Browsing by Subject "Drug repurposing"
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Item Open Access Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish light source "Turkish DeLight"(TÜBİTAK, 2023-01-01) Atalay, N.; Akcan, E. K.; Gül, M.; Ayan, E.; Destan, E.; Ertem, F. B.; Tokay, N.; Çakılkaya, B.; Nergiz, Z.; Karakadıoğlu, G.; Kepçeoğlu, A.; Yapıcı, İ.; Tosun, B.; Baldır, N.; Yıldırım, G.; Johnson, J. A.; Güven, Ö.; Shafiei, A.; Arslan, N. E.; Yılmaz, M.; Kulakman, C.; Paydos, S. S.; Çinal, Zeynep Sena; Şabanoğlu, K.; Pazarçeviren, A.; Yılmaz, A.; Canbay, B.; Aşcı, B.; Kartal, E.; Tavlı, S.; Çalıseki, M.; Göç, G.; Mermer, A.; Yeşilay, G.; Altuntaş, S.; Tateishi, H.; Otsuka, M.; Fujita, M.; Tekin, Ş.; Çiftçi, H.; Durdağı, S.; Dinler Doğanay, G.; Karaca, E.; Kaplan Türköz, B.; Kabasakal, B. V.; Katı, A.; Demirci, H.X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g., proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryocrystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, İstanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.Item Open Access Discovery of novel agents for liver cancer therapeutics and characterization of their bioactivities on cellular pathways(Bilkent University, 2015-12) Durmaz, İremHepatocellular carcinoma is the second deadliest and fifth most common cancer type worldwide. Due to the limited therapy options, it is crucial to develop novel targeted therapeutic agents that provide better prognosis and enhance life quality of patients. The specific aim of this thesis was to identify and characterize novel compounds with anticancer properties in liver cancer. Three groups of molecules were investigated. First group were cardiac glycosides extracted and purified from Digitalis Ferruginea. Extensive analysis Glycoside Lanatoside C revealed that these molecules induced ROS accumulation in liver cancer cells with differential downstream targets in mesenchymal-like PTENdeficient drug-resistant Mahlavu and epithelial-like PTEN-adequate drug-sensitive Huh7 liver cancer cells. Xenograft models on nude mice also confirmed the anticancer activities of Lanatoside C in vivo with decreased tumor volume and weight. The second group of compounds were novel molecules that contains triazolothiadiazine and triazolothiadiazole scaffold, derived from known NSAIDs (ibuprofen, naproxen and flurbiprofen). Results indicated that SubG1/G1 cell cycle arrest is induced in treated cells. In addition, extensive molecular analysis disclosed oxidative stress induction and COX activity inhibition leading to ASK1 activation and Akt inhibition. The levels of downstream elements GSK3β, β-catenin and CyclinD1 were also altered. Apoptosis was characterized as the cell death mechanism that is triggered by these molecules in liver cancer cells. Novel nucleobase/nucleoside analogues were the third group of molecules explored in this study. 24 of 127 investigated compounds showed significant cytotoxicity during initial screening. 6 molecules were selected for further molecular analysis upon real-time cytotoxicity assay. It was observed that the molecules induced SubG1/G1 cell cycle arrest through Src pathway inhibition. CyclinE-cdk2 complex formation was prevented then the inhibition of Rb leading to a decrease in cell growth and proliferation and induction of apoptosis in liver cancer cells. This thesis disclosed the mode of action of three groups molecules, glycosides are pure examples of drug repurposing. NSAID represent the modified small molecule compounds for novel targets and finally nucleobase analogs are novel compounds as anti metabolites.Item Open Access Drug repurposing and investigation of novel combinations for glioblastoma therapeutics using in vitro and zebrafish in vivo models(Bilkent University, 2024-01) Tok, GüneşGlioblastoma is the most common and aggressive brain cancer type with the survival rate less than 2 years after diagnosis. Yet, potent drug treatments used in patients are limited and the field is in need of development of new potential drugs. In this study, repurposing of approved drugs alone or in combination and novel drugs are investigated in terms of inhibition of cell viability, glial fluorescent signals and their effects on behavior in zebrafish larval models. The main aim of this study was to test whether phenothiazines, trifluoperazine and a novel molecule 10, could be repurposed for glioblastoma treatment with lower dosages and more potency when combined with Sorafenib, an approved drug, in glioblastoma cell lines and zebrafish larvae. Those drug combinations were not found as toxic in the dosages studied while acted on glia cells in zebrafish transgenic larval models. Last but not least, behavior and stress response of the wild type and heterozygous mutant ache larvae in comparison with homozygous siblings were tested upon drug administration to assess genotype by drug interactions. Combination treatments exhibited higher efficacies suggesting phenothiazines with sorafenib could have potential in glioblastoma treatment. Genotype specific effects of individual and combination treatments on larval light-dark behavior, stress response and recovery exhibited potential for passage of blood brain barrier by the tested drugs. The established protocols for genotype and drug interactions could be applied to other kinases in combination with phenothiazines.Item Open Access Identification of preclinical implications for novel indole-benzimidazoles and phenothiazines using in vitro cancer cell line and in vivo zebrafish models(Bilkent University, 2020-09) Yaman, MuratBreast cancer (BC) and hepatocellular carcinoma (HCC) are two major health problems with significant mortality rates. Although drug therapies are available, therapeutic success remains limited. Because of low bioavailability, high toxicity and recurring drug resistance, novel therapeutic options are essential. In the present thesis, a multitude of in vitro, in silico and in vivo approaches were executed to test anti-cancer effects and preclinical potentials of novel indole-benzimidazoles and phenothiazines in BC and HCC, respectively. In the first component of the thesis, I evaluated BC cell line toxicity and estrogen receptor (ER) relationship of novel indole-benzimidazole derivatives using in vitro cancer lines, in vivo zebrafish embryos/larvae, and in