Targeted small-molecule mediated degradation that stops... degradation!?

Small molecules, the compounds imagined and synthesized by chemists or by nature, form the basics of modern medicine. These molecules have an ability to enter cells, engage with cellular components that misfunction to cause disease, and correct the problem. Many of these molecules engage with their target only transiently – and these transient interactions are often sufficient to help and fix the functional problems. But: the transient nature of these interactions also means that the drug needs to be frequently replenished and that diseased cells have time to learn how to circumvent the effects of the drug – often by developing resistance. This also means that majority of drugs in current clinical use affect only a single functional aspects of the target, while not affecting other possible roles.

To address these shortcomings, chemists have recently developed a new strategy – small molecules that lead to complete removal of the target. These molecules bind to the target as well as recruit components of the cellular degradation machinery. The result is the protein target marked for permanent destruction through the proteasome, a large protein assembly that recognizes the destruction mark and wipes out the target. These small molecules are aptly called degrader molecules, as they result in target degradation. More technically, a subset of these molecules is known as PROTACs for proteolysis targeting chimeras as they are bifunctional (chimeric) molecules that lead to targeted proteolysis. The degrader molecules have another potential benefit – after the target is marked for destructions these molecules are thought to disengage from the given target molecule and become free to engage with the next one. This means that even low doses could be effective, and because the degradation is a more dramatic event than inhibition, these effects could be longer-lasting. All this makes targeted protein degradation very attractive modality for drug development, and the first degraders have recently entered clinical trials.

Another way to address issues surrounding transient nature of interactions between drugs and their targets is to develop drugs that bind their targets in a more permanent fashion. Those drugs that do that are called covalent drugs because they engage the target by forming a covalent bond. Whether covalent binding can be combined with degrader molecule design remains somewhat controversial. A degrader that remains permanently attached to the target may not be recycled – removing one of the key advantage features, and perhaps removing the degradation activity. Recent work has even gone on to show just that: a covalent inhibitor targeting BTK kinase developed into PROTACs did not result in BTK degradation, although it still inhibited the target via covalent binding.

But: there has also been a report of a successful covalent PROTAC targeting another kinase, ERK, leaving this question wide open. A new collaborative study by Kenneth C. Anderson, Jun Qi and Dharminder Chauhan laboratories at Dana-Farber Cancer Institute, co-led by Yan Song and Paul Park, now shows that a covalent inhibitor of a protein called RPN13 can be developed into an effective PROTAC – WL40. Song, Park and colleagues show that WL40 binds the E3 ubiquitin ligase cereblon (CRBN) via its CRBN-recruiting arm, a small molecule called thaliodomide, and RPN13 via the target-recruiting arm, a small molecules called RA190. The authors also show that WL40 leads to decrease in RPN13 levels in multiple myeloma cell lines (including MM.1S, RPMI-8226 and ANBL6.BR cells), and that this decrease is CRBN- and proteasome-dependent. All this supports the conclusions that WL40 is a small molecule degrader (PROTAC) that targets RPN13 for degradation. Moreover, WL40 displayed cytotoxic activity against a panel of multiple myeloma cell lines and patient-derived cells, including from patients with multiple myeloma that was refractory to current treatment options. WL40 administration was also found to prolong survival of mice bearing human MM.1S tumor xenografts.

Not only is this work interesting as another demonstration that covalent PROTACs are feasible, and of potential interest as therapeutic strategy for multiple myeloma, but the work is interesting from the target perspective as well. Rpn13 is not just any target worth marking for degradation – Rpn13 is a component of the proteasomal degradation machinery that recognizes the proteins that have been marked for degradation and shuttles them downstream. This means that Rpn13 removal affects the very same system that the PROTAC strategy hijacks, the ubiquitin-proteasome system. Remarkably, the authors document that Rpn13 degradation leads to global accumulation of polyubiquitinated species, leads to increase in ER stress, unfolded protein response and autophagy, and induction of apoptosis – all the well-documented hallmarks of clogged proteasomal degradation system. In this way, a hijacked proteasomal system turns the trigger on itself, effectively resulting in cellular destruction, which I find intriguing.

But, what was the most interesting aspect of this work from the authors perspective? I reached out to Paul Park, one of the two co-first authors on the study to find out more, and here is what Paul shared:

“Most notable about this study is the fact that we were able to covalently degrade the degrader. WL40 is a covalent degrader that targets RPN13, which recognizes polyubiquitinated proteins marked for proteasomal degradation. Interestingly, this permanent modification along with degradation of RPN13 allows for drug resistance to bortezomib in multiple myeloma to be overcome both in vitro and in vivo. Our study provides evidence for the development of covalent degraders as a novel therapeutic approach to combat drug resistance.”

All in all, this work is an interesting addition to the rapidly expanding space of small molecules that have the power to destroy - precisely and effectively. It is the area that recently included reports on tau degraders of broad interest to neurodegenerative disease community, and has captured my attention on more than one occasion. It's been a fascinating ride so far, and our journey continues...