Abstract Deciphering molecular targets and interaction networks of small molecules within native cellular contexts remains essential for both target-based and phenotypic drug discovery. This is particularly critical for molecular glues (MGs), which promote selective protein degradation through induced protein-protein interactions. Two mechanistic axes define MG action: (1) direct binding of the compound to an E3 ligase that subsequently recruits a neosubstrate, and (2) compound engagement with a primary protein target that promotes E3 ligase recruitment and ternary complex formation. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful, label-free approach for elucidating small-molecule target engagement and mapping binding sites in complex proteomes without chemical tagging or genetic manipulation. Here, we expand the application of LiP-MS to two complementary experimental formats that interrogate distinct stages of molecular glue activity. In the first scenario, an in-lysate LiP-MS workflow was implemented to identify primary drug-protein interactions across the proteome. Using quantitative data-independent acquisition mass spectrometry (DIA-MS) and a seven-point concentration series, we monitored conformational and accessibility changes across >250,000 peptides from >8,000 proteins. Machine learning-based LiP scoring enabled peptide-level resolution of binding sites and quantitative ranking of target engagement. In the second scenario, a live-cell LiP-MS assay was developed to capture compound-induced protein-protein interaction changes under physiological conditions. This live-cell format enables detection of secondary, compound-dependent protein recruitment events, including ternary complex formation with E3 ligases and other associated proteins. To evaluate LiP-MS performance in characterizing molecular glue mechanisms, we conducted global target identification experiments using two representative compounds: SR-4835, a cyclin K degrader that binds CDK12 and recruits DDB1, and MRT-2359, a GSPT1 degrader that engages CRBN. Live-cell LiP-MS experiments were performed at two time points (1 hour and 6 hours) to monitor compound-dependent conformational changes and protein recruitment dynamics associated with primary target engagement and potential ternary complex formation. Together, LiP-MS provides a comprehensive, high-resolution platform for mapping small-molecule target engagement and for characterizing dynamic protein recruitment events associated with molecular glue activity directly in the cellular environment. Citation Format: Martin Soste, Polina Shichkova, Matevz Stefancic, Daniel Redfern, Francesca Cavallo, Lorna Charge, Ka Ying Lee, Ricardo Canavate del Pino, Denise Swift, Roland Hjerpe, Stuart Thomson, Allan Jordan, Yuehan Feng. Proteome-wide target engagement and ternary complex mapping of molecular glues using LiP-MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2417.

Helicases have been implicated in the progression of microsatellite instability-high (MSI-H) tumours and therefore represent promising therapeutic targets. Our team has applied diverse techniques to expedite the discovery of novel small molecule inhibitors for this clinically relevant helicase target; delivering a development candidate in under 18 months. The strategy employed for fast and efficient hit finding harnessed our Hit Synergy platform, which combines distinct screening technologies to rapidly generate high-quality, structurally diverse hits. Deployment of multiple approaches in parallel, including high throughput screening (HTS), virtual screening (VS), and knowledge-based drug discovery (KBDD), while drawing upon our previous expertise in this target class, ensured we rapidly identified novel chemical hit matter. Distinct chemical matter from each strand of hit finding was successfully validated through orthogonal screening assays, tailored specifically to the target. In addition to this, a highly effective Direct-to-Crystallography strategy was employed using 10 mM HTS liquid stocks to quickly deliver bound structures, without re-synthesis or re-acquisition. Following hit validation, rapid hit expansion was achieved using high-throughput chemistry (HTC) and Direct-to-Biology approaches, closely integrated with traditional medicinal chemistry design. Idea generation for new chemical matter with optimized properties and prioritization of compound selection for library synthesis were supported by in silico approaches utilizing artificial intelligence (AI) models. Taken together, we have been able to deliver multiple compound series, showing cellular target engagement and picomolar activity in a clinically relevant MSI-H cell line, favourable ADME properties, and in vivo efficacy in a disease relevant animal model. Here, we will describe these approaches in more detail and demonstrate how the integrated use of these technologies can rapidly advance drug discovery projects toward clinical evaluation. Alexandra I J. Stowell, Megan Thompson, Graeme Walker, Denise Swift, Lyn Parkinson, Tarun Arya, Euan Fordyce, Kam Chohan, Jonathan Finlayson, Terence Wu, Daniel Clare, Allan Jordan, Raquel Faba Rodriguez, Christopher Pearce, Rebecca Morgan, Clare Wilson, Sarah Beck, Rachel Lawrence, Oliver Smith, Sam Forshaw. Through hit synergy and beyond! Integrating hit finding and direct to biology to shorten time to development candidate [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B118.

