Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions are fundamental in cell signaling and cancer progression. An increasing prevalent idea in cancer therapy is the development of small molecules to disrupt protein-protein interactions. Small molecules impart their action by binding to pockets on the protein surface of their physiological target. At protein-protein interactions, these pockets are often too large and tight to be disrupted by conventional design techniques. Residues that contribute a disproportionate amount of energy at these interfaces are known as hot spots. The successful disruption of protein-protein interactions with small molecules is attributed to the ability of small molecules to mimic and engage these hot spots. Here, the role of hot spots is explored in existing inhibitors and compared with the native protein ligand to explore how hot spot residues can be leveraged in protein-protein interactions. Few studies have explored the use of interface residues for the identification of hit compounds from structure-based virtual screening. The tight uPAR•uPA interaction offers a platform to test methods that leverage hot spots on both the protein receptor and ligand. A method is described that enriches for small molecules that both engage hot spots on the protein receptor uPAR and mimic hot spots on its protein ligand uPA. In addition, differences in chemical diversity in mimicking ligand hot spots is explored. In addition to uPAR•uPA, there are additional opportunities at unperturbed protein-protein interactions implicated in cancer. Projects such as TCGA, which systematically catalog the hallmarks of cancer across multiple platforms, provide opportunities to identify novel protein-protein interactions that are paramount to cancer progression. To that end, a census of cancer-specific binding sites in the human proteome are identified to provide opportunities for drug discovery at the system level. Finally, tumor genomic, protein-protein interaction, and protein structural data is integrated to create chemogenomic libraries for phenotypic screening to uncover novel GBM targets and generate starting points for the development of GBM therapeutic agents. / 2020-10-03

Bovine Infected Cell Protein 0 (bICP0) is an immediate-early protein encoded by Bovine Herpesvirus-1 that modulates host immune response, activates transcription for all viral promoters, and causes ubiquitin-dependent degradation of proteins. Presented herein is a bICP0 protein-protein interaction (PPI) profile, consisting of 98 cellular and 15 viral proteins, generated through co-immunoprecipitation of bICP0 and its binding partners. The PPI profile was analyzed computationally to identify potential sites of interaction with bICP0 and any cellular pathways that may be influenced by bICP0. Some interactors fall in conjunction with bICP0’s known roles during infection, and others are consistent with known associations of bICP0 homologs. However, some proteins in the PPI profile are involved in apoptosis signaling and mRNA spicing—processes both significant during viral infection and novel to the known functions of bICP0 and its homologs. The interaction and co-localization of some of these proteins with bICP0 was further examined.

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Protein-protein interactions (PPI) mediate numerous biological processes, but inhibiting these interactions with small molecules has been difficult to achieve in drug discovery. A small number of successes have shown that some PPIs are amenable to inhibition. Computational algorithms designed to measure the druggability of PPIs have been developed based on these successes. These algorithms have identified the interaction between the NF-κB essential modulator (NEMO) and I𝜅B kinase β (IKKβ) as a candidate for inhibition. Furthermore, in vivo peptide-based inhibition of the NEMO-IKKβ interface has shown benefits in attenuating the NF-𝜅B response in cellular and animal models. In addition to its intrinsic interest as a drug target, developing inhibitors against the NEMO/IKKβ interaction may help in the development of improved methods for PPI inhibition. In this thesis, the production of full-length, recombinant forms of soluble NEMO is described. This protein was used in a variety of biochemical assays to advance our understanding of NEMO structure and function. Furthermore, a fluorescence anisotropy (FA) assay was developed to screen for compounds inhibiting the NEMO/IKKβ PPI. Hits from the FA assay were tested by several methods to confirm true inhibition. Additionally, the FA assay was used to accurately measure the affinity of NEMO for IKKβ and to assess the degree of cooperativity in IKKβ binding. The oligomeric state of NEMO has been characterized through the development of a panel of NEMO cysteine to alanine mutants, using polyacrylamide gel electrophoresis analysis, analytical ultracentrifugation, and fluorescence anisotropy. These data represent the first comprehensive characterization of full-length human NEMO, and may provide a path toward development of drug-like inhibitors of the NEMO/IKKβ interaction.

