Issue:September 2025
TARGET DISCOVERY PLATFORM - Multi-Omics, SITESEEKER® Technology & the Future of Targeted Protein Degradatione
INTRODUCTION
The evolution of multi-omics-based approaches and large-scale sequencing for target discovery has contributed significantly to the identification of genes involved in currently incurable diseases, paving the way for the development of new therapies. The development of gene editing technologies, such as gene-trap, RNAi, and CRISPR, have allowed for genome-wide perturbation studies using high-throughput screening assays (arrayed and pooled) that permit the identification of previously unknown target candidates.1 However, there are challenges associated with these technologies as they do not directly provide functional characterization or inform on the presence of well-defined drug pockets or enzymatic activity of the target genes.
FUNCTIONAL GENOMICS & SITESEEKER®
The strong dependence on protein-protein interaction interfaces for signaling cascades and the necessity of identifying small molecule pockets remain significant challenges in early drug discovery. The advent of new technologies that can identify previously unknown druggable pockets on target proteins present innovative approaches for the discovery and development of small-molecule therapeutics. In recent years, we have introduced SITESEEKER. This cutting-edge target discovery platform utilizes programmatically controlled hyper-diverse libraries of highly active mini-proteins called PROTEINi®. These fragments are designed to maximize stability and present novel three-dimensional conformations and are delivered into cells via lentiviral vectors.
Following the screening process, cells are harvested and genomic DNA from populations of interest (selected based on phenotypic responses or markers of interest) isolated and enriched, or depleted PROTEINi identified through next- generation sequencing and bioinformatic data analysis (Figure 1). The application of SITESEEKER in high-throughput functional screening campaigns in disease-relevant cell models presents a promising avenue for the discovery of novel therapeutic targets that might be missed by conventional approaches. However, the potential of SITESEEKER extends beyond mere target identification. By integrating advanced computational tools for library design and experimental techniques for screening and reporter cell line generation for phenotypic assays, this technology also has the potential to map intricate protein interaction networks, revealing previously uncharacterized biological pathways and molecular mechanisms. These insights are crucial for understanding disease pathology at a deeper level and for developing targeted interventions that are both precise and effective.
The application of SITESEEKER technology to a broad range of therapeutic areas, such as oncology, neurodegeneration, and immunology, underscores its versatility and potential for delivering first-in-class drugs to a wide spectrum of patients in need. Its versatility has allowed expansion of the technology into the field of targeted protein degradation (TPD), an emerging area of biomedical research focused on developing therapeutic strategies that selectively degrade proteins of interest within cells using their own destruction machinery. The highly tunable library-based approach of this technology provides a unique ability to target the druggable space with alternative mechanisms of action to existing approved drugs, opening up new opportunities for the development of bivalent and monovalent degraders.
TARGETED PROTEIN DEGRADATION – HETEROBIFUNCTIONAL & MOLECULAR GLUE DEGRADERS
The concept of modulating the turnover of specific proteins of interest using the ubiquitin-proteasome system (UPS) has garnered significant attention as a mode of therapeutic intervention since the initiation of the first PROTAC clinical trials in 2019.2 The engineered recruitment of this cellular machinery to eliminate disease-related proteins has become a significant focus of drug development activities and clinical trials in recent years.
TPD discovery efforts can be divided into two categories: bivalent and monovalent degraders. Bivalent degraders are composed of two key moieties, one that recruits a ubiquitin ligase (E3 ligase) and one that binds to the target protein of interest, with a linker connecting the two parts. In contrast, monovalent degraders or molecular glues are not composed of two distinct ligands but rather are small molecules that bind to one protein, modifying the surface properties in such a way that enhances binding of an existing interactor or, more excitingly, creating a binding face for novel interactions.3 Through this modality, molecular glue degraders can bring E3 ligases into close proximity to neo-substrates, catalyzing their ubiquitination and subsequent degradation (Figure 2).
The generation and development of TPD drugs is now a well-defined process, generating a constant flow of molecules that have gradually increased in presence in clinical testing. Although more efficacy and safety evidence is needed, protein degraders are showing promising activity as cancer therapies.4 There are more than 600 different E3 ligases in the human proteome; however, only a limited number are currently being used for degrader-based therapies – mainly Cereblon (CRBN) and Von Hippel-Lindau (VHL). In both cases, there have been challenging limitations associated with tolerance, resistance, pharmacokinetic properties, and oral bioavailability.5 One of the problems associated with bifunctional degraders is that the rule of five (chemical properties that define druggability) for small molecules does not apply to these drugs, making it difficult to predict their properties. Advancements in computational modelling are needed to transition from static structures used for traditional small molecule development to conformational stabilization approaches, considering the dynamic nature of ternary complexes.6
The diversity of E3 ligase enzymes is a crucial biological feature, highlighting the huge therapeutic potential of utilizing novel ligases beyond CRBN and VHL to mitigate safety and resistance concerns. Expanding the E3 ligase toolbox to identify new ligases with differential expression and/or activity levels in healthy and diseased tissues is an important challenge to mitigate toxicity and the occurrence of resistance mechanisms due to reduced expression levels. Identifying alternative E3 ligase mechanisms therefore enhances the therapeutic potential of developing TPD drugs with improved chemical properties.7
SITESEEKER: LEVERAGING THE EXTENSIVE E3 LANDSCAPE
Our SITESEEKER platform is uniquely positioned to probe the unexplored proteome space within TPD. Approaches to discover ligands with the potential to direct degradation in a target or tissue specific manner include building of cis-degraders with an E3-recruiting PROTEINi library fused to a reporter protein leading to reporter degradation, or peptide heterobifunctional degraders comprising E3-recruiting PROTEINi libraries linked to a target-binding warhead.
