Tianxiong Mi, Lijun Fan, Amber Hackler, Diego B Diaz, Chang Qi, Errol L G Samuel, Qi Gao, Tao Meng, Xingjian Xu, Erwin G Abucayon, Edward N Dinunzio, Marcelo J Murai, Natalya Pissarnitski, Jordan De , Jesus Silva, Christopher Sondey, Jack D Scott

ChemRxiv

DOI: 10.26434/chemrxiv.15001086/v1

Abstract

Rapid, aqueous macrocyclization strategies that proceed in high conversion are valuable for ultra-large macrocyclic peptide library synthesis via display and DNA-encoded library (DEL) technology. Here we report an on-DNA macrocyclization based on sulfur(VI) fluoride exchange (SuFEx) that unites above features. This approach embeds a phenol and an aryl sulfonyl fluoride within a DNA-tagged peptide to accelerate SuFEx and trigger intramolecular cyclization immediately upon dissolution in basic aqueous buffer. Macrocycles ranging from 15-to 52-membered rings bearing diverse amino acids were synthesized efficiently. The reaction integrates seamlessly into DEL workflows and enabled discovery of potent, de novo macrocyclic RIPK1 inhibitors. The on-DNA cyclization conditions readily translate off-DNA to furnish sulfonate-and sulfonamide-linked macrocycles. NMR analyses showed SuFEx-derived biaryl linkers act as conformational elements: changes in aryl substitution and linker length tune backbone conformations from extended strands to rigid turns. These findings establish SuFEx cyclization as a robust, biocompatible strategy for programmable macrocycle design and drug discovery.

Xinyu Shi ,  Ze Liang ,  Wentao Meng ,  Guang Yang ,  Lei Yan

Molecules

DOI: 10.3390/molecules31050864

Abstract

CD38 is a multifunctional enzyme that plays a pivotal role in NAD+ metabolism and calcium signaling, and its abnormal activity is closely associated with multiple myeloma, age-related metabolic decline, neurodegenerative diseases, and other disorders. Although monoclonal antibodies such as daratumumab have been approved for clinical application, their inherent limitations necessitate the development of novel small-molecule CD38 inhibitors. In this study, we employed DNA-encoded library (DEL) technology for the high-throughput screening of CD38 inhibitors, using a DEL library containing more than 100,000 unique compounds to screen against recombinant human CD38. A total of 1043 enriched compounds were initially identified, and after rigorous validation and screening to exclude non-specific binding and previously reported active compounds, eight hit compounds with diverse chemical scaffolds were obtained, among which Fenbendazole-a clinically approved antiparasitic drug-was included. Surface plasmon resonance (SPR) assays confirmed the direct binding of these hit compounds to CD38, with dissociation constants (KD) ranging from 7.74 × 10-5 M to 2.15 × 10-4 M. Fluorescence-based enzymatic activity assays demonstrated that these compounds exert dose-dependent inhibitory effects on both the hydrolase (with ε-NAD as substrate) and cyclase (with NGD as substrate) activities of CD38. Further structure-activity relationship (SAR) analysis of Fenbendazole analogues revealed the critical structural features that regulate CD38 inhibitory potency, and Flubendazole was found to exhibit excellent inhibitory activity, with an IC50 of 14.78 ± 4.21 μM against CD38 hydrolase and 26.31 ± 3.40 μM against cyclase. Molecular docking and 100 ns molecular dynamics (MD) simulations further elucidated the molecular mechanism of CD38 inhibition by lead compounds, confirming that van der Waals interactions are the main driving force for the binding of small-molecule ligands to CD38, with conserved aromatic residues in the active site mediating ligand recognition. This study validates DEL technology as an efficient and reliable platform for the discovery of CD38 inhibitors, and the identified lead compounds-especially Fenbendazole and its analog Flubendazole-provide valuable molecular scaffolds for the further structural optimization of CD38 inhibitors. These findings lay a solid foundation for the development of novel therapeutic agents for the treatment of CD38-associated diseases.

