Ananthan Sadagopan, Anurag Sodhi, William J. Gibson

Journal of Medicinal Chemistry

DOI: 10.1021/acs.jmedchem.6c00249

Abstract

Ligand–protein binding prediction remains a central challenge, yet the contribution of ligand-side information to performance is unclear. We combined pretrained molecular embeddings with TabPFNv2 to build per-target classifiers without protein features. Across 159 BindingDB targets, models assigned higher probabilities to annotated binders and achieved >10-fold enrichment at the top 1% for 42 targets and >50-fold enrichment for three. Fragment- and atom-level interpretability analyses recovered established pharmacophores and nominated concise target-associated substructures. In a BRD9 DNA-encoded library screen, the model distinguished hits from nonhits from the same experiment (AUC = 0.913) and recovered the 2-pyridone chemotype. Supporting analyses separated carbonic anhydrase actives from matched DUD-E decoys, recovered primary and off-targets for compounds in DepMap, and guided the synthesis of a structurally simplified compound that measurably inhibited ACC2 ATPase activity. These results establish ligand-only models as interpretable screening tools and motivate their use as a baseline for assessing the added value of protein representations.

Yuichi Onda, Yurika Ochi, Toshihiro Araki, Miho Kageoka-Takahashi, Shuzo Takeda, Kazunori Yamada, Takehiko Ueda, Ken Ohno, Minoru Tanaka, Daiki Sakai, Miki Hasegawa, Yoshihito Tanaka

ACS Chemical Biology

DOI: 10.1021/acschembio.6c00234

Abstract

Targeted protein degradation (TPD), including proteolysis targeting chimeras (PROTACs) and molecular glue degraders (MGDs), is a promising therapeutic approach. However, systematic discovery of such small molecules remains a major challenge. Here, we present PhenoDEL, a novel phenotypic DNA-encoded library (DEL) screening platform that integrates one-bead one-compound DEL (OBOC-DEL) with the Beacon optofluidic system for single-cell analysis. By coculturing individual OBOC-DEL beads and engineered reporter cells in nanoliter-scale chambers, PhenoDEL enables time-resolved, single-cell phenotypic evaluation and direct linkage between compound identity and intracellular response. As a proof-of-concept, we demonstrate discrimination of active and inactive on-bead compounds using an FKBP12F36V-EGFP degradation reporter in PC-3 cells, followed by DNA barcode decoding.

Baiyang Mu, Yiwei Zhang, Xudong Wang, Rui Jin, Weiwei Lu, Nidhal Selmi, Sixiu Liu, Avinash Bhat, Sara Pahlén, Giulia Bergonzini, Zhiqiang Duan, Yinan Song, Xiaojie Lu

Bioconjugate Chemistry

DOI: 10.1021/acs.bioconjchem.4c00581

Abstract

The C­(sp²)–C­(sp³) cross-coupling reaction is an effective way to increase the C­(sp³) content in compound collections for drug discovery, enhancing molecular diversity and offering a unique chemistry starting point. In this study, we report a mild, DNA-compatible, and off-DNA-inert photochemical cross-coupling reaction inspired by the amino radical transfer strategy. This method demonstrates broad substrate scopes for DNA-encoded library (DEL) constructions, utilizing commonly available structures on DNA and diverse alkyl boronate ester building blocks, which have not been widely applied in the current DEL chemical space.

For years, a robust and general method for DNA compatible "reverse" Buchwald-Hartwig-type amination – coupling DNA-conjugated amines with external aryl halides – has been long sought, yet this transformation remained elusive due to the prohibitive challenge of DNA nucleobase interference. To overcome this long-standing technical bottleneck, the Ritter group at the Max-Planck-Institut für Kohlenforschung reported an air-stable ruthenium-mediated η6 π-arene activation strategy in the Journal of the American Chemical Society (JACS). This breakthrough finally enables the precise N-arylation of DNA-linked amines, unlocking a vast and previously inaccessible chemical space for drug discovery.