silico comparative transcriptomics analyses. In the second part, antipsychotic compounds phenothiazines (PTZ) were repurposed for HCC therapy. Therefore, generic PTZ derivatives alone or in combination with sorafenib (SFB) were tested using in vitro cancer lines followed by zebrafish developmental assays and embryonic stage xenografts. In addition, RNAseq analyses were performed on trifluoperazine (TFP), SFB, and TFP+SFB combination treated Hep3B cells to understand synergistic/antagonistic effects of the drugs at gene expression level. Lastly, anti-HCC potential of novel PTZ derivatives were explored by in vitro and in vivo screenings. Moreover, effects of the novel and generic derivatives on neural pathways were evaluated by cholinesterase assays and motor response measurements. The findings of the dissertation present potential leads for conducting further preclinical studies tailored towards novel BC and HCC therapies.Item Open Access Mineralocorticoid and glucocorticoid receptors as novel targets in breast and liver cancer therapies(Bilkent University, 2020-12) Güneş, DamlaCell signaling is a complex phenomenon and is maintained through intertwined signal transmissions within and in-between the cells. Anti-cancer therapies are often challenged by this fact due to crosstalk-associated activation of alternative survival routes. Hence, development of new treatment strategies and identification of novel prognostic markers depends on in-depth knowledge on cell signaling routes altered in cancer and possible crosstalk paths. Herein, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling, two closely related members of steroid receptor hormone family, and their possible crosstalk were studied across breast and liver cancer cell lines. In breast cancer cell lines, estrogen responsive and MR expressing T47D was used in order to study possible crosstalk among Estrogen receptor (ER) and MR. MR-GR ligand aldosterone (ALDO) and ER ligand estrogen (E2) administered to breast cancer cells alone and in combination and, MR, ER and GR and their downstream signaling members were studied employing qRT-PCR and Western blot assays. Furthermore, ALDO, E2, ALDO-E2 hormone administrations were also used for cell viability assessments. Our results implied possible interactions of ALDO-E2 signaling at the level of cell viability, and at mRNA levels of progesterone receptor. In liver cancer cell lines, MR and GR was investigated as targets of a novel treatment. Liver cancer subtype hepatocellular carcinoma (HCC) has high mortality rate with limited treatment options. Multi-kinase inhibitor Sorafenib (SFB) with mild effectivity is most known systemic therapy against HCC. To potentiate the effectiveness of SFB and overcome to the crosstalk associated limitations, combinatorial drug treatment approach targeting multiple signaling modalities has been adopted in literature. Previously in our lab, SFB was combined with repurposed anti-psychotic drug TFP as a novel combinatorial treatment against hepatocellular carcinoma (HCC) and liver cancer cell lines. Cellular viability was synergistically reduced by SFB-TFP in HCC cell line Hep3B, while antagonistic effects on viability in SkHep1 was apparent. Herein, two liver cancer cell lines Hep3B and SkHep1, were used in comparison to unravel mechanism of action of SFB-TFP combination at the protein level. Apoptosis, cell cycle, PI3K/AKT/mTOR and MAPK pathways were investigated in addition to MR and GR. Our results revealed several markers indicating success of drug combinations and targeted pathways at protein level which needs to be pursued further.Item Open Access Zebrafish as an in vivo model of drug screens for liver cancer: the role of phenothiazines(Bilkent University, 2021-08) Korkmaz, BüşraHepatocellular carcinoma (HCC) is one of the most dangerous cancer types and sorafenib (SFB) is a commonly used drug against HCC, being the only FDA-approved medication until 2018. However, its limited efficacy and severe side effects emphasize the importance of developing alternative approaches. Regulation of cholesterol has been implicated in the progression of various cancers including HCC. Phenothiazines, which are in use as antipsychotic drugs and with effects on cholesterol biosynthesis, have drawn recent attention as anti-cancer drugs against different cancers. The elucidation of how phenothiazines exert their effects at cellular level might pave the way for proposing better strategies for HCC treatment. Zebrafish serves as a good cancer model for in vivo validations, since it enables efficient tumor formation and tracking. Its conserved genes and similar organ system to human make it even better of a model to reveal both molecular and morphological alterations caused by drugs. Based on previous cell viability studies from our lab, the combination of trifluoperazine (TFP) with SFB was found to exhibit synergism in Hep3B cells, while it was antagonistic in SkHep1 cells. Moreover, RNA-seq analysis on Hep3B cells has demonstrated that steroid biosynthesis and cholesterol metabolism was among the modulated pathways. Therefore, in the present thesis, the regulation of cholesterol levels in response to phenothiazine derivatives was investigated in HCC cell lines SkHep1 and Hep3B. The combinatorial approach was examined on Huh7 cells in terms of the expression of cholesterol biosynthesis genes based on RT-qPCR. After showing the ability of phenothiazine derivatives and TFP-SFB combinations to modulate cellular cholesterol, zebrafish drug screens were performed to determine applicable derivative and combination doses. Assessment of safe doses for TFP, SFB and their combination directed us for xenograft studies, which resulted in enhanced Hep3B cell survival by the combination of low dose (6 μM) TFP and 1 μM of SFB. This finding was further validated based on Alu-based DNA quantifiation, a powerful method we have established. With the help of RNA-seq analysis results, orthologous zebrafish genes, involved in cholesterol biosynthesis were identified and used in cross-species testing. The impact of TFP on liver vasculature was evaluated using transgenic (Fli1a:EGFP) zebrafish and found insignificant. Overall, the findings presented in this thesis highlight that TFP alone or in combination was able to change cellular cholesterol levels and tumor growth.