Targeted Protein Degradation: From Drug Discovery to the Clinic

Bispecific protein degraders are an emerging therapeutic modality that can engage the ubiquitin-proteasome pathway to catalytically degrade intracellular proteins through the formation of ternary complexes with the target proteins and E3 ubiquitin ligases. The “event-driven” pharmacology resulting from this catalytic degradation mechanism challenges traditional drug development paradigms and requires a novel approach for understanding pharmacokinetic (PK)-pharmacodynamic (PD) relationships. Therefore, a mechanistic PK-PD model framework was developed for bispecific protein degraders that includes the reaction network governing ternary complex formation and subsequent target ubiquitination and degradation. In this presentation, I will provide an overview of this model framework along with examples to illustrate how it can be used to understand the impact of system- and drug-specific parameters on the dynamics of induced protein degradation both in vitro and in vivo. Such a model-guided approach is envisioned to help accelerate the design and translational development of potent and selective bispecific protein degraders.

Vistara has built a new capability for targeting proteins via the ubiquitin proteasome system using Peptides (called PINTACs) as the active component. When expressed as a DNA construct, the PINTAC process accomplishes selective intracellular degradation (or polyubiquitination) of target. PINTAC technology does not utilize ligands to achieve proximity-mediated engagement of the selected protein, and is therefore much more widely applicable to E3L – Target binary pairs wherein at least one protein is undruggable. PINTACs can be designed for targeting proteins of interest in any disease area, while the current emphasis has been in Cancer. A proprietary back-end process using a combination of bench science and informatics identifies active Peptides, and determines their specificities. Vistara has a current capability of designing PINTACs targeting nearly 4,000 unique proteins from humans, engaged by any of 150 E3Ls. Proof of concept data with targeting human Erk2 and MEK1/2 in cell culture models will be presented. Data on targeting selected E3Ls will also be presented, to accomplish simultaneous targeting of multiple proteins. Applications of PINTACs in upstream pharmaceutical discovery, such as, Target ID, Chemoproteomics, Toxicoproteomics, Mutation screening, Lead discovery and optimization, will be outlined. Approaches for optimizing PINTACs and applying them with assays aimed at achieving selected biological end points will be discussed.

Only a fraction of human proteome is druggable, and this constraint creates a major hurdle to treat many diseases. The latest technological advancement in the area of targeted protein degradation withholds a great promise to take onto undruggable targets. E3 ubiquitin ligases at the heart of this enzymatic process is one of the key determinants of the targeted protein degradation. However, laboratory-based approaches are very labor intensive and time consuming. Predicting the ternary complex of a heterobifunctional degrader with a pathogenic protein of interest (POI) and the corresponding E3 ligase in silico reliably is crucial for rational data-driven degrader design. We achieve this by the combination of complementary modules: ligand-protein interactions are evaluated by a deep interaction prediction using graph representations. Bayesian optimization loops are utilized to assess the arrangement of POI and E3 ligase complexed with the respective ligand. Based on the optimal arrangement a linker is automatically generated using the available chemical space and evaluating all possible conformations. This approach has been filed for patenting by Celeris Therapeutics. By means of a synergistic approach between trained deep learning algorithms, in silico validation and human experience in drug discovery, we designed heterobifunctional degraders for alpha synuclein, a hard-to-drug target for the treatment of Parkinson’s Disease.

PROTACs® are heterobifunctional molecules belonging to the beyond-Rule-of-5 (bRo5) chemical space, made of a warhead, an E3 ligand and a linker coupling both regions. Their success as oral drugs is limited by their large and flexible structure, responsible for drug metabolism and pharmacokinetics (DMPK) challenges that hinder permeability and thus oral bioavailability. Here we provide a first PROTAC® chemical space in which it is possible to identify subregions of permeable/oral bioavailable degraders. The chemical space is based on three computed descriptors: the number of carbon atoms (nC), the molecular flexibility (PHI) and the topological polar surface area (TPSA). Oral bioavailable degraders (ARV-110 and ARV-471, red dots) tend to be closer to the Ro5 region. Permeable PROTACs® could be located in three regions: 1) the subregion having the “best drug” like properties (smaller size, lower polarity and flexibility, e. g. PROTAC-14) 2) an intermediate region with acceptable molecular properties (BI-3663 and ACBI1) and 3) a region occupied by molecular chameleons; this situation is verified for PROTAC-1 for which chameleonicity was demonstrated by NMR studies.

