Browsing by Subject "drug discovery"

Now showing 1 - 4 of 4
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    Analysis and interpretation of high-throughput chemical-genetic interaction screens.
    (2018-08) Simpkins, Scott
    Screening chemical compounds against genome-wide mutant arrays identifies genetic perturbations that cause sensitivity or resistance to compounds of interest. The resulting chemical-genetic interaction profiles contain information on the cellular functions perturbed by compounds and can be used to elucidate their modes of action. When performed at high throughput, chemical-genetic interaction screens can be used to functionally profile entire libraries of chemical compounds in an unbiased manner to identify promising compounds with diverse modes of action. My contributions to the field of chemical-genetic interaction screening come primarily in the form of two software pipelines, called BEAN-counter and CG-TARGET, that were developed to interface with the large-scale datasets generated from screens of thousands of compounds performed by collaborators. The former pipeline processes the raw data into chemical-genetic interaction scores and provides tools to remove systematic biases and other unwanted signals from large-scale datasets. The latter provides for interpretation of chemical-genetic interaction profiles via a compendium of reference genetic interaction profiles, with a focus on controlling the false discovery rate and prioritizing the highest-confidence predictions for further study. Enabled by the tools I developed to analyze and interpret these data, our collaboration characterized novel compounds, identified general trends surrounding the interactions between compounds and biological systems, and demonstrated the value of performing chemical-genetic interaction screens to functionally annotate compounds at high throughput.
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    Biochemical and Biophysical Assay Development for Screening and Characterization of Small Molecules and Synthesized Analogues Targeting Human Cytosine Deaminases
    (2023-01) Grillo, Michael
    The APOBEC3 family of enzymes converts cytosine to uracil in single-stranded DNA as a part of the innate immune defense against viruses. A large body of work has demonstrated that when dysregulated, APOBEC3s contribute to mutagenesis of somatic DNA in cancer. These mutational events lead to poor clinical outcomes such as tumor recurrence, metastasis, and therapeutic resistance. Because of this, we are interested in targeting APOBEC3s for inhibition by small molecules with the goal of improving the outcome of treatment with current cancer therapies. Chapter 1 introduces APOBEC3s as targets of interest with commentary on current and potential biochemical, biophysical, cellular, and in vivo assay technologies to evaluate potential inhibitors. Chapter 2 discusses careful in silico reconstruction of APOBEC3B followed by molecular dynamics simulations and druggability analysis identifying putative allosteric sites. Virtual screening was performed, and compounds were validated in biochemical and biophysical assays to serve as potential starting points for hit to lead optimization. Chapter 3 focuses on the development of sensitive biophysical assays and implementation in fragment screening. One fragment was validated during triage and served as a starting point for preliminary structure-activity relationship studies on two potentially divergent chemical series. Chapter 4 discusses the development of a real-time fluorescence-based activity assay for human cytidine deaminase utilizing a fluorogenic substrate. This assay was validated with known small molecule inhibitors and implemented in a fragment screen to discover novel non-nucleoside inhibitors of cytidine deaminase. The long-term goal of this work is to apply the same technology to measure APOBEC3 activity. Chapter 5 outlines several attempts at synthesizing a rationally designed covalent sulfur (VI) fluoride exchange probe targeting conserved hydroxyl-containing residues in the APOBEC3 active site. A variety of standard, as well as novel, approaches to nucleoside chemistry were explored with the goal of eventually incorporating a covalent warhead into a DNA oligonucleotide. Finally, Appendix A describes attempts at expressing and purifying A3B containing 5-fluorotryptophan as a tool for protein-observed 19F-NMR experiments.
