Eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF), promotes protein synthesis by charging translation initiation factor 2 (eIF2) with GTP. Stress-induced phosphorylation of eIF2 on its α-subunit [eIF2(αP)] inhibits this reaction triggering a protective Integrated Stress Response (ISR). A DNA-encoded chemical library (DEL) screen for modulators of eIF2B, led to the identification of a chemical series that stabilises the inactive state of eIF2B, stimulating the ISR. Cryo-EM of compound-bound eIF2B reveals a conformational switch to the inactive state engaged by eIF2(αP). In cells, compound activity is sensitive to eIF2's phosphorylation state and to a competing eIF2B ligand (ISRIB) that activates the GEF allosterically. These findings establish the feasibility of targeting eIF2B with a drug-like allosteric inhibitor, that serves as an ISR activator (ISRAC), paving the way to explore the therapeutic potential of eIF2B-directed ISR activation.

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.

Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplars exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant that rapidly generated bispecific nanomolar degraders of ERα, with PROTACs inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization. Citation Format: Anthony D. Keefe, Jeremy S. Disch, Jennifer Duffy, Esther C. Lee, Diana Gikunju, Betty Chan, Benjamin D. Levin, Michael I. Monteiro, Sarah A. Talcott, Anthony Lau, Fei Zhou, Anton Kozhushnyan, Neil E. Westlund, Patrick B. Mullins, Yan Yu, Moritz von Rechenberg, Junyi Zhang, Yelena Arnautova, Yanbin Liu, Ying Zhang, Andrew J. McRiner, Anna Kohlmann, Matthew A. Clark, John W. Cuozzo, Christelle Huguet, Shilpi Arora. Discovery of new targeted protein degraders using DNA-encoded chemistry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5346.

Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library (DECL) screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function (GOF) mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplar 7 exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant of 7 rapidly generated bispecific nanomolar degraders of ERα, with PROTACs 18 and 21 inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

DNA-encoded small molecule libraries (DELs) have enabled discovery of novel inhibitors for many distinct protein targets of therapeutic value through screening of libraries with up to billions of unique small molecules. We demonstrate a new approach applying machine learning to DEL selection data by identifying active molecules from a large commercial collection and a virtual library of easily synthesizable compounds. We train models using only DEL selection data and apply automated or automatable filters with chemist review restricted to the removal of molecules with potential for instability or reactivity. We validate this approach with a large prospective study (nearly 2000 compounds tested) across three diverse protein targets: sEH (a hydrolase), ER{\alpha} (a nuclear receptor), and c-KIT (a kinase). The approach is effective, with an overall hit rate of {\sim}30% at 30 {\textmu}M and discovery of potent compounds (IC50 <10 nM) for every target. The model makes useful predictions even for molecules dissimilar to the original DEL and the compounds identified are diverse, predominantly drug-like, and different from known ligands. Collectively, the quality and quantity of DEL selection data; the power of modern machine learning methods; and access to large, inexpensive, commercially-available libraries creates a powerful new approach for hit finding.

Abstract K-Ras is the most frequently mutated oncogene in cancer. Most mutations cluster in codons 12, 13 and 61, and are involved in the development and progression of a wide range of cancers. Drugging K-Ras has been challenging using traditional drug discovery strategies. Covalent inhibition is a viable drug discovery strategy for drugging difficult targets where identification of non-covalent small molecule inhibitors has been challenging. Marketed covalent inhibitors have been identified for various targets including EGFR and BTK. The K-Ras G12C mutation accounts for 12% of all observed K-Ras mutations in cancer and is most prevalent in non-small lung (NSCLC), pancreatic and colorectal cancers. It has recently been shown that this mutated cysteine can be harnessed for the development of specific covalent inhibitors. We performed covalent based-selections for K-Ras G12C utilizing a 1011 member covalent DNA-encoded library. These selections led to the identification of three novel and chemically distinct series of K-Ras G12C-GDP specific covalent inhibitors. Direct target engagement was demonstrated using FAM-labeled probes in cells over-expressing G12C K-Ras. No target engagement was observed with wild-type or other mutants of K-Ras indicating that these compounds are selective for G12C. Co-crystal structures for all 3 series have been obtained validating the covalent linkage to G12C and also demonstrating “series specific” binding modes for all 3 series of inhibitors. Hit to lead efforts have led us to a tool compound, X-498, which is potent and mutant selective inhibitor of K-Ras G12C. X-498 has a biochemical potency <10 nM (2h) in an HTRF based assay and demonstrates a dose dependent inhibition of phospho-ERK (pERK) in the NCI-H358 (G12C) NSCLC cell line after 4h of compound treatment. No effect on pERK is seen in a G12S containing NSCLC (A549) cell line. In vitro cellular viability assessment of X-498 in K-Ras G12C and non-G12C cancer cell lines demonstrate mutant specific killing of cell lines harboring a K-Ras G12C mutation with GI50 <50 nM and a >400-fold selectivity over a wild-type K-Ras cell line. Structure based drug design is currently being used to optimize chemical properties and potency. Assessment of ADME and PK is ongoing. In summary, we have identified novel chemical series selectively targeting K-Ras G12C mutant that are currently being optimized with the goal of developing treatment for cancer patients harboring the K-Ras G12C mutation. Citation Format: Shilpi Arora, Chris Hupp, Sarah Talcott, Usha Narayanan, Diana Gikunju, Mortiz Von Rechenberg, Michael I Monteiro, John P Giulinger, Kyle Denton, Matt Clark, Andrew Ferguson, Andrew J McRiner, John Cuozzo, Christelle Huguet, Anthony D Keefe. Identification of novel K-Ras G12C inhibitors using a DNA encoded library platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C049. doi:10.1158/1535-7163.TARG-19-C049

