Exploring the Features and Challenges of Proteolysis Targeting Chimera (PROTAC) Development

Abstract

Small molecule inhibitors offer a powerful approach to modulate biological systems, having been successfully used as probes to interrogate protein functions and as drug therapies. However, many proteins of therapeutic interest are difficult to target with traditional small molecules due to either a lack of conventional drug-binding pockets or because they possess scaffolding activities that are not addressable by enzymatic inhibition. In contrast, targeted protein degradation (TPD), a pharmacological approach in which an E3 ligase is recruited into close proximity to a target of interest to induce its ubiquitination and subsequent proteasomal degradation, has emerged as a therapeutic modality with the potential to pursue targets previously deemed ‘undruggable’. Here, we explore different features of TPD, ranging from assessing the degradability of different targets, redirecting the neo-substrate specificity of immunomodulatory imide drugs (IMiDs) and expanding the E3 ligase toolbox for proteolysis targeting chimera (PROTAC) development. Chapter 2 describes the development of INY-03-041, the first reported small molecule AKT degrader. While AKT is an attractive therapeutic target with several ATP-competitive and allosteric inhibitors, these inhibitors have displayed a lack of efficacy or tolerability in clinical trials. As an alternative approach to inhibiting AKT activity, we conjugated the AKT inhibitor GDC-0068 to lenalidomide, a cereblon (CRBN)-recruiting ligand, to generate INY-03-041 as a potent and highly selective AKT degrader. The relatively slow kinetics of INY-03-041-induced AKT degradation also prompted us to generate INY-05-040, an AKT degrader with improved degradation kinetics, through conjugating GDC-0068 with a Von Hippel-Lindau (VHL) ligand. Both INY-03-041 and INY-05-040 not only displayed enhanced anti-proliferative effects, but also induced more potent and durable inhibition of downstream signaling relative to AKT inhibitors, demonstrating the potential advantages of AKT-targeted degradation. In Chapter 3, we employ small molecule degraders to interrogate the discrepancies observed between ERK5 kinase inhibition and genetic ERK5 ablation. As selective ATP-competitive ERK5 inhibitors were unable to recapitulate the anti-proliferative or anti-inflammatory effects observed from genetic ERK5 knockdown, it has been proposed that kinase-independent functions of ERK5 may play key roles in ERK5 signaling. Thus, we developed INY-06-061, a heterobifunctional degrader of ERK5, to investigate the therapeutic potential of inhibiting ERK5 signaling. While our studies suggest that acute pharmacological degradation of ERK5 does not inhibit cellular proliferation or immune response, INY-06-061 offers a valuable chemical probe to further explore the biological functions of ERK5. In Chapter 4, we rationally redirect the neo-substrate specificity of IMiD-conjugated PROTACs to target the transcription factor Helios (IKZF2) as a proof-of-concept study. Through conjugation of the CDK4/6 inhibitor palbociclib to the IMiD-based Helios degrader DKY709, we generate ALV-07-082-03, a triple degrader of CDK4/CDK6/Helios. ALV-07-082-03 exhibited heightened immunostimulatory effects relative to palbociclib or DKY709 alone, highlighting the potential to synergistically target and degrade multiple proteins with one compound. Chapter 5 employs covalent ligand discovery to identify ZNL-06-031 as a covalent DCAF11 binder. Through the incorporation of ZNL-06-031 as an E3 ligase recruiter for PROTACs, we explore the degradable target space of DCAF11-based PROTACs. In addition, we also develop DCAF11 degraders as potential chemical probes to study DCAF11 function. In sum, this work not only highlights the opportunities that come with PROTAC development, but also the challenges to overcome in the rapidly developing field.