Data_Sheet_1_DeCoST: A New Approach in Drug Repurposing From Control System Theory.DOCX

<p>In this paper, we propose DeCoST (Drug Repurposing from Control System Theory) framework to apply control system paradigm for drug repurposing purpose. Drug repurposing has become one of the most active areas in pharmacology since the last decade. Compared to traditional drug development, drug repurposing may provide more systematic and significantly less expensive approaches in discovering new treatments for complex diseases. Although drug repurposing techniques rapidly evolve from “one: disease-gene-drug” to “multi: gene, dru” and from “lazy guilt-by-association” to “systematic model-based pattern matching,” mathematical system and control paradigm has not been widely applied to model the system biology connectivity among drugs, genes, and diseases. In this paradigm, our DeCoST framework, which is among the earliest approaches in drug repurposing with control theory paradigm, applies biological and pharmaceutical knowledge to quantify rich connective data sources among drugs, genes, and diseases to construct disease-specific mathematical model. We use linear–quadratic regulator control technique to assess the therapeutic effect of a drug in disease-specific treatment. DeCoST framework could classify between FDA-approved drugs and rejected/withdrawn drug, which is the foundation to apply DeCoST in recommending potentially new treatment. Applying DeCoST in Breast Cancer and Bladder Cancer, we reprofiled 8 promising candidate drugs for Breast Cancer ER+ (Erbitux, Flutamide, etc.), 2 drugs for Breast Cancer ER- (Daunorubicin and Donepezil) and 10 drugs for Bladder Cancer repurposing (Zafirlukast, Tenofovir, etc.).</p

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<p>Shiga toxin (Stxs) is a family of structurally and functionally related bacterial cytotoxins produced by Shigella dysenteriae serotype 1 and shigatoxigenic group of Escherichia coli that cause shigellosis and hemorrhagic colitis, respectively. Until recently, it has been thought that Stxs only inhibits the protein synthesis and induces expression to a limited number of genes in host cells, but recent data showed that Stxs can trigger several signaling pathways in mammalian cells and activate cell cycle and apoptosis. To explore the changes in gene expression induced by Stxs that have been shown in other systems to correlate with cancer progression, we performed the simulated analysis of cDNA dataset and found differentially expressed genes (DEGs) of human THP1-monocytic cells treated with Stxs. In this study, the entire data (treated and untreated replicates) was analyzed by statistical algorithms implemented in Bioconductor packages. The output data was validated by the k-fold cross technique using generalized linear Gaussian models. A total of 50 DEGs were identified. 7 genes including TSLP, IL6, GBP1, CD274, TNFSF13B, OASL, and PNPLA3 were considerably (<0.00005) related to cancer proliferation. The functional enrichment analysis showed 6 down-regulated and 1 up-regulated genes. Among these DEGs, IL6 was associated with several cancers, especially with leukemia, lymphoma, lungs, liver and breast cancers. The predicted regulatory motifs of these genes include conserved RELA, STATI, IRFI, NF-kappaB, PEND, HLF, REL, CEBPA, DI_2, and NFKB1 transcription factor binding sites (TFBS) involved in the complex biological functions. Thus, our findings suggest that Stxs has the potential as a valuable tool for better understanding of treatment strategies for several cancers.</p

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<p>Shiga toxin (Stxs) is a family of structurally and functionally related bacterial cytotoxins produced by Shigella dysenteriae serotype 1 and shigatoxigenic group of Escherichia coli that cause shigellosis and hemorrhagic colitis, respectively. Until recently, it has been thought that Stxs only inhibits the protein synthesis and induces expression to a limited number of genes in host cells, but recent data showed that Stxs can trigger several signaling pathways in mammalian cells and activate cell cycle and apoptosis. To explore the changes in gene expression induced by Stxs that have been shown in other systems to correlate with cancer progression, we performed the simulated analysis of cDNA dataset and found differentially expressed genes (DEGs) of human THP1-monocytic cells treated with Stxs. In this study, the entire data (treated and untreated replicates) was analyzed by statistical algorithms implemented in Bioconductor packages. The output data was validated by the k-fold cross technique using generalized linear Gaussian models. A total of 50 DEGs were identified. 7 genes including TSLP, IL6, GBP1, CD274, TNFSF13B, OASL, and PNPLA3 were considerably (<0.00005) related to cancer proliferation. The functional enrichment analysis showed 6 down-regulated and 1 up-regulated genes. Among these DEGs, IL6 was associated with several cancers, especially with leukemia, lymphoma, lungs, liver and breast cancers. The predicted regulatory motifs of these genes include conserved RELA, STATI, IRFI, NF-kappaB, PEND, HLF, REL, CEBPA, DI_2, and NFKB1 transcription factor binding sites (TFBS) involved in the complex biological functions. Thus, our findings suggest that Stxs has the potential as a valuable tool for better understanding of treatment strategies for several cancers.</p