Tumors utilize many different escape mechanisms to evade anti-tumor immune responses. Xios Therapeutics has screened X-Chem’s proprietary 200-billion molecule DNA-encoded library against several immuno-oncology (IO) targets addressing T-cell centric, myeloid immunity and onco-metabolite pathways. Adenosine, for example, is a potent immunosuppressive metabolite, and the ecto-5-nucleotidase (a.k.a. CD73), which catalyzes the conversion of AMP to adenosine, is the rate limiting enzyme for the production of extracellular adenosine in the tumor microenvironment. Hence, CD73 and/or the downstream adenosine receptors are considered attractive targets for IO drug discovery. Likewise, the characteristics of tumor-associated macrophages (TAMs) have fueled interest in therapeutically targeting the colony-stimulating factor 1 axis (CSF1R). Here, we exemplify and enumerate the diversity, selectivity and physiochemical properties of selected hit-to-lead compounds identified from our DNA-encoded library screens of CD73, the adenosine A2A receptor and CSF1R. In the context of the immune-suppressive purinergic pathway, we have integrated structural biology, medicinal chemistry and clinical pathology evaluation of target expression across tumor types and developed both A2A selective and dual A2A/A2B selective receptor antagonists. From a DNA-encoded library screen, we have identified novel sub-micromolar ligands, which binds to CD73 in an ‘open conformation’ revealed by the co-crystal structure of X6034 (EC50 = 310 nM) in complex with CD73. Interestingly, an inorganic phosphate molecule (Pi) that is structurally shown to be co-present in the active site, illustrates the novelty of these inhibitors, which are chemically distinct from currently reported ADP/AMP substrate analogs. Finally, using tumor tissue microarrays and in situ cell hybridization (RNA-Scope; Advanced Cell Diagnostics), we have explored the co-expression pattern of pathway targets across a subset of immune cells. Informed by the tissue expression pattern of A2AR (primarily CD3+ T-Cells) and A2BR (primarily CD33+ myeloid cells), we compared the ability of equipotent A2A and dual A2A/A2B adenosine receptor antagonists to reverse the effect of NECA, a nonhydrolyzable analog of adenosine, on the maturation and activation of dendritic cells (DC). Starting with the differentiation of human immature DC, we demonstrate the added benefit of dual A2A/A2B inhibitors vs A2A selective inhibitors on relieving immunosuppression of myeloid cells. To conclude, the combination of all these data, together with the productivity of the DNA-encoded library screens for identifying novel, drug like chemical matter on challenging targets like CD73, provide a unique opportunity for potentially harnessing the full power of immunotherapy for cancer. Citation Format: Andrew J. McRiner, Jannik N. Andersen, Lynette A. Fouser, Junyi Zhang, Kaan Certel, John Cuozzo, Betty Chan, Ragunath Chandran, Matt Clark, Diana Gikunju, Christopher D. Hupp, Anthony D. Keefe, Julie Liu, Yanbin Liu, Michael Monteiro, Allison Olszewski, Moritz Von Rechenberg, Daniel Resnicow, Heather A. Thomson, Dawn M. Troast, Zooey Wang, Neil Westlund, Ying Zhang, Fei Zhou, Xiaotian Zhu, Michael Briskin, Diala Ezzeddine. Novel, potent, and selective small-molecule inhibitors modulating immuno-oncology targets CD73, A2A/A2B adenosine receptors and CSF1R discovered via DNA-encoded library screening [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 4453.

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets.

A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.