Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.

The current inhibitor-based approach to therapeutics has inherent limitations owing to its occupancy-based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off-target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event-driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small-molecule-based proteolysis-targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.

Inhibition of Bruton's tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide a basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL.

Proteolysis targeting chimera (PROTAC) technology, the recruitment of E3 ubiquitin ligases to induce the degradation of a protein target, is rapidly impacting chemical biology, as well as modern drug development. Here, we explore the universality of this approach by evaluating different E3 ubiquitin ligases, engineered in their substrate binding domains to accept a recruiting ligand. Five out of six E3 ligases were found to be amenable to recruitment for target degradation. Taking advantage of the tight spatiotemporal control of inducing ubiquitination on a preselected target in living cells, we focused on two of the engineered E3 ligases, βTRCP and parkin, to unravel their ubiquitination characteristics in comparison with the PROTAC-recruited endogenous E3 ligases VHL and cereblon.

A new series of Proteolysis Targeting Chimeras (PROTACs) targeting Bruton's Tyrosine Kinase (BTK) was synthesized, with the goal of improving the pharmacokinetic properties of our previously reported PROTAC, MT802. We recently described the ability of MT802 to induce degradation of both wild-type and C481S mutant BTK in immortalized cells and patient-derived B-lymphocytes. However, the pharmacokinetic properties of MT802 were not suitable for further in vivo development. Therefore, we undertook a systematic medicinal chemistry campaign to overcome this issue and made a series of PROTACs with structural modifications to the linker and E3-recruiting ligand; more specifically, the new PROTACs were synthesized with different von Hippel-Lindau (VHL) and cereblon (CRBN) ligands while keeping the BTK ligand and linker length constant. This approach resulted in an equally potent PROTAC, SJF620, with a significantly better pharmacokinetic profile than MT802. This compound may hold promise for further in vivo exploration of BTK degradation.

The immunomodulatory drugs (IMiDs) thalidomide, pomalidomide, and lenalidomide have been approved for the treatment of multiple myeloma for many years. Recently, their use as E3 ligase recruiting elements for small-molecule-induced protein degradation has led to a resurgence in interest in IMiD synthesis and functionalization. Traditional IMiD synthesis follows a stepwise route with multiple purification steps. Herein we describe a novel one-pot synthesis without purification that provides rapid access to a multitude of IMiD analogues. Binding studies with the IMiD target protein cereblon (CRBN) reveals a narrow structure-activity relationship with only a few compounds showing sub-micromolar binding affinity in the range of pomalidomide and lenalidomide. However, anti-proliferative activity as well as Aiolos degradation could be identified for two IMiD analogues. This study provides useful insight into the structure-degradation relationships for molecules of this type as well as a rapid and robust method for IMiD synthesis.

In eukaryotic cells, organelle-specific stress-response mechanisms are vital for maintaining cellular homeostasis. The Golgi apparatus, an essential organelle of the secretory system, is the major site of protein modification and sorting within a cell and functions as a platform for spatially regulated signaling. Golgi homeostasis mechanisms that regulate organelle structure and ensure precise processing and localization of protein substrates remain poorly understood. Using a chemical biology strategy to induce protein unfolding, we uncover a Golgi-specific transcriptional response. An RNA-sequencing profile of this stress response compared with the current state-of-the-art Golgi stressors, nigericin and xyloside, demonstrates the enhanced precision of Golgi targeting achieved with our system. The data set further reveals previously uncharacterized genes that we find to be essential for Golgi structural integrity. These findings highlight the Golgi’s ability to sense misfolded proteins and establish new aspects of Golgi autoregulation.