Inhibiting protein function selectively is a major goal of modern drug discovery. Here, we report a previously understudied benefit of small molecule proteolysis-targeting chimeras (PROTACs) that recruit E3 ubiquitin ligases to target proteins for their ubiquitination and subsequent proteasome-mediated degradation. Using promiscuous CRBN- and VHL-recruiting PROTACs that bind >50 kinases, we show that only a subset of bound targets is degraded. The basis of this selectivity relies on protein-protein interactions between the E3 ubiquitin ligase and the target protein, as illustrated by engaged proteins that are not degraded as a result of unstable ternary complexes with PROTAC-recruited E3 ligases. In contrast, weak PROTAC:target protein affinity can be stabilized by high-affinity target:PROTAC:ligase trimer interactions, leading to efficient degradation. This study highlights design guidelines for generating potent PROTACs as well as possibilities for degrading undruggable proteins immune to traditional small-molecule inhibitors.

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.

PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model.

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.

Highlights d TRAFTACs are heterobifunctional chimeric oligos d Transcription factors are recruited to the double-stranded DNA of the chimeric TRAFTACs d TRAFTACs are generalizable d Brachyury-TRAFTAC induces no tail phenotype in zebrafish embryos

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low  nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable with degradation-based approaches by targeting mutant BRAF-driven cancers while sparing BRAFWT, providing an anti-tumor drug modality that expands the therapeutic window.

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.