Student Theses and Dissertations
Date of Award
2018
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Tarun Kapoor
Keywords
dynein, ATPase, ciliobrevins, dynapyrazoles, inhibitors, microtubules.
Abstract
Cells utilize energy to maintain order within the cytoplasm. Motor proteins are the enzymes that convert the chemical energy contained in adenosine triphosphate (ATP) into directed movement along polarized filaments of actin and tubulin within cells. Dyneins are the primary enzymes that drive motion toward the stable "minus ends" of tubulin-containing filaments known as microtubules. This protein family is divided into two sub-families. Axonemal dyneins drive flagellar beating while cytoplasmic dyneins (hereafter, dyneins) are required a wide array of cellular processes including moving RNAs, proteins, and whole organelles and for the formation, maintenance, and positioning of the mitotic spindle, the protein apparatus that ensures proper cell division. Dyneins move their cargos at velocities > 1µm/sec in cells and contribute to processes that occur on timescales of minutes and seconds. Perturbations that act on a comparable timescale to dynein are best suited to study this dynamic motor protein. Small molecules (molecular weight <1000 Daltons) can generally engage their target within minutes. However the first cell-permeable small molecule antagonists of dynein, the ciliobrevins, have only recently been identified. As these compounds have suboptimal chemical properties and low potency, their use as probes for studying dynein has been limited. The work presented here describes a chemistry-based approach to develop new antagonists of dyneins and the resulting identification of three new classes of dynein inhibitors. The first chapter "Chemical probes for dynein" motivates the need for cellpermeable dynein antagonists. It summarizes the available antagonists for dynein and describes their discovery and utility. Where possible, the features of these dynein inhibitors are considered in light of the concepts of selectivity and principles of proteinligand binding. In the outlook, prospects for future development of dynein inhibitors are presented in the context of recent advances in design and development of potent and selective inhibitors for other members of the ATPases Associated with diverse cellular Activities (AAA+) protein family, to which dynein belongs. In the second chapter, "Chemical structure-guided design of dynapyrazoles, cellpermeable dynein inhibitors with a unique mode of action," I present a rational approach to the development of derivatives of ciliobrevins that have improved potency and several improved chemical properties relative to the ciliobrevins. Structural analysis of the ciliobrevins suggested the hypothesis that replacing the isomerizable core of these compounds with a rigid tricyclic heterocycle and synthesis of such compounds led to the identification of two compounds that had 6-8 fold improved potency compared to ciliobrevin D (named the dynapyrazoles). An analysis of the mechanism of inhibition of dynein 1 by dynapyrazole A revealed that it inhibited the microtubule-stimulated ATPase activity of dynein, but did not potently inhibit the basal ATPase activity. This finding and further biochemical analyses revealed that dynapyrazole A likely inhibits the ATPase activity of dynein 1 that arises at just one of its four ATP-binding sites, AAA1. Taken together, these findings suggest that dynapyrazole A is likely to be a useful probe for studying dynein. Subsequent chapters extend this chemical structure-guided approach to the identification and characterization of two other compound scaffolds that inhibit dynein— pyrazolopyrimidinone-based derivatives of dynapyrazoles and structurally-unrelated diaminoquinazolines. The mechanisms and sites of inhibition of these compound classes were analyzed using biochemical and structural techniques. Finally, I present an outlook chapter in which I discuss the trends emerging from the dynein inhibitors I discovered during the course of my PhD. Most dynein inhibitors do not act in a substrate-competitive mechanism and I posit that this may be a consequence of the complex chemomechanical cycle of dynein, which involves allosteric communication between distinct ATPase sites. All compounds with identified sites of inhibition act at the AAA1 ATPase site. I propose that this may be due to the low apparent affinity of this site for ATP. In conclusion, I suggest experiments that are likely to be valuable to more clearly understand the dynein inhibitors presently available and to develop improved dynein inhibitors in the future.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Steinman, Jonathan Baruch, "Chemical Approaches to Dynein Inhibition" (2018). Student Theses and Dissertations. 482.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/482
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy