Discovery Of Host-targeted Anti-adenovirus Drugs Based On Drug Repositioning Strategy

Objective:To discover potential therapeutic host targets and drugs with anti-adenovirus activity.Metheds:1.Prediction of drug repositioning for anti-adenovirus:(1)Methods based on association network:we constructed association network between the Humanadenovirus(HAdV)and drugs(FDA-approved drugs and Natural product-derived drugs)through integrating the information of HAdV-target associations and drug-target associations based on the accumulated knowledge on EHFPI(Essential Host Factors for Pathogenic Infection)database,VirHostNet database and DrugBank database,HIT(Herbal Ingredients&Targets)database to explore possible host-directed anti-adenovirus drugs.(2)Methods based on transcriptional expression profile analysis:we constructed time-course transcriptional expression profile of HAdV infected human lung epithelial cells(A549 cells)through high-throughput sequencing.Identification of key host targets through Upstream regulator analysis of differentially expressed genes(DEGs).(3)Methods based on Expression Signatures Analysis:Comparison of Ad5 infected A549 cells expression profile and drug disturbance expression profile from Library of Integrated Network-based Cellular Signatures(LINCS)database to predict anti-adenovirus drugs through Gene Set Enrichment Analysis Based on Expression Signatures.2.Host target verification:Verification of the relationship between the identified host target and adenovirus replication via gene overexpression technology and siRNA interference technology.3.Evaluation of antiadenovirus activity:In vitro:Cytopathic effect(CPE)inhibition assay,plaque reduction assay and determination of DNA virus copy number were carried out to evaluate the activity of target drugs and explore the possible mechanism.In vivo:Established the viable animal model of HAdV infection,and evaluated the activity of the target drug in vivo.Results:1.A antitumor drug Roscovitine which in phase Ⅱ clinical trials was screened out based on association networkMethods andshowed good antiadenovirus activity in vitro,which activity was superior to positive drug cidofovir.The median effective concentration(IC50)for inhibiting the titer of Ad5,Ad7 and Ad55 infected A549 cells were 4.36 μM、6.49 μMand 3.93 μM,respectively.In addition,the IC50 of four Roscovitine derivatives for inhibiting the viral titer in Ady infected A549 cells were about 2 μM.2.The functional annotations of DEGs in time-course transcnptional expression profile of HAdV infected A549 cells were the functions of body injury,tumorigenesis,cell cycle regulation,immunoreaction,coagulation,angiogenesis and so on.3.We predicted the upstream regulatory factors of these DEGs through Upstream regulator analysis through the IPA tool.Based on the consistency between the predicted activation status and the corresponding gene differentially expression,seven upstream regulatory factors at 6 hpi and 16 upstream regulatory factors at 12 hpi were screened out.In contrast,five upstream regulators,namely,CD24,PIMI,MAP3K14,RARp,and NEDD9,were screened out at two points and their functions were inhibited.The RARp(retinoic acid receptor p)molecule,one of the upstream regulatory factors of differentially expressed genes(DEGs),played important roles in HAdV replication.The results of reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting showed that RARp mRNA and protein were downregulated by HAdV infection in the A549 cells.The knockdown of RARp by RARp siRNA increased the HAdV production and the overexpression of RARp decreased the HAdV production.Furthermore,FDA-approved Tazarotene,which is an RAR selective agonist with relatively more selectivity for RARp,was found to reduced HAdV production and inhibited HAdV DNA replication in a dose-dependent manner in vitro.Further study suggested that the blockade of HAdV replication by Tazarotene happened after the expression of HAdV early proteins and Tazarotene prevented adenovirus infection from progressing into the late stage.Taken together,a key host molecule in adenovirus infection,which could be developed as a potential host target to an anti-adenovirus drug.4.Rosiglitazone,an FDA-approved for the treatment of type 2 diabetes,could be potentially re-exploited for therapeutic applications in adenovirus infection.The drug could activate the PPAR gamma receptor directly and promote the phosphorylation of STAT1 protein with the synergistic effect of PGC-1 alpha oofactors.Induced activation of type I interferon signal pathway after combining with IRF9,subsequent induction of antiviral interferon stimulation gene expression to inhibit HAdV replication.5.A model of type 5 adenovirus infected SCID mice was established.The model mice showed severe pulmonary tissue lesions.Type 5 adenovirus infection involved abnormal changes in a variety of inflammatory cytokines.Virus DNA proliferate in mice’s lung,liver and kidney tissues.High infection virus titer could still be maintained in the lung tissue of mice after 8 days of infection.The model was applied to evaluate the efficacy of rosiglitazone in vivo,results show that rosiglitazone treatment after administration of 25mg/kg/d reflected the significant inhibition of adenovirus replication and reduced pathological changes of lung tissue caused by infection.Conclusion:In summary,three FDA-approved drugs were screened out and validated to be potentially re-exploited for therapeutic applications in adenovirus infection based on drug repositioning strategy through combined with time-course transcriptional expression profile of HAdV infected A549 cells and multiple knowledge databases.Four derivatives of roscovitine were also shown significant anti adenovirus activity,which provided candidate structures for the development of new antiadenovirus drugs.Retinoic acid receptor β,a potential therapeutic target in the inhibition of adenovirus replication was found,which provided rich clues and theoretical support for the development of anti-HAdV drugs.