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    Development of Small-Molecule Inhibitors and Biophysical Tools to Study the Epigenetic Protein BPTF
    (2022-06) Zahid, Huda
    Bromodomains are protein-protein interaction modules involved in epigenetic regulation of gene expression, typically through the recognition of acetylated lysine residues in histones. Bromodomain-containing proteins are involved in several disease processes, including cancer, inflammation and viral replication. There are 61 known human bromodomains and due to their high structural similarity, developing small-molecule inhibitors that bind with high affinity to specific proteins is a major challenge in the field. While numerous small molecule probes have been developed for the BET (bromodomain and extra terminal) family, few have been reported for the 53 non-BET bromodomains. Potent and selective chemical probes are, therefore, required to enhance our biological understanding of the non-BET bromodomain family. Among them, the understudied protein Bromodomain and PHD finger Transcription Factor (BPTF) is known to play an important role in chromatin remodeling and is the focus of this dissertation. BPTF is overexpressed in several cancers such as melanoma, breast cancer and high-grade gliomas. It has been identified as a potential target for developing cancer therapeutics and BPTF bromodomain inhibitors are being explored for combination treatment with chemotherapeutics. Given the emerging significance of BPTF as an anticancer target, small-molecule inhibitors for this bromodomain-containing protein are needed. This dissertation details efforts to develop potent chemical inhibitors of the BPTF bromodomain through systematic structure-activity relationship analyses. Starting from a reported pyridazinone scaffold and using structure-based design approaches, the lead compound BZ1 is developed with a Kd of 6.3 nM for BPTF, making it one of the most potent inhibitors reported to date for this bromodomain. These efforts are assisted by the optimization of a bead-based competition assay AlphaScreen (described in Chapter 2) which is used to quantify affinity values and cross-validate other biophysical methods for studying BPTF interactions. Several of the first cocrystal structures of BPTF bound to small-molecule ligands are also reported, which are key to rational design of potential drug candidates. We further show that these compounds sensitize breast cancer cells to the chemotherapeutic doxorubicin, indicating that inhibition of the BPTF bromodomain can overcome chemoresistance in these cells. Efforts are now underway to evaluate the drug-like properties of pyridazinone inhibitors for in vivo studies. The development of potent BPTF inhibitors paved the way to heterobifunctional molecules described in Chapter 4 of this dissertation. In this study, degraders based on a pyridazinone scaffold are designed and evaluated using an in-cell NanoBRET assay. As first-generation degraders, these compounds induce ternary complex formation of the BPTF bromodomain with the cereblon E3 ligase, evident through both in vitro and in-cell assay formats. Using an optimized NanoBRET assay, we show that they also efficiently degrade a Nanoluciferase-BPTF bromodomain construct. While preliminary western blotting studies indicate that these pyridazinone-based degraders do not show any degradation activity for endogenous BPTF, future work will look at other cell lines and various linkers to optimize the degradation efficiency of these compounds. In another project, detailed in Chapter 5, a fundamental study of noncovalent and highly stabilizing sulfoxide-aromatic interactions in proteins is described. This will provide insight into the effects of oxidation of methionine residues in α-synuclein, a modification known to be involved in neurodegenerative disorders such as Parkinson’s disease. β-hairpin peptides are used as model systems to study these motifs and quantify the interaction of oxidized methionine with aromatic side chains of amino acids. In this system, no additional stabilization is observed when interactions of phenylalanine with methionine and oxidized methionine are compared. Further analysis with tryptophan indicates a slight destabilization of the β-hairpin peptide motif with oxomethionine. Follow-up studies for this project will look at other aromatic side chains such as tyrosine and the application of this approach in the broader context of α-synuclein.
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    http://z.umn.edu/INNOVATIONS 2013, Vol. 4, No. 3, Article 121 INNOVATIONS in pharmacy 1 Design and Implementation of an Interdisciplinary Elective Course in Drug Discovery, Development, and Commercialization
    (University of Minnesota, College of Pharmacy, 2013) Ettouati, William S.; Hirsch, Jan D.; Ma, Joseph D.
    Objective: To describe the design and implementation of an elective course in drug discovery, development, and commercialization for pharmacy, medical, biomedical graduate, business, and law students. Case Study: This course included didactic lectures, student group discussions, a longitudinal assignment, and a question and answer panel session. A 9-item instrument using a 5-point response scale was used for course evaluation. The longitudinal assignment was the creation and presentation of a product lifecycle strategic plan (PLSP). Respondents rated ‘agree’ and ‘strongly agree’ in the course providing useful information on drug discovery (39% and 53%), drug development (39% and 60%), and drug commercialization (33% and 60%). The majority of student-reported overall understanding of the drug discovery and drug development process was rated ‘very good’ (49% and 46%), while the drug commercialization process was rated ‘good’ (46%). Conclusions: An elective course on drug discovery, development, and commercialization included enrollment of students with diverse educational training. The course provided useful information and improved overall student understanding.