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Browsing by Subject "Protein−protein interaction"
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Item Design and Synthesis of Fragment Derivatives with a Unique Inhibition Mechanism of the uPAR·uPA Interaction(American Chemical Society, 2020) Bum-Erdene, Khuchtumur; Liu, Degang; Xu, David; Ghozayel, Mona K.; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine;There is substantial interest in the development of small molecules that inhibit the tight and highly challenging protein-protein interaction between the glycophosphatidylinositol (GPI)-anchored cell surface receptor uPAR and the serine protease uPA. While preparing derivatives of a fragment-like compound that previously emerged from a computational screen, we identified compound 5 (IPR-3242), which inhibited binding of uPA to uPAR with submicromolar IC50s. The high inhibition potency prompted us to carry out studies to rule out potential aggregation, lack of stability, reactivity, and nonspecific inhibition. We designed and prepared 16 derivatives to further explore the role of each substituent. Interestingly, the compounds only partially inhibited binding of a fluorescently labeled α-helical peptide that binds to uPAR at the uPAR·uPA interface. Collectively, the results suggest that the compounds bind to uPAR outside of the uPAR·uPA interface, trapping the receptor into a conformation that is not able to bind to uPA. Additional studies will have to be carried out to determine whether this unique inhibition mechanism can occur at the cell surface.Item Inflect: Optimizing Computational Workflows for Thermal Proteome Profiling Data Analysis(American Chemical Society, 2021-04-02) McCracken, Neil A.; Peck Justice, Sarah A.; Wijeratne, Aruna B.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of MedicineThe CETSA and Thermal Proteome Profiling (TPP) analytical methods are invaluable for the study of protein–ligand interactions and protein stability in a cellular context. These tools have increasingly been leveraged in work ranging from understanding signaling paradigms to drug discovery. Consequently, there is an important need to optimize the data analysis pipeline that is used to calculate protein melt temperatures (Tm) and relative melt shifts from proteomics abundance data. Here, we report a user-friendly analysis of the melt shift calculation workflow where we describe the impact of each individual calculation step on the final output list of stabilized and destabilized proteins. This report also includes a description of how key steps in the analysis workflow quantitatively impact the list of stabilized/destabilized proteins from an experiment. We applied our findings to develop a more optimized analysis workflow that illustrates the dramatic sensitivity of chosen calculation steps on the final list of reported proteins of interest in a study and have made the R based program Inflect available for research community use through the CRAN repository [McCracken, N. Inflect: Melt Curve Fitting and Melt Shift Analysis. R package version 1.0.3, 2021]. The Inflect outputs include melt curves for each protein which passes filtering criteria in addition to a data matrix which is directly compatible with downstream packages such as UpsetR for replicate comparisons and identification of biologically relevant changes. Overall, this work provides an essential resource for scientists as they analyze data from TPP and CETSA experiments and implement their own analysis pipelines geared toward specific applications.