@article{BoulosSaeedChatterjeeetal.2021, author = {Boulos, Joelle C. and Saeed, Mohamed E. M. and Chatterjee, Manik and B{\"u}lb{\"u}l, Yagmur and Crudo, Francesco and Marko, Doris and Munder, Markus and Klauck, Sabine M. and Efferth, Thomas}, title = {Repurposing of the ALK inhibitor crizotinib for acute leukemia and multiple myeloma cells}, series = {Pharmaceuticals}, volume = {14}, journal = {Pharmaceuticals}, number = {11}, issn = {1424-8247}, doi = {10.3390/ph14111126}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250258}, year = {2021}, abstract = {Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G\(_2\)M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib's ability to prevent mitotic exit. However, cells accumulated in the sub-G\(_0\)G\(_1\) fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.}, language = {en} } @phdthesis{Busch2021, author = {Busch, Albert Franz Jakob}, title = {Modification of angiogenesis to abrogate abdominal aortic aneurysm growth}, doi = {10.25972/OPUS-24135}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-241356}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Introduction: Abdominal aortic aneurysm (AAA) is a pathological saccular enlargement most often of the infrarenal aorta. Eventual rupture is fatal, making preemptive surgical therapy upon a diameter threshold of >50mm the treatment of choice. The pathophysiology, especially the initial trigger aortic remodeling is still largely unknown. However, some characteristic features involved in aneurysm growth have been established, such as medial angiogenesis, low-grade inflammation, vascular smooth muscle cell (VSMC) phenotype switch, extracellular remodeling, altered hemodynamics and an eventual humoral immune answer. Currently, no medical treatment options are available. RNA therapeutics and drug repurposing offer new possibilities to overcome this shortage. Using such to target angiogenesis in the aneurysm wall and investigate their potential mechanisms is the aim of this thesis. Material and Methods: We test our hypothesis by targeting the long non-coding RNA H19 and re-use the anti-cancer drug Lenvatinib in two murine inducible AAA models and one preclinical large animal model in the LDLR-/- pig. Furthermore, a H19-/- mouse is included to verify the results. AAA and control samples from a human biobank along with a primary human cell culture are used to verify results ex vivo by qPCR, WesternBlot, live cell imaging, histo- and immunohistochemistry along with gene array analysis, RNA knockdown, pull-down- and promotor assays. Results: H19 is significantly upregulated in AAA mice models and its knockdown limited aneurysm growth. It is well known that H19 interacts with several transcription factors. We found that cytoplasmic interaction between H19 and hypoxia-inducible factor 1-alpha (HIF1α) increased apoptosis in cultured SMCs associated with sequential p53 stabilization. In contrast, the knockdown of H19 was associated with markedly decreased apoptotic cell rates. Our data underline that HIF1α was essential in mediating the pro-apoptotic effects of H19. Secondly, Lenvatinib was applied both systemically and locally by endovascular means in mice with an established AAA. The drug significantly halted aneurysm growth and array analysis revealed myosin heavy chain 11 (MYH11) as the most differentially regulated target. This was shown to be up regulated after Lenvatinib treatment of primary AAA smooth muscle cells suggesting a salvage mechanism to obtain a contractile phenotype based on gene expression and immunohistochemistry. The same results were shown upon a local endovascular Lenvatinib-coated balloon angioplasty in the established aneurysmatic lesion of a novel atherosclerotic LDLR-/- Yucatan minipig model. Decreased phosphorylation of extracellular-signal regulated kinases 1-2 (ERK1-2) is the downstream effect of Lenvatinib-specific blockage of the vascular endothelial growth factor receptor (VEGFR2). Conclusion: Taking into account the heterogeneity of the disease, inhibition of VSMC phenotype switch, extracellular remodeling and angiogenesis seem promising targets in some if not all AAA patients. Together with surveillance and surgical therapy, these new non-invasive treatment strategies would allow for a more personalized approach to treat this disease.}, subject = {Aortenaneurysma}, language = {en} } @article{OsmanogluGuptaAlmasietal.2023, author = {Osmanoglu, {\"O}zge and Gupta, Shishir K. and Almasi, Anna and Yagci, Seray and Srivastava, Mugdha and Araujo, Gabriel H. M. and Nagy, Zoltan and Balkenhol, Johannes and Dandekar, Thomas}, title = {Signaling network analysis reveals fostamatinib as a potential drug to control platelet hyperactivation during SARS-CoV-2 infection}, series = {Frontiers in Immunology}, volume = {14}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2023.1285345}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-354158}, year = {2023}, abstract = {Introduction Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear. Methods We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved. Results Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients. Discussion Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/.}, language = {en} } @phdthesis{Ramirez2024, author = {Ramirez, Yesid A.}, title = {Structural basis of ubiquitin recognition and rational design of novel covalent inhibitors targeting Cdu1 from \(Chlamydia\) \(Trachomatis\)}, doi = {10.25972/OPUS-19168}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191683}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors.}, subject = {Ubiquitin}, language = {en} } @article{ShityakovSkorbFoersteretal.2021, author = {Shityakov, Sergey and Skorb, Ekaterina V. and F{\"o}rster, Carola Y. and Dandekar, Thomas}, title = {Scaffold Searching of FDA and EMA-Approved Drugs Identifies Lead Candidates for Drug Repurposing in Alzheimer's Disease}, series = {Frontiers in Chemistry}, volume = {9}, journal = {Frontiers in Chemistry}, issn = {2296-2646}, doi = {10.3389/fchem.2021.736509}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248703}, year = {2021}, abstract = {Clinical trials of novel therapeutics for Alzheimer's Disease (AD) have consumed a significant amount of time and resources with largely negative results. Repurposing drugs already approved by the Food and Drug Administration (FDA), European Medicines Agency (EMA), or Worldwide for another indication is a more rapid and less expensive option. Therefore, we apply the scaffold searching approach based on known amyloid-beta (Aβ) inhibitor tramiprosate to screen the DrugCentral database (n = 4,642) of clinically tested drugs. As a result, menadione bisulfite and camphotamide substances with protrombogenic and neurostimulation/cardioprotection effects were identified as promising Aβ inhibitors with an improved binding affinity (ΔGbind) and blood-brain barrier permeation (logBB). Finally, the data was also confirmed by molecular dynamics simulations using implicit solvation, in particular as Molecular Mechanics Generalized Born Surface Area (MM-GBSA) model. Overall, the proposed in silico pipeline can be implemented through the early stage rational drug design to nominate some lead candidates for AD, which will be further validated in vitro and in vivo, and, finally, in a clinical trial.}, language = {en} }