Jie Li, Ying Yao, Shuning Zhang, Wei Wang, Wanting Bi, Jian Lv, Wei Hou, Peixiang Ma, Hongtao Xu

Chem

DOI:10.1016/j.chempr.2026.102945

Abstract

To address the challenges associated with the construction and selection of single-stranded DNA-encoded libraries (ssDELs), an efficient and precise short-splint RNA-mediated single-stranded DNA (ssDNA) ligation system has been developed. This system exhibits superior discrimination of single-base mismatches at oligonucleotide ligation junctions and requires only 12–14 nucleotides to achieve optimal ligation. By leveraging these advantages, we have established a concise ssDEL-encoding platform, as evidenced by the efficient synthesis and selection of pilot libraries ranging from 10⁴ to 2.95 × 10⁶ members. Additionally, capitalizing on the high sensitivity of this ligation system, a SplintR ligase-mediated proximity ligation (SIMPL)-based polymerase chain reaction (PCR) method has been developed. This method achieves remarkable sensitivity, enabling the linking of chemical and phenotypic information at detection limits as low as zeptomole levels. This capability facilitates significant hit enrichment ratios and enables the identification of a novel carbonic anhydrase XII (CAXII) binder with picomolar affinity through label-free, live-cell-based ssDEL selection.

The recent Cell publication by Xavier et al. (2026) demonstrates a groundbreaking “binder-first” strategy to target the immune scaffold protein CARD9 – a historically "undruggable" protein due to its coiled-coil (CC) domain architecture. This work highlights how DNA-encoded library (DEL) technology, provided by HitGen, played an important role in identifying a ligandable site and enabling the discovery of functional inhibitors with therapeutic potential for Crohn’s disease and inflammatory bowel disease (IBD).

1. Drugging the "Undruggable" Protein: CARD9

CARD9 is a key immune scaffold protein that regulates the NF-κB signaling pathway and participates in inflammatory responses. Its structure is mainly composed of CC domains, lacking traditional small-molecule binding pockets, and has long been regarded as an “undruggable” target. Human genetic studies have revealed that the truncated CARD9 variant (CARD9 Δ11) is associated with protective effects against Crohn’s disease, indicating potential therapeutic value in targeting CARD9. However, the absence of well-defined binding pockets within the CC domains poses a significant challenge for developing inhibitors against this structurally unique protein.

2. HitGen DEL Technology for Targeting a Specific Domain of CARD9

The study leveraged HitGen’s distinct DEL libraries, comprising over 22.4 billion molecules, to screen against full-length CARD9 and its truncations. This massive chemical space exploration was critical because CARD9’s structure lacks traditional binding pockets, making it inaccessible to conventional small-molecule approaches. By establishing distinct screening setups, DEL screening can yield molecules targeting specific structural domains of the target protein (Figure 1A, B). Additional resolution of the potential binding site was obtained using hydrogen/deuterium exchange mass spectrometry (HDX-MS). In the presence of compound 1, reduced deuterium uptake was observed within CC-1 domain residues E167-A186 (Figure 1C). The DEL screening successfully identified a benzimidazole series as initial binders to the CC domain of CARD9.

Figure 1. (A) Illustration of CARD9 DEL screening and structures of CARD9 binders. (B) CARD9 domain structure and truncations used for the DEL screening. (C) HDX-MS. Data represent regions of CARD9 having reduced H/D exchange in the presence of compound 1.

Crucially, X-ray crystallography revealed that these compounds bind to a unique hydrophobic cavity at the CC-1 domain dimer interface (Figure 2A, B, C) – a site never previously characterized for CARD9. This discovery proved CARD9 is “ligandable”, overturning assumptions about its druggability.

Figure 2. X-ray co-crystal structure of compound 1 and CARD9K156-K214. (A) Apo X-ray crystal structure of CARD9K156-K214. (B) Highlight of X-ray co-crystal structure of CARD9K156-K214 and compound 1. (C) Specific contacts between compound 1 and CARD9 residues.

3. From Binders to Functional Inhibitors: DEL-Driven Innovation

While the initial benzimidazole compounds did not directly inhibit CARD9 signaling, the team performed further optimization based on the defined ligandable site. Using a TR-FRET displacement assay with a benzimidazole-derived probe (Figure 3A), the team screened additional 800,000 compounds, leading to the discovery of benzodiazepine inhibitors (Figure 3B). Compound 6 uniquely engaged both CC-1 and CC-2 domains (Figure 3C). Critically, the benzodiazepines demonstrated potent anti-inflammatory activity in human dendritic cells (Figure 3D), mirroring the protective effects of the CARD9 Δ11 variant linked to reduced Crohn's disease risk.

