической активации и примесей лигнина на реакционную способность образцов целлюлозы, выделенных методом гидротропной варки из мискантуса, соломы пшеницы и плодовых оболочек овса, в процессе их гидролиза-дегидратации в присутствии твердых кислотных катализаторов, полученных сульфированием и окислением углеродного материала Сибунит-4. Показано, что активация существенно увеличивает реакционноспособность всех субстратов и выходы целевых продуктов реакции (моносахаридов и фурфуролов). Наличие примесей лигнина приводит к уменьшению выходов продуктов в процессе деполимеризации целлюлозыIn the presence of acidic catalysts based on the Sibunit-4 material the effects of mechanical activation and a lignin impurities on both the reactivity of cellulose and yields of products (monosaccharides and furfurals) being achieved during the hydrolysis of the polysaccharide isolated by hydrotropic cooking of miscanthus, wheat straw, and fruit shells of oats were studied. Activation was shown to significantly increase the reactivity of all substrates and the yields of the target products. Impurities of lignin led to decreasing product yield

PP2A-based triple-strike therapy overcomes mitochondrial apoptosis resistance in brain cancer cells

Mitochondrial glycolysis and hyperactivity of the phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway are hallmarks of malignant brain tumors. However, kinase inhibitors targeting AKT (AKTi) or the glycolysis master regulator pyruvate dehydrogenase kinase (PDKi) have failed to provide clinical benefits for brain tumor patients. Here, we demonstrate that heterogeneous glioblastoma (GB) and medulloblastoma (MB) cell lines display only cytostatic responses to combined AKT and PDK targeting. Biochemically, the combined AKT and PDK inhibition resulted in the shutdown of both target pathways and priming to mitochondrial apoptosis but failed to induce apoptosis. In contrast, all tested brain tumor cell models were sensitive to a triplet therapy, in which AKT and PDK inhibition was combined with the pharmacological reactivation of protein phosphatase 2A (PP2A) by NZ-8-061 (also known as DT-061), DBK-1154, and DBK-1160. We also provide proof-of-principle evidence for in vivo efficacy in the intracranial GB and MB models by the brain-penetrant triplet therapy (AKTi + PDKi + PP2A reactivator). Mechanistically, PP2A reactivation converted the cytostatic AKTi + PDKi response to cytotoxic apoptosis, through PP2A-elicited shutdown of compensatory mitochondrial oxidative phosphorylation and by increased proton leakage. These results encourage the development of triple-strike strategies targeting mitochondrial metabolism to overcome therapy tolerance in brain tumors.Peer reviewe

Inhibition of Influenza A Virus Infection <i>in Vitro</i> by Saliphenylhalamide-Loaded Porous Silicon Nanoparticles

Influenza A viruses (IAVs) cause recurrent epidemics in humans, with serious threat of lethal worldwide pandemics. The occurrence of antiviral-resistant virus strains and the emergence of highly pathogenic influenza viruses have triggered an urgent need to develop new anti-IAV treatments. One compound found to inhibit IAV, and other virus infections, is saliphenylhalamide (SaliPhe). SaliPhe targets host vacuolar-ATPase and inhibits acidification of endosomes, a process needed for productive virus infection. The major obstacle for the further development of SaliPhe as antiviral drug has been its poor solubility. Here, we investigated the possibility to increase SaliPhe solubility by loading the compound in thermally hydrocarbonized porous silicon (THCPSi) nanoparticles. SaliPhe-loaded nanoparticles were further investigated for the ability to inhibit influenza A infection in human retinal pigment epithelium and Madin-Darby canine kidney cells, and we show that upon release from THCPSi, SaliPhe inhibited IAV infection <i>in vitro</i> and reduced the amount of progeny virus in IAV-infected cells. Overall, the PSi-based nanosystem exhibited increased dissolution of the investigated anti-IAV drug SaliPhe and displayed excellent <i>in vitro</i> stability, low cytotoxicity, and remarkable reduction of viral load in the absence of organic solvents. This proof-of-principle study indicates that PSi nanoparticles could be used for efficient delivery of antivirals to infected cells