Student Theses and Dissertations

Date of Award

2020

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Cytoplasmic dynein is a AAA (ATPase Associated with various Activities) motor protein that transports cellular cargoes towards the microtubule minus-end. Despite its essential role in intracellular transport, dynein remains the least understood cytoskeletal motor. With speeds >25 μm/min in cells, dynein’s cellular functions are challenging to study using genetic approaches, such as CRISPR and RNA interference, as the perturbation timescales far exceed those of dynein action. Fast-acting small molecule probes can be powerful tools to study dynein’s many cellular mechanisms, but the design of potent and selective inhibitors of dynein remains challenging. Inhibitors of dynein’s motor domain have been reported, such as ciliobrevins, dynapyrazoles and dynarrestin, but the inhibitor-binding site(s) have yet to be elucidated. Though inhibitor-bound dynein structures have been long-sought after, efforts have been curtailed by difficulties in obtaining high-resolution dynein structures and the lack of an inhibitor with the requisite chemical properties, such as compound solubility and stability, for structural studies. The work presented here describes the design and characterization of a class of dynein probes, one of which we use to obtain an inhibitor-bound structure of dynein. The first chapter “Chemical inhibitors of AAA proteins” provides a brief overview of AAA proteins in general and the inhibitors that have been designed. The chapter emphasizes on the design and characterization of ciliobrevins, dynapyrazoles and dynarrestin, three classes of dynein inhibitors with distinct mechanisms of action. Their limitations as cellular probes are discussed and motivates the need for compounds with improved potency and selectivity. In the second chapter, “Dynapyrazoles acutely inhibit intraflagellar transport”, I characterize dynapyrazole-A in a cell-based assay that can measure the effect of the compound on intraflagellar transport (IFT). Dynapyrazole-A is an acute reversible inhibitor of dynein 2-dependent retrograde transport and can be a useful probe to study IFT function. However, as dynapyrazole-A inhibits both isoforms of dynein, its use as a chemical probe is limited due to its cytotoxic effects at treatment times >1 hour. This chapter motivates the need for dynein-2 specific inhibitors. Finally, I present a third chapter, “Structural insights into the chemical inhibition of dynein”, that discusses the design of a dynapyrazole derivative, compound 20, that inhibits the basal ATPase activity of human and S. cerevisiae dynein. I used cryo-EM to obtain a structure of S. cerevisae dynein’s motor domain in the presence of the dynapyrazole derivative and find that the compound binds to the regulatory ATPase sites in the AAA3 and AAA4 domains, rather than the main catalytic site in the AAA1 domain. This finding addresses a major gap in our knowledge, as inhibitors of dynein’s ATPase activity have been assumed to target the AAA1 domain. Inhibitor design efforts can now be focused on the regulatory ATPase sites to obtain potent and selective small molecule probes of dynein.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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