RNA protection assay.

<p>HCV RNA was denatured by incubation at 65°C for 5 min. The threshold and a 10-fold higher Benzonase doses were added to the SSB protein–HCV RNA mixture. HCV RNA with no SSB proteins was used as the control.</p

SSB proteins bind ssRNA.

<p>TAE agarose gel electrophoresis (1.2%) and an <i>in vitro</i>–transcribed HCV RNA were used to assess SSB protein-binding activity. Non-denatured RNA and denatured RNA were mixed with 240 and 600 min heat-treated SSB proteins. Zero minutes represents SSB proteins that were not heated, and nude RNA (denatured at 65°C for 5 min) represents RNA alone.</p

SSB proteins modulated HCV RNA metabolism.

<p>A. <i>In vitro</i>–transcribed HCV RNA (500 ng) was transfected into 1×10⁶ Huh7 cells with or without 10 or 20 ng of SSB proteins. The BSA was used as random protein control. After the 72-h incubation, the cells were harvested for RNA expression analysis. A HCV-infected patient serum was used as the primary antibody to probe HCV proteins expressed by transfected HCV RNA. The positive cells were visualised using a FITC-labelled goat anti-human antibody followed by FACS analysis. All <i>p</i> values marked by star are less than 0.05. B. The mean fluorescence intensity of the HCV protein positive cells were calculated by FACS. All <i>p</i> values marked by star are less than 0.05. C. cellular localization and co-localization analysis, the above cells were double stained with anti-HCV serum and anti-His (all SSB proteins were labelled with His-tag) antibody, localization of SSB proteins and co-localization of SSB and HCV protein were evaluated by a cofocal microscope.</p

SDS-PAGE and native PAGE analysis of SSB proteins.

<p>A. Purity and protein molecular weight were visualised by SDS-PAGE in a 12.5% polyacrylamide gel containing 0.1% SDS under reducing conditions. B. Functional SSB protein polymers were notarised by 3.5–5–12% non-denaturing gel electrophoresis.</p

Primary sequence analysis of four SSB proteins.

<p>The amino-acid sequences of TTH, BL21, KOD and SSOB SSB proteins (NCBI access number: AP008226, AM946981, GM017008, and NP_343725, respectively) were aligned using a multiple alignment program for amino acid or nucleotide sequences (<a href="http://mafft.cbrc.jp/alignment/server/" target="_blank">http://mafft.cbrc.jp/alignment/server/</a>). The structure of the DNA-binding domain was marked according to reference 3. The conserved DNA-binding sites (amino acids labelled in red) were identified by the NCBI CD-Search & Batch CD-Search online programs. One dot represents a semi-conserved amino acid change, two dots represent a conserved amino acid change, and stars represent identical amino acids. All SSB proteins were abbreviated by the nomenclature of their ancestor bacteria.</p

Heat-resistance analysis of four SSB proteins.

<p>Four SSB proteins were heated at 95°C for 1, 2, 3, 4, 5, 120, 240, and 600 min, respectively, and their binding activities were assessed by native PAGE. Black arrow indicates the SSB protein–ssDNA binding complex. Non-heat-treated SSB proteins were used as controls.</p

Effect of SSB proteins on long-fragment human genome amplification.

<p>Human beta globin gene fragments (5, 9, and 13 kb) were used as templates to assess the effect of SSB proteins on PCR amplification under optimised conditions.</p