Date of Award
Doctor of Philosophy (PhD)
Chemotype-specific resistance is a major factor limiting the efficacy of molecularly targeted therapeutics. Analyses of resistance mechanisms to drugs reveal that mutations conferring resistance often arise in the drug’s binding sites. As selective binding of inhibitors to their protein targets is mediated by specific interactions of the inhibitor with the protein backbone and side chains, mutations that disrupt these interactions can lead to resistance. Identifying resistance-conferring alleles can thus reveal and suggest the biochemical determinants of the drug’s selectivity and potency. In this thesis, I explore how mutations in active sites of proteins can be leveraged to understand inhibitor binding and to design selective chemical inhibitors. In my thesis work I focus on the design of chemical probes for proteins from the AAA (ATPase Associated with diverse cellular Activities) superfamily, for which only a few inhibitors are available. As the number of inhibitor-bound models of AAA proteins is also limited, the key protein-inhibitor interactions needed for design of probes for these proteins are not known. I have developed an approach - Resistance Analysis During Design - that involves testing selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations that retain enzymatic activity. These analyses, along with computational docking, can guide optimization of inhibitor potency and selectivity. We used this approach to design the pyrazolylpyrrolopyrimidine-based spastazoline - the first potent and selective chemical probe for spastin, a microtubule-severing AAA protein needed for cell division and intracellular vesicle transport. I confirmed the predicted binding mode of spastazoline analogs by X-ray crystallography and used these high-resolution structural models, along with biochemical analyses of spastin mutant alleles, to design an allele-specific inhibitor of spastin. Further, I show that RADD can be used to analyze the binding modes of diaminotriazole-based chemical inhibitors that are chemically unrelated to spastazoline. I also identified a more potent, diaminotriazole-based analog and used our approach to show that it binds spastin’s active site in a different orientation in comparison to the starting compound. The distinct binding modes of these compounds predicted by RADD also match the high-resolution models I generated using X-ray crystallography. Together, these data show how analyses of resistance can be useful at the early stages of the inhibitor design process. In summary, the work in my thesis outlines how mutations in active sites of proteins can be identified and how they could facilitate inhibitor design. I discuss how the binding models of spastin inhibitors I developed can inform on the design of new inhibitors for other AAA proteins and suggest experiments that could be valuable to advance these efforts. I also propose how analyses of resistance to chemical inhibitors could be valuable for designing drugs against which resistance might be less likely to arise. Věnováno babičce Anně Sochnové.
Pisa, Rudolf, "Analyzing Resistance to Design Chemical Inhibitors of AAA Proteins" (2020). Student Theses and Dissertations. 707.