The Nomura Research Group is focused on reimagining druggability using chemoproteomicplatforms to develop transformative medicines. One of the greatest challenges that we face indiscovering new disease therapies is that most proteins are considered “undruggable,” in thatmost proteins do not possess known binding pockets or “ligandable hotspots” that smallmoleculescan bind to modulate protein function. Our research group addresses this challengeby advancing and applying chemoproteomic platforms to discover and pharmacologically targetunique and novel ligandable hotspots for disease therapy. We currently have three majorresearch directions. Our first major focus is on developing and applying chemoproteomicsenabledcovalent ligand discovery approaches to rapidly discover small-molecule therapeuticleads that target unique and novel ligandable hotspots for undruggable protein targets andpathways. Our second research area focuses on using chemoproteomic platforms to expandthe scope of targeted protein degradation technologies. Our third research area focuses onusing chemoproteomics-enabled covalent ligand discovery platforms to develop new inducedproximity-based therapeutic modalities. Collectively, our lab is focused on developing nextgenerationtransformative medicines through pioneering innovative chemical technologies toovercome challenges in drug discovery.

Targeted protein degradation (TPD) takes advantage of the ubiquitin proteasome system by using degrader molecules to hijack activity of the E3 ubiquitin ligases and achieve degradation of a protein target of interest. TPD has been heralded as an approach that can take small molecule drug discovery out of the current druggable zone and expand the reach into parts of the proteome that are often referred to as undruggable. Although TPD undoubtedly represents an exciting technology, every new technology usually goes through a cycle of inflated expectation followed by disillusionment leading to a lot of efforts to better understand, characterize, and eventually improve the technology in order for it to reach its full potential. In this talk, I will provide a broad overview of the field and discuss potential strategies to address current challenges for the field.

Targeted protein degradation is a promising new therapeutic strategy consisting of small molecules, most commonly molecular glues or Proteolysis Targeting Chimeras (PROTACs), which elicit degradation of a target protein. Significant challenges persist to characterize the cellular mechanism of action and the highly dynamic interactions required for degradation. One of the keys steps in the pathway is the formation of an induced ternary complex consisting of the target protein:degrader:E3 ligase component. Here we will present several research stories investigating the role and types of formation of cellular ternary complexes, and how this is correlated with degradation outcome for several different targets and E3 ligase combinations. We will also demonstrate how ternary complex ensemble modeling can predict target lysine positioning and whether mutations to these residues in the cellular environment impacts PROTAC-mediated target ubiquitination. The approaches highlighted will further understanding of cellular mechanism of action and can be used to advance discoveries in the area of targeted protein degradation and induced proximity.

The 26S proteasome degrades ubiquitin-tagged proteins in several well-coordinated steps that include ubiquitin binding, substrate engagement, de-ubiquitination, mechanical unfolding, translocation, and proteolytic cleavage. Our recently developed single-molecule FRET measurements, relying on the fluorescent labeling of incorporated unnatural amino acids, provide unprecedented insights into the conformational changes of the proteasome and the progression of individual substrates through the ATPase motor. We found that ubiquitin binding to an allosteric trigger affects the proteasome conformational switching between engagementcompetent and processing-competent states, and accelerates motor engagement of the substrate for faster, more efficient degradation. Furthermore, mutational studies revealed important details about the mechanisms of degradation initiation and the role of individual ATPase subunits in substrate sensing and inducing the conformational switch for multi-step processing. These studies indicate how the proteasome may utilize the “ubiquitin code” to prioritize substrates in a complex and crowded cellular environment.

Arvinas aims to treat cancer and neurological disorders by specifically targeting disease-causing proteins for degradation by the ubiquitin-proteasome system using PROteolysis-TArgeting Chimeras (PROTAC® protein degraders). A PROTAC protein degrader is a bifunctional small molecule that recruits a specific ubiquitin ligase (E3) to a target protein of interest, thereby inducing proteasome-mediated degradation of the ubiquitin-modified target. The estrogen receptor (ER)-targeting PROTAC protein degrader ARV-471 is being evaluated for treatment of ER+/HER2- locally advanced or metastatic breast cancer. Preclinical and early clinical data for this program will be presented. The Arvinas Discovery Engine is also employed to develop a robust pipeline beyond ARV-471. Data will be presented on our discovery of PROTAC protein degraders that recruit an E3 not previously reported for use with protein degrader technology. Topics discussed will include approaches for target and E3 selection, target and E3 ligand identification, and considerations for PROTAC design for human therapeutics.

Proteolysis-targeting chimeras (PROTACs) are next-generation small molecule therapeutics that exploit the ubiquitin-proteasome system to selectively degrade target proteins. These degraders have shown promise in pre-clinical models but their translation to the clinic may be hindered by their un-druglike characteristics and on-target off-tumor toxicities. Recent work in the Heller lab has enabled the design of nanoparticles that target solid tumors by binding to P-selectin, a cell adhesion molecule upregulated in the tumor vasculature. These self-assembling nanoparticles encapsulated drugs at high loadings and showed fewer side effects and longer survival in vivo compared to free drug. In these studies, we engineered self-assembling PROTAC nanoparticles (nanoPROTACs) to target various solid tumors by incorporating polysaccharides with high affinity to P-selectin. We demonstrated that a variety of PROTACs can be predictably encapsulated with high drug loadings and therapeutically relevant sizes (~100-200 nm). Initial tumor regression studies showed therapeutic benefit of nanoformulated dBET6, a degrader of the oncogenic regulator BRD4, in nut midline carcinoma xenografts. We next aim to validate other nanoPROTACs by investigating biodistribution, PK/PD, tumor regression and overall survival. The successful development of a nanoformulation for PROTACs will expand the utility of this exciting next-generation therapeutic for the treatment of solid tumors in the clinic.

The serine/threonine kinase AKT, a well-characterized node in the PI3K pathway, is involved in several critical cellular pathways, including proliferation, survival, metabolism, and apoptosis. PI3K/AKT pathway mutations are common across many cancer types, often leading to hyperactivation of AKT. Thus, AKT has become valued as a therapeutic target in cancer therapy. Several ATP-competitive, allosteric, and covalent pan-AKT inhibitors have been developed, but have displayed only limited therapeutic benefits as monotherapies. We hypothesized that reducing cellular AKT levels via targeted protein degradation would be an attractive alternative to AKT inhibition. Here, we report the development and extensive characterization a heterobifunctional degrader molecule that selectively degrades AKT1, AKT2, and AKT3 both in vitro and in vivo. We observed long-lasting AKT degradation more potent anti-proliferative effects than AKT inhibition. We employed a multi-omics approach to uncover deregulated regulation of nucleoside metabolism and stress kinase response as factors that differentiate AKT inhibition and degradation. We propose that these heterobifunctional degraders can be used as novel tools to decode the pleiotropic mechanisms that govern AKT signaling in human cancer, and as potential cancer therapeutics.

Until recently the world of drug discovery was partitioned: the druggable and the undruggable proteome. The rules that defined this landscape were developed based on whether a protein had been previously liganded and its function blocked or attenuated as required. A new modality has subsequently entered the fray, and Targeted Protein Degradation radically alters classic rules of druggability which has been by the understanding of Thalidomide (an Imid), and its related analogs in modulating the endogenous ubiquitin proteosome system. The molecule serves as a glue to direct the binding of proteins to the ubiquitin ligase substrate receptor CRBN leading to ubiquitination and degradation. To exploit this mechanism for drug hunting, an expansive library of imids and related CRBN binders were elaborated and screened using a mammalian two hybrid system developed at Orionis Biosciences which identifies novel neosubstrates that are “glued” and recruited to the small molecule CRBN binary complex. Thus far 150 proteins of interest have been screened in this system leading to the identification of novel glues that lead to the degradation of proteins previously considered described undruggable. We continue to advance the science of glues to look for addition ligases as well as degradation-independent opportunities.