Inhibition of protein-protein interactions is a promising therapeutic strategy for targeting non-enzymatic proteins. One strategy to inhibit protein-protein interactions is to design inhibitors specifically toward the protein-protein interaction interface. To achieve this objective using structure-aided design, knowledge of the structure of the interface of the protein-protein interaction is needed. Augmenting the utility of NMR to determine the structure of larger proteins, and of protein complexes, than is currently achieved with conventional approaches, expands structure-aided design and to evaluate protein-protein interactions for target proteins relevant to human disease such as cancer. A second approach to bring new therapies to the clinic is to identify lead molecules using high-throughput screening. High-throughput screening (HTS) offers the potential to more rapidly and less expensively identify promising molecules that may inhibit a specific protein-protein interaction.
To improve efforts at structure-aided design, we hypothesize that we can expand the utility of fluorescence- and NMR-based approaches to monitor protein-protein interactions by incorporating fluorescent or paramagnetic labels, site-specifically, into a protein of interest. This technology will provide much more structural detail regarding the binding site of a candidate small molecule protein-protein interaction inhibitor. This structural information can then be used in downstream medicinal chemistry efforts to improve inhibitor affinity and specificity. Additionally, site-specific fluorescent and paramagnetic labels can be used in sensitive HTS assays for protein-protein interaction inhibitors.
The lanthanide series of elements, which possess intriguing spectroscopic and paramagnetic properties, was chosen to accomplish these efforts. Lanthanides exhibit luminescence and have excitation and emission maxima unique from biological fluorophores, broad Stokes shifts, and narrow emission bands. In the context of NMR, some lanthanides impart long-range perturbations in the form of paramagnetic relaxation enhancements, pseudocontact chemical shifts, and residual dipolar couplings, which are used to facilitate protein structure determination. Site-specific labeling of the protein of interest was achieved using site-directed mutagenesis followed by incorporation of the unnatural amino acid para-azidophenylalanine (paF). Synthesis of the small-molecule lanthanide chelator amenable to copper-free click chemistry-mediated incorporation is described herein.
To improve efforts at identification of candidate protein-protein interaction inhibitors, we hypothesized that a high-throughput fluorescence polarization screening assay could be established. Inhibitors identified in this assay would be further characterized using the lanthanide labeled Survivin protein. A recently-identified cancer target, the Survivin protein, was evaluated using the methodologies described herein. The results demonstrate we were able to fluorescently label the Survivin protein site-specifically using a click chemistry approach. In addition, preliminary fluorescence results indicate that synthesis of a clickable lanthanide chelator was also successful. Finally, a high-throughput fluorescence polarization-based screening platform for analyzing Survivin protein-peptide interactions was evaluated.
Taken together, the methodologies described herein attempt to augment efforts at structure-aided drug design as well as to produce new technologies to identify promising protein-protein interaction inhibitors, with the ultimate goal of improving our understanding of protein-protein interactions implicated in many human diseases, including cancer.
|Advisor:||Lampe, Jed N.|
|Commitee:||Chen, Qi, Fisher, Mark, Hagenbuch, Bruno, Reed, Greg|
|School:||University of Kansas|
|Department:||Pharmacology, Toxicology & Therapeutics|
|School Location:||United States -- Kansas|
|Source:||DAI-B 76/11(E), Dissertation Abstracts International|
|Subjects:||Pharmacology, Biochemistry, Organic chemistry|
|Keywords:||Copper-free click chemistry, Fluorescence, Lanthanide chelation, Nuclear magnetic resonance, Paramagnetism, Unnatural amino acid incorporation|
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