Development of a versatile system for evaluating the target protein degradation activity of novel ubiquitin ligases utilizing existing PROTACs.

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

Introduction The von Hippel-Lindau (VHL) E3 ubiquitin ligase has seen extensive research due to its involvement in the ubiquitin proteasome system and role as a tumor suppressor within the hypoxia signaling pathway. VHL has become an attractive target for proteolysis targeting chimeras (PROTACs), bifunctional molecules that can induce degradation of neo-substrate proteins. The development of VHL inhibitors and PROTACs has seen rapid development since disclosure of the first non-peptidic VHL ligand (2012). Areas covered Due to the demand for more diverse and sophisticated VHL ligands that can be applied to PROTACs, the number of patents disclosed has risen significantly in the past 5 years. Herein, the wide range of VHL modifications that have been patented since 2019 is covered. Specifically, any new or unique chemical modification to established VHL ligands or PROTACs will be discussed. Expert opinion The VHL chemical space continues to expand within the patent literature. There are exciting new modifications that can enhance the physiochemical properties of VHL PROTACs and other alterations can improve the affinity of the VHL ligand itself. Further optimization of the VHL chemical space will no doubt lead to the development of more VHL-based therapies and clinical candidates.

Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that can bring an E3 ligase (e.g. VHL) into the vicinity of a protein of interest (POI). The E3 ligase poly-ubiquitinates the POI, marking it for degradation via a proteasome. Uniquely, PROTACs have the ability to bind to any chosen amino acid region, meaning there is tailored targeting of the POI, followed by subsequent degradation. This means that PROTACs have the potential to degrade targets previously considered ‘undruggable’, such as the oncogene, KRAS, which is historically difficult to therapeutically target due to lack of deep binding pockets. Typically, PROTACs utilize small molecules to target the POI, however, our work aims to uncover the potential of nanobodies to replace these small molecules. The gold standard for protein targeting remains monoclonal antibodies, however, nanobodies possess the same antigen binding site specificity, whilst possessing a tertiary structure only a tenth of the size. Our work focuses on identifying whether the nanobody-PROTAC has the potential for cellular entry, and thus degradation of its POI. For proof of concept, we utilized an anti-GFP nanobody tagged with AZdye647, synthesized using the Michael addition reaction, followed by SDS-PAGE analysis to assess successful conjugation. This conjugate was then used to treat fixed and live cells expressing enhanced green fluorescent protein (EGFP), followed by fluorescent microscopy to confirm cellular entry and POI targeting. Initial SDS-PAGE analysis revealed the successful conjugation of the AZDye647 to the nanobody. Specific binding within fixed cells was confirmed using fluorescent microscopy, as co-localization of EGFP and the AZdye647-tagged anti-GFP nanobody was revealed. Our data also confirmed that liposomal encapsulation of the AZdye647-tagged anti-GFP nanobody yielded efficient delivery into live EGFP expressing cells. Together, this data provides confirmation that a nanobody-PROTAC would be capable of passing through the cellular membrane, maintaining its integrity and degrading its POI. Further understanding of whether the replacement of AZDye647 with an E3 ligase ligand could result in the degradation of ‘undruggable’ oncogenes such as KRAS, in vitro, will confirm whether this is a viable therapeutic approach to patients with KRAS-mutant cancers. Citation Format: Caroline Cope, Cristina Tufarelli, Robert Britton. Nanobody-PROTACs: Method development towards using single-domain antibodies to target undruggable proteins for degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7026.

Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into proximity. The resulting non-native protein-protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the screening of the linkers to induce favorable non-native PPIs between POI and E3 ligase. Here, we present a physics-based computational protocol to predict noncanonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the ternary complex and enhance degradation. Specifically, we build the non-Markovian dynamic model using the Integrative Generalized Master equation (IGME) method from ∼1.5 ms all-atom molecular dynamics simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue protein and the von Hippel-Lindau E3 ligase. Our protocol revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included thermodynamic and kinetic stabilities of PPIs and the accessibility between the solvent-exposed sites on the warheads and E3 ligand. One selected PPIs closely matches a recent cocrystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our protocol has significant potential for widespread application in predicting metastable POI-ligase interfaces that can enable rational design of PROTACs.

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, 'fit-for-purpose' PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

PO-449 Optimisation of an AlphaLISA assay for the characterisation of PROTAC-induced ternary complexes within cell lysates

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins

Using PROteolysis TArgeting Chimeras (PROTACs) to degrade proteins that are important for tumorigenesis has emerged as a potential therapeutic strategy for cancer. PROTACs are heterobifunctional molecules consisting of one ligand for binding to a protein of interest (POI) and another to an E3 ubiquitin (E3) ligase, connected via a linker. PROTACs recruit the E3 ligase to the POI and cause proximity-induced ubiquitination and degradation of the POI by the ubiquitin proteasome system (UPS). PROTACs have been developed to degrade a variety of cancer targets with unprecedented efficacy against a multitude of tumor types. To date, most of the PROTACs developed have utilized ligands to recruit E3 ligases that are ubiquitously expressed in both tumor and normal tissues. These PROTACs can cause on-target toxicities if the POIs are not tumor-specific. Therefore, identifying and recruiting the E3 ligases that are enriched in tumors with minimal expression in normal tissues holds the potential to develop tumor-specific/selective PROTACs. In this review, we will discuss the potential of PROTACs to become anticancer therapeutics, chemical and bioinformatics approaches for PROTAC design, and safety concerns with a special focus on the development of tumor-specific/selective PROTACs. In addition, the identification of tumor types in terms of solid versus hematological malignancies that can be best targeted with PROTAC approach will be briefly discussed.

An E3 ligase guide to the galaxy of small-molecule-induced protein degradation

Recent years have seen great advancement of targeted protein degradation technology, in particular, proteolysis targeting chimeras (PROTACs) are now widely used in developing therapeutics for treating cancer. A PROTAC is a heterobifunctional small molecule with three distinct moieties: a ligand to bind a targeted protein of interest (POI), a second ligand to recruit E3 ubiquitin ligase to form a ternary complex, and a linker for bridging the two ligands. Through the E3 ubiquitin ligase pathway, properly designed PROTACs can effectively degrade POIs with high specificity, which can be pathogenic proteins, thus regulate related pathways and inhibit tumor growth. Currently, there is no high-throughput biochemical assay to measure the POI degradation in vitro, hindering the application of the PROTAC technology. Here we report the development of a high-throughput cell based assay to quantitatively measure the endogenous drug target degradation induced by PROTACs. This assay encompasses over 800 cancer cell lines, many of which are CRISPR-engineered ones on common drug targets. A unique feature of our assay is the incorporation of HiBiT short sequence tags on either N-terminal or C-terminal of endogenous protein in E3 ligase matched cell line through site-specific gene homologous recombination technology. By highly specific and sensitive detection of HiBiT tag content in cell lysates with biochemical assay, we can determine target protein degradation by PROTAC treatment. We demonstrate the utility of the assay on a wide array of drug targets including RAS, LDHA, BTK and HPK1, and show that it can accelerate PROTAC drug discovery. Citation Format: Yunpeng Zhai, Ming Tan, Defu Liu, Guoqian Wang, Jinying Ning, Feng Hao. Cell line panel with HIBIT tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6181.

Development and Characterization of Third Generation MDM2 PROTACs for the Treatment of Acute Myeloid Leukemia Utilizing an Achiral Ligand for E3 Ligase Cereblon

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, ‘fit-for-purpose’ PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable. Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.

Bridged PROTAC, DNA-based PROTAC and hydrophobic tag technologies for targeted protein degradation.