Large scale genome wide CRISPR screens identified WRN helicase as a promising synthetic lethal target for MSI-H cancers, independent of tumor type. Here we describe the discovery of the novel clinical WRN helicase inhibitor GSK4418959 (IDE275), which recapitulates the MSI-H synthetic lethality of WRN genetic inhibition in vitro and in vivo. GSK4418959 (IDE275) engages a unique allosteric site in the WRN helicase domain, competing with ATP binding and inducing an inhibitory conformation distinct from previously reported WRN inhibitors. It selectively inhibits the ATPase and DNA unwinding activities of WRN, but not other RecQ helicase family members, including BLM helicase. GSK4418959 (IDE275) binds directly to WRN in cells, inducing DNA damage selectively in MSI-H cancer cells in a concentration-dependent manner. GSK4418959 (IDE275) shows strong anti-proliferative effects in MSI-H cell lines and patient-derived organoids across multiple tumor types with no measurable effects in MSS models. In vivo, GSK4418959 (IDE275) causes tumor regressions and induces DDR markers in several MSI-H CDX and PDX models harboring different oncogenic drivers and tumor suppressor mutations, without affecting MSS models. One of these models was an MSI-H CRC PDX from a patient that had failed 3 previous lines of therapy, including the immune checkpoint inhibitor Nivolumab. Due to its unique binding mode, GSK4418959 (IDE275) also induced regressions in an MSI-H CDX CRC tumor model that had developed resistance to treatment with other reported WRN inhibitors. These findings demonstrate GSK4418959 (IDE275)'s potent and selective preclinical activity against MSI-H cancer models, indicating its potential as a promising clinical treatment for MSI-H cancer patients, including those that have failed existing therapies. All studies were conducted according to GSK's Policy on the Care, Welfare and Treatment of Animals and reviewed by the Institutional Animal Care and Use Committee at GSK or by the ethical review process at the institution where the work was performed. Yanhua Rao, Brian T. Jones, Edward J. Brnardic, Joshua E. Cottom, Yang Lee, Diana M. Munoz, Claire Neilan, Sunjay Sethi, Robert A. Reid, Lisa M. Shewchuk, Ethan D. McSpadden, Daniel L. Severance, Kira Campbell, Philip Landis, Jay Prakash Jain, Richard Zang, Leng Nickels, Dennis J. Murphy, H. Christian Eberl, Muzaffar Alam, Melissa Fleury, Lara K. Leister, Tessa Lynch-Colameta, James P. Phelan, Michael D. VanHeyst, Amberly B. Sanford, Ann M. Rowley, Hongyi Yu, Anna Rutkowska-Klute, Thilo Werner, Xin Linghu, Ian S. Young, An D. Nguyen, Sabrina Bédard, Eldridge N. Nartey, Nanhua Deng, Yang Peng, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Gabriele Picco, Mathew J. Garnett, Jessica L. Schneck, Geeta Sharma, Joshua P. Taygerly, Michael P. DeMartino, Yujiro S. Hata, Paul A. Barsanti, Michael A. White, Benjamin Schwartz. An innovative and reversible WRN helicase inhibitor, GSK4418959 (IDE275), emerges as a promising clinical candidate for MSI-H cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr ND10.

Recent large-scale genome wide CRISPR screens identified Werner (WRN) Helicase as uniquely essential to cell survival specifically in the microsatellite instability-high (MSI-H) cancer setting. This synthetic lethal relationship elevated WRN as an attractive precision therapeutic target for the treatment of MSI-H cancers, irrespective of tumor type. High-throughput screening identified a hit characterized by low potency inhibition of ATPase function. This poster describes the systematic optimization of this hit resulting in the discovery of clinical candidate GSK4418959, a novel, potent and selective small molecule that binds to WRN in an allosteric pocket and inhibits ATPase and DNA unwinding functions in an ATP-competitive manner. GSK4418959 phenocopies the genetic silencing studies in cells and organoids and accordingly, elicits complete tumor regression in murine xenograft MSI-H models (CDX and PDX) following oral dosing. Importantly, no response is observed in analogous microsatellite stable (MSS) models, exemplifying the precision effect of the WRN inhibition mechanism. GSK4418959 will be investigated as a potential treatment for patients with MSI-H cancer, including those who have been treated with and failed at least one line of checkpoint immunotherapy for their advanced disease. Joshua P. Taygerly, Michael P. DeMartino, Brian T. Jones, Edward J. Brnardic, Joshua E. Cottom, Yang Lee, Diana M. Munoz, Claire Neilan, Sunjay Sethi, Robert A. Reid, Apirat Chaikuad, Lisa M. Shewchuk, Ethan McSpadden, Daniel L. Severance, Kira Campbell, Philip Landis, Jay P. Jain, Richard Zhang, Leng Nickels, Dennis K. Murphy, Hans C. Eberl, Muzaffar Alam, Melissa Fleury, Lara K. Leister, Tessa Lynch-Colameta, James P. Phelan, Michael D. VanHeyst, Amberly B. Sanford, Ann M. Rowley, Hongyi Yu, Anna Rutkowska-Klute, Thilo Werner, Stephan Gade, Damian Hruszkewycz, Tyler Higgins, Xin Linghu, Ian S. Young, An D. Nguyen, Sabrina Bedard, Eldridge N. Nartey, Nanhua Deng, Yang Peng, Jessica L. Schneck, Geeta Sharma, Yanhua Rao, Paul A. Barsanti, Michael A. White, Benjamin Schwartz. Discovery of GSK4418959 (IDE275): A novel, non-covalent, reversible Werner Helicase inhibitor and a new potential therapeutic for the treatment of MSI-H cancers [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 5750.