1. The Challenge: The DNA Nucleobase Interference

In DEL synthesis, C–N cross-coupling is a cornerstone for building structural diversity. While conventional Buchwald-Hartwig amination (DNA-linked aryl halides reacting with external amines) was well-established, the reverse approach has been hindered by the presence of multiple amino groups within DNA nucleobases (adenine, guanine, and cytosine).

1) Limitations of Traditional Catalysts: Conventional palladium-based methods often lead to undesired arylation of the DNA backbone rather than the intended terminal amine.

2) Chemoselectivity Hurdles: Because the amino groups on nucleobases are typically more acidic than aliphatic amines, palladium-catalyzed pathways (which involve deprotonated amine intermediates) preferentially target the DNA nucleobases.

2. Ruthenium-Mediated η6 π-Arene Activation

To overcome these limitations, the Ritter group developed a bench-stable, DNA-compatible acetyl-substituted cyclopentadienyl (CpAc) ruthenium complex (1).

Figure 1. N-arylation of DNA-conjugated amines in aqueous media. (a) SNAr of DNA-conjugated aliphatic amines with η6 arene complexes. (b) Electrophilicity of Ru π-arene complexes 5−7 and N-arylation of DNA-conjugate 2.

• Mechanism of Action: The ruthenium reagent activates commercially available haloarenes in situ through η6 π-arene coordination. This coordination significantly increases the electrophilicity of the arene ring, allowing for nucleophilic aromatic substitution (SNAr) directly with the DNA-conjugated amine.
• Operational Simplicity: Much like amide coupling reagents (e.g., HATU), the protocol involves simple activation of haloarenes by complex 1 followed by addition to the DNA conjugate. The final aniline product was released via photolysis at 390 nm, a wavelength safe for DNA integrity.

3. Superior Chemoselectivity and Validation

The core innovation lies in the complementary selectivity of ruthenium compared to traditional palladium catalysts.

1. Selective Amine Attack: Unlike palladium, the ruthenium-mediated pathway involves the attack of a neutral amine, which naturally favors the more nucleophilic aliphatic amine over the less nucleophilic DNA nucleobases.

Figure 2. Chemoselectivity of DNA N-arylation.

2. Substrate Scope: The method demonstrates exceptional tolerance for diverse functional groups, including carbonyls, sulfonamides, and even Lewis-basic heterocycles when treated with HBF4⋅Et2O to prevent metal coordination. It successfully arylated a wide array of primary and secondary amine-DNA conjugates, including natural and non-natural amino acid.

Figure 3. Substrate scope of N-arylation for various amine-DNA conjugates. Aryl halide (c = 10 mM) and Ru complex 1 (c = 1.0 mM) in DMC, 80oC, 2 h; then amine-DNA conjugate (c = 0.10 mM), in sodium borate buffer (c = 0.50 M, pH 9.4): DMSO (1:9), 40°C, 2−16 h; then 390 nm irradiation in water (c = 0.10 mM). aHBF4·Et2O was used for the complexation step.

3. Preservation of DNA Integrity: High-throughput compatibility was confirmed through qPCR analysis and ligation tests, proving that the DNA barcode remains stable and readable throughout the reaction sequence.

4. Paradigm Shift in DEL Synthesis

This ruthenium-mediated strategy reconfigures the drug discovery workflow for DELs in three key ways:

1) Unlocking "Reverse" Disconnections: Researchers can now use DNA-linked amines as a starting point for C–N coupling with a vast library of commercially available aryl halides, drastically expanding structural diversity.

2) Orthogonal Reactivity: By providing a method that is chemoselective for aliphatic amines over DNA nucleobases, it offers a "surgical" precision that palladium-based methods lack for this specific disconnection.

3) Establishing a Bench-Stable Standard: Complex 1 provides a practical, scalable, and single-reagent tool for both academic research and industrial DEL production.