Recent discoveries have highlighted that small monovalent molecules can leverage a variety of cellular mechanisms to induce degradation of a specific target protein. This perspective exemplifies and contextualizes these mechanisms within drug discovery, with a focus on identification of novel monovalent degraders via cell-based high-throughput screening (HTS) methodologies. Considerations for screening strategies, assay technologies and mechanistic deconvolution are discussed, with emphasis on identification of degrader compounds from diverse compound collections.

G-protein-coupled receptors (GPCRs) are the largest and most versatile cell surface receptor family with a broad repertoire of ligands and functions. We've learned an enormous amount about discovering drugs of this receptor class since the first GPCR was cloned and expressed in 1986, such that it's now well-recognized that GPCRs are the most successful target class for approved drugs. Here we take the reader through a GPCR drug discovery journey from target to the clinic, highlighting the key learnings, best practices, challenges, trends and insights on discovering drugs that ultimately modulate GPCR function therapeutically in patients. The future of GPCR drug discovery is inspiring, with more desirable drug mechanisms and new technologies enabling the delivery of better and more successful drugs.

Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.

Abstract The concept of harnessing the immune system to target cancer cells has been an active area of research for decades. The advent of antibodies targeting the checkpoint receptors CTLA-4 and PD-1/PDL-1, have now provided definitive clinical validation for this approach. Since these discoveries the search for small molecule immuno-oncology agents have intensified. Here we present data on HPK1 (hematopoietic progenitor kinase 1), a novel immuno-oncology kinase involved in the negative regulation of T-cell receptor (TCR) signalling and describe a high throughput screen to identify novel chemical starting points to develop potent and selective inhibitors of this kinase. HPK1 is a member of the Ste-20 family of Serine/Threonine protein kinases. It is expressed highly in cells of haematopoietic lineage, including T-cells and is activated upon engagement of the TCR with cell surface MHC complexes. Active HPK1 leads to phosphorylation of an adaptor protein SLP76, triggering a signalling cascade that results in the downregulation of TCR signalling and thus downregulation of T-cell function. Recent reports have demonstrated that the kinase activity is essential for the function of HPK1 in T-cells and support the hypothesis that small molecule inhibitors of HPK1 kinase activity will result in sustained activation of T-cells. To identify inhibitors of HPK1 we developed an enzyme activity assay that could be used in a high throughput assay to screen Sygnature Discovery's proprietary screening library. We utilised the intrinsic ATPase activity of HPK1 to develop an ADP product fluorescent polarisation assay suitable for an HTS screen. We validated the assay using a series of known inhibitors with a broad range of potencies versus HPK1 activity. Next, we developed a cell-based assay encompassing anti-CD3 TCR mediated induction of phosphorylation of SLP76 in Jurkat cells. We tested the activity of our panel of HPK1 tool inhibitors in this assay and demonstrated a strong correlation between the biochemical and cell potencies of the compounds, further validating the robustness and cell potency predictivity of the biochemical assay. The biochemical assay was then used to screen our small molecule chemical library to identify potential inhibitors of HPK1 kinase activity. This library is biased toward lead-like chemical space and has undergone significant triage to remove unwanted chemotypes and PAINs moieties. The initial hits were confirmed and IC50 values determined for a select group of chemotypes. These compounds were subsequently tested in the Jurkat pSLP76 cell assay for cellular activity versus HPK1. In summary we present the results of a successful HTS and hit evaluation to identify novel and attractive chemical starting points for the development of inhibitors of the immuno-oncology target HPK1. Citation Format: Denise Swift, Samantha Hitchin, Chris Tomlinson, Anindita Sengupta, Callum Taylor, Alec O'Keeffe, Grace Boden, John Unitt, Stuart Thomson, Allan M. Jordan. A high throughput screen of the novel immuno-oncology target HPK1 identifies a range of chemical starting points for the development of potent and selective inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5317.

The nonselective glucocorticoid receptor (GR) antagonist mifepristone has been approved in the U.S. for the treatment of selected patients with Cushing's syndrome. While this drug is highly effective, lack of selectivity for GR leads to unwanted side effects in some patients. Optimization of the previously described fused azadecalin series of selective GR antagonists led to the identification of CORT125134, which is currently being evaluated in a phase 2 clinical study in patients with Cushing's syndrome.

We report the further optimization of our series 1H-pyrazolo[3,4-g]hexahydro-isoquinoline sulfonamides as GR antagonists. By incorporating a heteroaryl ketone group at the ring junction, we have obtained compounds with excellent functional GR antagonism. Optimization of the sulfonamide substituent has provided compounds with a very desirable overall profile, including minimal hERG activity, good bioavailability and in vivo efficacy.

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as KD, kon, koff, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand–p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in Tm values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.

We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [γ-33P]ATP filter wash assay. Determined pIC50s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation `FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.