Supplementary Text, Tables 1-4 from Impact of Common Epidermal Growth Factor Receptor and HER2 Variants on Receptor Activity and Inhibition by Lapatinib

E1A binding protein (p300) and CREB binding protein (CBP) are two highly homologous and multidomain histone acetyltransferases. These two proteins are involved in many cellular processes by acting as coactivators of a large number of transcription factors. Dysregulation of p300/CBP has been found in a variety of cancers and other diseases, and inhibition has been shown to decrease Myc expression. Herein, we report the identification of a series of highly potent, proline-based small-molecule p300/CBP histone acetyltransferase (HAT) inhibitors using DNA-encoded library technology in combination with high-throughput screening. The strategy of reducing ChromlogD and fluorination of metabolic soft spots was explored to improve the pharmacokinetic properties of potent p300 inhibitors. Fluorination of both cyclobutyl and proline rings of 22 led to not only reduced clearance but also improved cMyc cellular potency.

A series of diarylurea inhibitors of the cardiac-specific kinase TNNI3K were developed to elucidate the biological function of TNNI3K and evaluate TNNI3K as a therapeutic target for the treatment of cardiovascular diseases. Utilizing a structure-based design, enhancements in kinase selectivity were engineered into the series, capitalizing on the established X-ray crystal structures of TNNI3K, VEGFR2, p38α, and B-Raf. Our efforts culminated in the discovery of an in vivo tool compound 47 (GSK329), which exhibited desirable TNNI3K potency and rat pharmacokinetic properties as well as promising kinase selectivity against VEGFR2 (40-fold), p38α (80-fold), and B-Raf (>200-fold). Compound 47 demonstrated positive cardioprotective outcomes in a mouse model of ischemia/reperfusion cardiac injury, indicating that optimized exemplars from this series, such as 47, are favorable leads for discovering novel medicines for cardiac diseases.

Reducing the required frequence of drug dosing can improve the adherence of patients to chronic treatments. Hence, drugs with longer in vivo half-lives are highly desirable. One of the most promising approaches to extend the in vivo half-life of drugs is conjugation to human serum albumin (HSA). In this work, we describe the use of AlbuBinder 1, a small-molecule noncovalent HSA binder, to extend the in vivo half-life and pharmacology of small-molecule BMP1/TLL inhibitors in humanized mice (HSA KI/KI). A series of conjugates of AlbuBinder 1 with BMP1/TLL inhibitors were prepared. In particular, conjugate c showed good solubility and a half-life extension of >20-fold versus the parent molecule in the HSA KI/KI mice, reaching half-lives of >48 h with maintained maximal inhibition of plasma BMP1/TLL. The same conjugate showed a half-life of only 3 h in the wild-type mice, suggesting that the half-life extension was principally due to specific interactions with HSA. It is envisioned that conjugation to AlbuBinder 1 should be applicable to a wide range of small molecule or peptide drugs with short half-lives. In this context, AlbuBinders represent a viable alternative to existing half-life extension technologies.

ABSTRACTIndoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of established and novel IDO1 inhibitors remains of great interest. Comparison of a newly-developed IDO1 inhibitor (GSK5628) to the existing best-in-class compound, epacadostat (Incyte), allows us to report on a unique inhibition mechanism for IDO1. Here, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1) while epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition may help design superior inhibitors or may confer a unique competitive advantage over other IDO1 inhibitorsvis-à-visspecificity and pharmacokinetic parameters.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.

Structure-guided progression of a purine-derived series of TNNI3K inhibitors directed design efforts that produced a novel series of 4,6-diaminopyrimidine inhibitors, an emerging kinase binding motif. Herein, we report a detailed understanding of the intrinsic conformational preferences of the scaffold, which impart high specificity for TNNI3K. Further manipulation of the template based on the conformational analysis and additional structure-activity relationship studies provided enhancements in kinase selectivity and pharmacokinetics that furnished an advanced series of potent inhibitors. The optimized compounds (e.g., GSK854) are suitable leads for identifying new cardiac medicines and have been employed as in vivo tools in investigational studies aimed at defining the role of TNNI3K within heart failure.

The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.

A series of selective TNNI3K inhibitors were developed by modifying the hinge-binding heterocycle of a previously reported dual TNNI3K/B-Raf inhibitor. The resulting quinazoline-containing compounds exhibit a large preference (up to 250-fold) for binding to TNNI3K versus B-Raf, are useful probes for elucidating the biological pathways associated with TNNI3K, and are leads for discovering novel cardiac medicines. GSK114 emerged as a leading inhibitor, displaying significant bias (40-fold) for TNNI3K over B-Raf, exceptional broad spectrum kinase selectivity, and adequate oral exposure to enable its use in cellular and in vivo studies.

Starting from the micromolar 8-quinoline carboxamide high-throughput screening hit 1a, a systematic exploration of the structure-activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai, were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL/6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD.