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