5. Conclusion

This study marks the first successful "reverse" Buchwald-Hartwig-type amination on DNA. While "normal" pathways (DNA-halides + external amines) are well-established, the reverse route (DNA-amines + external halides) was historically hindered by nucleobase interference. Due to the lower pKa of nucleobase amines compared to aliphatic amines, traditional palladium catalysis often leads to undesired DNA backbone modification. Ruthenium reagent 1 overcomes this via η6 π-arene activation, targeting neutral amines with "surgical" precision to bypass nucleobase competition. This paradigm shift significantly expands the accessible chemical space for DEL-driven drug discovery while ensuring rigorous DNA integrity

References

1. Kanoo, S., de Pedro Beato, E., et al. (2025). Ruthenium-Mediated N-Arylation for DNA-Encoded Libraries. J. Am. Chem. Soc. https://doi.org/10.1021/jacs.5c11842

Raphael M. Franzini

Molecular Informatics

DOI: 10.1002/minf.70045

Abstract

Linkers play a central role in many areas of medicinal chemistry, including proximity inducers, small‐molecule conjugates, and DNA‐encoded libraries. However, little is known about the accessibility of molecules to linker attachment when bound to proteins. Here, we analyze linker accessibility across protein–ligand complexes in cocrystal structures. A computational workflow was developed to evaluate the linkerability of modifiable positions on molecules based on solvent accessibility, local steric space for introduction of a linker atom, and the geometry of solvent‐directed escape paths approximated as conical frustums. Analysis of 8,228 protein–ligand cocrystal structures with 131 431 modifiable positions shows that approximately 22% of positions can accommodate linkers without significant geometric restriction. Limited linkerability of positions influences DEL data and may confound efforts to use such data for lead prediction.

Slavko Rast, Marie Morgan-Fisher, Sarah D. Blomquist, Jorge Peiró Cadahía, Sanne Cowland, Thomas Franch, Emil Glibstrup, Alex Gouliaev, Margit Haahr Hansen, Aleksejs Kontijevskis, Titi Kronborg, Loris Moretti, Anna Nadali, Søren Nielsen, Sebastian Leth-Petersen, Michael Rabe, Adili Alafate, Jennifer R. Allen, Abhisek Banerjee, Shon K. Booker, John R. Butler, Imelda Hot, David Huang, Matthew R. Kaller, Rajiv Kapoor, Qingyian Liu, Patricia Lopez, Vu Ma, Francesco Manoni, Jose M. Medina, Alexander J. Pickrell, Hui-Ling Wang, Jingjing Xie, Wenhan Zhang, Christopher Mohr, Kui Chen, Anne Y. Saiki, Paul Wang, Monica Leavitt, Karen Rex, Guo Zhong, Ling Zou, Julie Lade, Upendra P. Dahal, Nashid Farhan, Prashant Agarwal, Borna Zandkarimi, Kai Zhu, Gitte Husemoen, Nuria A. Tamayo, Brian A. Lanman

Journal of Medicinal Chemistry

DOI: 10.1021/acs.jmedchem.6c01357

Abstract

Activating mutations in the Kirsten rat sarcoma (KRAS) gene are prevalent oncogenic drivers in nonsmall cell lung cancer (NSCLC). Patients harboring KRAS-mutant lung cancers frequently develop central nervous system (CNS) metastases. Although approved KRAS G12C inhibitors (i.e., sotorasib and adagrasib) show promising clinical CNS activity, these agents demonstrate low preclinical brain-to-plasma ratios, raising the question of whether compounds with elevated preclinical Kp,uu,brain values might show enhanced clinical performance. Here, we report the first successful application of DNA-encoded library (DEL) screening technology to the identification of CNS-penetrant covalent inhibitors of KRAS G12C. In this effort, a property-biased covalent DEL-screening approach enabled the discovery of a structurally novel series of hydrogen bond donor-free KRAS G12C inhibitors with improved CNS exposure. Leveraging structure-based design, we refined this hit series to deliver lead compound AM-8719, a CNS-penetrant, orally efficacious KRAS G12C inhibitor exhibiting 200-fold improved potency with respect to initial screening hits.