Browsing by Subject "high-throughput screening"

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  • Wang, Linping; Saarela, Jani; Poque, Sylvain; Valkonen, Jari P. T. (2020)
    The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.
  • Peltonen, Kaisa (Helsingin yliopisto, 2018)
    New alternative feedstocks are needed for biofuel production to fulfil the growing demand in the coming years. The industry is moving away from second-generation biofuels, produced from food and feed crops, to using waste streams from industrial processes. An abundant, cheap and attractive waste stream for processing in Europe is the pectin-rich pulp from sugar beet processing and fruit juice industry. Sugar beet pulp is particularly rich in D-galacturonic acid and arabinose, but neither are naturally used by the yeast Saccharomyces cerevisiae, which would be an interesting candidate for the microbial fermentation of the biomass. S. cerevisiae is one of the most used organisms in the industrial biotechnology, and methods for the genetic engineering of the organism are highly developed. To overcome the natural limitations of the yeast for D-galacturonic acid fermentation, the metabolic pathways present in other organisms could be integrated in the yeast genome. Two bacterial and one fungal pathway are known to convert D-galacturonic acid into metabolites of the yeast glycolytic and ethanol fermentation pathways, and are thus considered promising for engineering in yeast. A major engineering challenge in integrating the fungal pathway in yeast is the redox imbalance caused by the two NADPH-specific reducing enzymes. The aim of this thesis was to review the potential of different D-galacturonic acid pathways for yeast fermentation. S. cerevisiae is a well-characterised organism for heterologous protein expression, but at times functional expression of foreign proteins is not achieved. One approach to study the pathways was to clone and express enzymes of the bacterial isomerase and dehydratase pathways in S. cerevisiae, and to test their activity in culture lysates. In addition, to overcome the redox imbalance in the eukaryotic pathway, two approaches were used to obtain an NADH-spesific D-galacturonic acid reductase. First, a mutant library of the Trichoderma reesei gar1 reductase was designed with the structure-guided cofactor specificity reversal tool CSR-SALAD. An automated high-throughput screening method for expression in Escherichia coli was developed, and the library was screened for enzymatic activity. The second approach was to try to identify the sequence for the characterised NADH-utilising reductase from the single-cell algae Euglena gracilis. A cDNA library of the algae was made and screened with PCR and in vivo methods. The reductase uxaB of the isomerase pathway and dehydrogenase kduD of the dehydratase pathway were functionally expressed in S. cerevisiae, with specific activities of 1.1 µmol min-1 mg-1 and 0.22 µmol min-1 mg-1 , respectively. The enzymes dehydratase uxaA and isomerase kduI did not exhibit activity in activity assays. The galurD of the dehydratase pathway was expressed in E. coli, and the purified enzyme was successfully used to convert D-galacturonate to 5-keto-4-deoxy galacturonate. The approaches to change the cofactor specificity of the NADPH-specific reductase of the eukaryotic pathway did not lead to a discovery of a NADH-specific enzyme. More research is needed for engineering active enzymes for S. cerevisiae expression and constructing a fully functional D-galacturonic acid pathway for feasible D-galacturonic acid fermentation.
  • Sassetti, Elisa; Cruz, Cristina Durante; Tammela, Päivi; Winterhalter, Mathias; Augustyns, Koen; Gribbon, Philip; Windshügel, Björn (2019)
    The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for Escherichia coli ClpP inhibitors. The approved drugs bortezomib, cefmetazole, cisplatin, as well as the investigational drug cDPCP, and the protease inhibitor 3,4-dichloroisocoumarin (3,4-DIC) emerged as ClpP inhibitors with IC50 values ranging between 0.04 and 31 mu M. Compound profiling of the inhibitors revealed cefmetazole and cisplatin not to inhibit the serine protease bovine -chymotrypsin, and for cefmetazole no cytotoxicity against three human cell lines was detected. Surface plasmon resonance studies demonstrated all novel ClpP inhibitors to bind covalently to ClpP. Investigation of the potential binding mode for cefmetazole using molecular docking suggested a dual covalent binding to Ser97 and Thr168. While only the antibiotic cefmetazole demonstrated an intrinsic antibacterial effect, cDPCP clearly delayed the bacterial growth recovery time upon chemically induced nitric oxide stress in a ClpP-dependent manner.
  • Wang, Linping; Pogue, Sylvain; Laamanen, Karoliina; Saarela, Jani; Poso, Antti; Laitinen, Tuomo; Valkonen, Jari P. T. (2021)
    Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta. In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases. IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.
  • Rebane, Anni (Helsingin yliopisto, 2015)
    Given the success of first-line treatment in chronic myeloid leukemia (CML), the prevalence of the disease is estimated to increase and more patients are expected to develop resistance to therapy. Thus, even relatively rare point mutations are likely to become more common. In CML, the uncontrollable division of myeloid cells is caused by a reciprocal translocation of chromosomes 9 and 22, resulting in the Philadelphia chromosome. At the meeting point of the two chromosomes, breakpoint cluster region (BCR) and Abelson proto-oncogene 1 (ABL1) fuse together to form the chimeric fusion oncogene BCR-ABL1, the latter of which, the non-receptor tyrosine kinase ABL1, is the driver of the disease. Since the tyrosine kinase inhibitor (TKI) imatinib became available in 2001, the success of first-line therapy has significantly improved the prognosis of CML patients. However, up to 50% of patients with imatinib-refractory disease develop resistance due to point mutations in ABL1, and the most common mutation to emerge is BCR-ABL1 T315I. The broad-range TKI ponatinib is the only approved TKI that inhibits the kinase activity of BCR-ABL1 T315I, but adverse side effects leave patients with this mutation in need of a better, safer, and more effective treatment. The kinase inhibitor axitinib was shown to be selective for BCR-ABL1 T315I, but mutations that emerge as a consequence of axitinib-resistance have yet to be explored. Moreover, patients with the T315I mutation treated with ponatinib have been reported to develop highly drug-resistant mutations in BCR-ABL1 such as T315M and the E255V/T315I compound mutation. The purpose of this study was to identify mutations that enable cells to develop resistance to the kinase inhibitor axitinib and to find new, potential inhibitors for cells expressing the drug-resistant mutations BCR-ABL1 T315I, BCR-ABL1 T315M, and BCR-ABL1 E255V/T315I. To this end, mouse hematopoietic cell lines were constructed prior to determining cell viability in response to inhibitors in combinations and as independent agents. As a novel finding, cells stably expressing T315M were found to exhibit sensitivity to inhibitors of topoisomerase II and mTOR. Moreover, synthetic lethality occurred in these cells in response to the combined treatment of the allosteric inhibitor asciminib and the TKI ponatinib, although not in clinically relevant doses. The highly resistant cells expressing BCR-ABL1 E255V/T315I, like cells expressing T315I and T315M, showed sensitivity to conventional chemotherapy. Notably, however, three SMAC mimetics displayed selectivity to cells expressing BCR-ABL1 E255V/T315I over cells expressing only the single T315I mutation. Considering that CML is expected to become increasingly prevalent, more patients are estimated to develop resistance to therapy. As even relatively rare mutations in BCR-ABL1 become more common, finding an effective treatment for cells expressing these highly resistant mutations takes us one step closer to identifying a safe and effective treatment for CML patients carrying those mutations.