Figure 3. Discovery and validation of a family of benzodiazepines. (A) Description of the TR-FRET assay used to screen for chemical matter. (B) SPR results and TR-FRET inhibition curves of compound 6. (C) HDX-MS of CARD9M1-S536 with compounds 1 or 6. (D) Inhibition of cytokine secretion from MDDCs by compound 6. Primary human monocyte-derived dendritic cells (MDDCs) were stimulated with two different fungal-derived Dectin-1 agonists (DZ and HKCA) or theTLR2/6 agonist FSL-1

4. Conclusion

The study demonstrates DEL technology’s key role in accelerating drug discovery for challenging targets. By establishing distinct screening setups, DEL screening identified molecules targeting specific structural domains of CARD9, a historically "undruggable" protein due to its CC domain architecture. This initial binder enabled subsequent discovery of benzodiazepine inhibitors via displacement screening, which effectively modulated inflammatory signaling. This "binder-first" approach exemplifies DEL's power to unlock novel therapeutic strategies for complex proteins, bridging genetic insights to functional modulators with clinical potential.

5. How DEL Technology Reconfigures the Drug Discovery Workflow

• Genetically-guided precision targeting: Integrating human genetics discoveries, the CARD9 truncated variant (CARD9 Δ11) is associated with protective effects against Crohn’s disease, while DEL technology enables screening of binders that precisely target disease-relevant truncated/mutated proteins.
• Transcending conventional target paradigms: First demonstration that the CC domain of CARD9 harbors druggable sites, providing novel insights for targeting other scaffold proteins.
• Establishing a new "binder-to-functionalization" paradigm: Proof that even initial binders lacking functional activity can be rapidly converted into functional inhibitors through DEL-derived probes combined with high-throughput screening approaches.

1. Rahman, J. S., Wang, J. D., Guo, S. D., Graham, D. B., Hu, K., Venables, J. D., ... & Xavier, R. J. (2026). Human genetics guides the discovery of CARD9 inhibitors with anti-inflammatory activity. Cell. Advance online publication. https://doi.org/10.1016/j.cell.2025.12.013

Julien Poupart,Sunit Kumar Jana,Sasmita Tripathy,Anne Marinier

Bioconjugate Chemistry

DOI: 10.1021/acs.bioconjchem.5c00661

Abstract

While DNA-encoded libraries (DELs) are well recognized as valuable tools for the development of novel small-molecule therapeutics, their significant potential for developing new imaging probes initially received less attention. However, as DEL technology develops and novel screening modalities are introduced, several robust strategies for generating imaging probes from DEL screening campaigns have emerged. The current topical review aims to provide an overview of DEL technology as it relates to the discovery of new molecular probes and to present recent contributions that highlight innovative ways DELs are advancing the field. Approaches to harnessing DEL-derived probes for therapeutic applications, including their conversion into integrated theranostic modalities, will also be discussed.

Wenhao Xie,Yuehong Chen,Lulu Jiang,Xinyuan Wu,Zhiyuan Qi,Tianbai Shuai,Mingzhong Yang,Zhiyang Wang,Jinghan Gao,Yingjie Zhu,Xiaojie Lu,Min Huang,Zhibei Qu,Wenfu Tan,Jian Ding,Lu Zhou

Journal of Medicinal Chemistry

DOI: 10.1021/acs.jmedchem.5c02841

Abstract

Gastric cancer is commonly diagnosed at advanced stages and frequently develops multidrug resistance (MDR). Clinical evidence highlights the overexpression of glutathione S-transferase pi 1 (GSTP1) in gastric cancer, which is closely associated with tumor progression and the development of MDR. However, highly potent and selective GSTP1 inhibitors remain scarce. Guided by our previously reported covalent DNA-encoded library (DEL) hit, a series of warhead-removed 1-(azetidin-3-ylmethyl)-1H-benzo[d]imidazole derivatives were obtained as noncovalent GSTP1 inhibitors. The most potent compound, 16n, inhibited GSTP1 enzymatic activity with an IC50 value of 0.79 ± 0.05 μM and demonstrated improved isoform selectivity. In human gastric cancer cells (AGS, HGC27, and NUGC-3), 16n dose-dependently suppressed proliferation and increased intracellular reactive oxygen species levels while decreasing glutathione levels. Importantly, 16n exhibited favorable systemic exposure and achieved 58% tumor growth inhibition with good tolerability in HGC27 xenograft mouse models. Collectively, 16n represents a promising noncovalent GSTP1 inhibitor for the treatment of gastric cancer.