Understanding targeted protein degradation for design of optimized therapeutic strategies

A new type of therapeutic molecule has shaken up drug discovery since 2015. Traditional approaches have relied on discovering drugs that bind to 'rogue' proteins that cause disease and switch off their activity. These so-called inhibitor drugs bind tightly to their target to switch them off, and they need to be permanently present at high doses to keep the rogue protein inactive. The discovery of molecules that can bind to elements of the protein degradation machinery of the cell has opened up the possibility of a new mode of therapy altogether. Bifunctional compounds created by linking an inhibitor to a molecule that recruits protein degradation machinery can lead to complete destruction of the rogue protein. This approach has been shown to work in eliminating a number of therapeutic targets and is now in clinical trials as a potential treatment for prostate cancer (through destruction of the Androgen Receptor) and other diseases. One great advantage of this approach is that a drug no longer needs to be present at all times to keep its target blocked permanently: Since a single binding event can destroy the target protein, these drugs can be used at much lower doses than conventional drugs and may have fewer side-effects.
Despite the excitement generated by the therapeutic possibilities of these bifunctional molecules, there is still a lot that we don't understand about how they work - and therefore about what makes a successful drug. Deciphering the biology that underlies the action of these bifunctional molecules, widely referred to as 'PROTACs', will therefore be key to designing future strategies that take into account which proteins can be degraded and by what type of drug, and whether there are additional parameters that can be modified to enhance the efficiency of target destruction. Our lab has spent 10 years researching an important cellular protein, Aurora kinase A (AURKA), known to be one rogue player in cancer, and a favourite target in cancer drug development. We have recently discovered a PROTAC that works to degrade AURKA, and propose to use this PROTAC, and our extensive knowledge of the cellular properties and behaviour of AURKA, to uncover some of the subcellular parameters that regulate its activity. We aim to identify parameters contributing to PROTAC activity, both specific features that will optimize targeted degradation of AURKA and general features of this class of drug. We will test degradation of additional PROTAC targets (with overlapping and distinct properties to AURKA), and alternative degradation tools against AURKA (that harness different bits of the cellular degradation machinery) to establish the general and specific rules of PROTAC activity.

The project will examine the molecular cell biology of Targeted Protein Degradation (TPD) tools against the cancer-associated mitotic kinase Aurora A (AURKA) and other cellular targets. Despite the clear potential for PROTACs to be powerful tools for cell biology (as well as potential therapeutic agents) they are not yet in general use as cell biology tools, and PROTAC development is mostly driven by the drug discovery field. Because of this, there is a marked lag in our understanding of the molecular and cellular detail underpinning the successful PROTACs that have so far been described in the literature. This lag is hindering the development of PROTACs, since many characteristics that define a successful PROTAC have not been systematically defined: we don't know which molecular and cellular features are important for targeting or degradation of substrates (such as subcellular localization, binding partners, ubiquitin site availability, deubiquitinases).
We propose to examine such features using as a reference tool a small molecule PROTAC (Proteolysis Targeting Chimera) that combines ligands specific to AURKA (MLN8237) and to the E3 ligase containing Cereblon (CRBN). The PROTAC functions to clear AURKA from the cell by ubiquitin-mediated proteolysis, but detailed study of the differential cell biology induced by PROTAC vs MLN8237 treatment reveals marked differential responses of different subcellular pools of the target. We will extract the identity of parameters influencing PROTAC-mediated AURKA destruction using our existing knowledge of AURKA and benefiting from the extensive collection of AURKA tools and assays available in the lab. We will test new hypotheses on cellular parameters influencing AURKA sensitivity to PROTAC and other TPD tools harnessing different E3s, and to PROTAC-substrate pairs for which PROTACs are now commercially available or can be synthesized upon request.