Factor Quinolinone Inhibitor 1 (FQI1), discovered as a small molecule inhibitor of the transcription factor LSF, causes cell death in many cancer cell lines and inhibits tumor growth in tumor xenografts and an endogenous hepatocellular carcinoma model in mice. Significantly, multiple animal studies have shown minimal to no toxicity after FQI1 treatment, making it a promising potential lead chemotherapeutic for multiple cancer types. In determining how FQI1 causes cancer cell death, it was previously shown that FQI1 treatment, like knockdown of LSF expression by siRNA, produced a mitotic arrest with condensed but unaligned chromosomes, but with no clearly observable transcriptional dysregulation. In this thesis, I establish that introducing FQI1 to cells already in mitosis induces a mitotic arrest in colorectal cancer cells, demonstrating that FQI1 inhibits mitotic processes directly while these processes are occurring. This mitotic arrest is characterized by defects in the mitotic spindle and limited connections of mitotic spindles to the kinetochores, as indicated by a dramatic decrease cold-stable microtubules in mitosis. Additionally, in a dose-dependent manner, FQI1 treatment resulted in supernumerary γ-tubulin-containing mitotic centrosomes and γ-tubulin-deficient aster-like bodies, indicating a defect in centrosome stability. As FQI1 is known to be a specific inhibitor of LSF, with its dose dependence for LSF inhibition directly proportional to its ability to inhibit cell proliferation, these findings suggested the novel hypothesis that LSF regulates mitosis through non-transcriptional mechanisms by interacting with key mitotic proteins required for proper spindle formation and metaphase alignment. By mass spectrometry, multiple proteins were identified that interact with biotinylated LSF in mitosis in a FQI1-sensitive manner, with several related to the formation and stability of the mitotic spindle. Proximity ligation assays validated endogenous LSF interactions with CKAP5, a processive microtubule polymerase that protects kinetochore microtubules from depolymerization, and MISP, a requirement for proper mitotic spindle positioning. However, in this assay these interactions were not demonstrably FQI1-sensitive. In conclusion, FQI1 treatment results in defects in kinetochore-microtubule attachment and centrosome stability, triggering a mitotic arrest. Combined with the target specificity of FQI1, this suggests the hypothesis that LSF is required for proper mitotic spindle formation through its protein interactions in mitosis. / 2022-10-16T00:00:00Z

The cytosolic iron sulfur cluster assembly (CIA) pathway is responsible for the maturation of >40 cytosolic and nuclear iron sulfur (FeS) proteins critical for fundamental processes such as DNA replication, transcription, and translation. The final stages of the pathway require the CIA targeting complex, which is composed of Cia1, Cia2, and Met18. This large multiprotein complex is proposed to recognize apo-enzyme substrates and insert their FeS clusters. However, it is unclear how these substrates are identified and how the CIA targeting complex mediates cofactor insertion. In this thesis, I mapped the protein-protein interaction sites critical for formation of the CIA targeting complex and discovered the first peptide motif that is both necessary and sufficient for recognition of a subset of FeS proteins by the CIA system. Cia1’s seventh beta-propeller blade was found to bind to Cia2, while Cia2’s fifth conserved region mainly interacts with Cia1, via an in vitro affinity co-purification assay. A quantitative MicroScale Thermophoresis assay supported these findings, in addition this approach affirmed that Cia2’s N-terminal intrinsically disordered domain and hyperreactive cysteine are dispensable for CIA targeting complex assembly. In collaboration with the Drennan Lab at MIT, Met18 was discovered to form a hexamer via cryo-EM. Met18 is proposed to arrange into a hexamer before its CIA-related function. Hexamer formation and Cia2 binding depend on Met18’s C-terminus, whereas Leu1 recognition relies on Met18’s N-terminus. A C-terminal W motif was demonstrated as both necessary and sufficient for identification of a subset of FeS proteins by the CIA targeting complex. A bioinformatics analysis revealed roughly 20% of CIA client proteins, including substrates, factors, and adaptors, terminate in a conserved [LTQ]-[DE]-[W]-COO- motif. CIA recognition depends on the C-terminal aromatic side chain and the carboxy terminus. This tripeptide motif is also sufficient for identification by the CIA system when attached to SUMO. Moreover, a series of competition experiments showed that the CIA targeting complex contains distinct, non-overlapping binding sites for client proteins where Cia1 serves as the docking site for the C-terminal W motif. Altogether, the first recognition motif is defined for one in five of CIA client proteins. / 2024-11-03T00:00:00Z

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