SITESEEKER TPD screening campaigns have revealed a host of novel E3s that can be recruited for degradation of therapeutic targets and the PROTEINi degrader motifs able to induce their proximity (Figure 3). These peptide degraders form the basis for design of small molecule heterobifunctional degraders, with the aid of computational modelling.
A number of degraders targeting novel E3 ligases have progressed through drug discovery with successful demonstration of protein degradation, including in vivo degradation of the protein of interest in tumor-bearing mice.
The successful development of small molecule degraders directing targeted protein degradation via novel E3 ligases demonstrates the exciting capability of SITESEEKER to drive the creation of heterobifunctional degrader therapeutics, expanding the range of therapeutics in the field with the potential to overcome the shortcomings of CRBN-based bivalent degraders.
GLUESEEKER®: THE NEXT GENERATION OF MOLECULAR GLUE DISCOVERY
Molecular glues have emerged as a promising new strategy for TPD, addressing some of the chemistry challenges associated with bivalent molecules. However, most discoveries in this area have been serendipitous, such as the IMiDs thalidomide and lenolidomide. Thalidomide’s mechanism of action as a molecular glue degrader was discovered when it was found to bind to CRBN promoting the formation of a neomorphic interface for interaction with the neo-substrates Ikaros (IKZF1) and Aiolos (IKZF3), leading to their ubiquitination and degradation.8 Monovalent degraders, or molecular glues, have smaller molecular sizes than bivalent degraders and function by creating a new interface between the E3 ligase and the protein of interest or neo-substrate. However, innovative approaches are needed to drive the rational design of these molecules. PhoreMost’s new GlueSEEKER platform offers a novel method to tackle this challenge and redirect E3 ligases to novel targets.
Key concepts such as protein-protein interfaces and compound-induced proximity are crucial for understanding and characterizing molecular glue interactions. These concepts have helped in rationalizing the design of successful phenotypic screening campaigns. However, computational methods alone are not providing sufficient sensitivity in identifying interfaces and surface contact points between an E3 ligase and a protein of interest.
GlueSEEKER technology leverages advances in machine-learning and protein structure predictive models to identify novel binding sites within an E3 ligase. This identification permits the insertion of high-complexity libraries of small amino acid sequences that enhance affinity to known targets or induce neomorphic protein-protein interactions (Figure 4). The combination of these tools with high-throughput screening and NGS deconvolution for degradation of the protein of interest in a real biological model offers a great opportunity for molecular glue degrader discovery. The identification of new induced pockets that drive the degradation of a protein of interest is a starting point for the computational design of new molecules that mimic the side-chain interactions found in protein-protein interactions.
THE FUTURE OF DEGRADER DISCOVERY
The world of degrader drug discovery is rapidly expanding and evolving, providing new promise for disease targets classically considered “undruggable.” PhoreMost’s SITESEEKER technology combines the benefits of high-throughput screening typically associated with genetic perturbation screens with highly translatable output enabling the development of small molecule degraders. The versatility of PROTEINi library design and implementation has not only allowed SITESEEKER to be exploited for the development of heterobifunctional degraders, but has also permitted its adaptation to the discovery of molecular glues.
With the first wave of cereblon-based degraders showing promise in the clinics, there is much anticipation for the impending improvement and expansion of degrader molecules targeting a diverse array of diseases, with recruitment of novel E3 ligases playing a big role in the advancement of these therapeutics.
REFERENCES
- Bock C 2022 Nature Reviews https://doi.org/10.1038/s43586-021-00093-4.
- Fang Y 2023 Trends in Pharmacological Sciences https://doi.org/10.1016/j.tips.2023.03.003, Hamilton E 2022 Journal of Clinical Oncology https://doi.org/10.1200/JCO.2022.40.16_suppl.TPS1120 Gao X 2022 Journal of Clinical Oncology https://doi.org/10.1200/JCO.2022.40.6_suppl.017.
- Bouvier C 2024 Cells https://doi.org/10.3390/cells13070578.
- Chirnomas D 2023 Nature Reviews Clinical Oncology https://doi.org/10.1038/s41571-023-00736-3.
- Fang Y 2023 Trends in Pharmacological Sciences https://doi.org/10.1016/j.tips.2023.03.003.
- Mostofian B 2023 JCIM https://doi.org/10.1021/acs.jcim.3c00603.
- Bekes M 2022 Nature Reviews Drug Discovery https://doi.org/10.1038/s41573-021-00371-6.
- Konstantinidou M 2024 Cell Chemical Biology https://doi.org/10.1016/j.chembiol.2024.04.002.

Dr. Alberto Moreno is Associate Director, SITESEEKER® Screening, He earned his PhD in Biochemistry and Molecular Biology (Madrid) and continued his post-doctoral training in Dundee working in DNA replication and DNA damage. He joined PhoreMost in 2016 as Senior Scientist helping to develop the SITESEEKER platform and running the first ever PROTEINi® screens.

Dr. Laura Butler is Associate Director, Target ID & Alliances. She joined PhoreMost as a team leader from Atrin Pharma in 2018, where she was working on novel ATR inhibitors. Prior to this, she completed post-doctoral training at the University of Pennsylvania and Birmingham, UK. At PhoreMost, she leads on the deconvolution of hits to their cognate cellular targets and coordinating alliance relationships across SITESEEKER.
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