Baljit Kaur, Ashraf Abdo, Sungwoo Cho, Moustafa Gabr

bioRxiv - Pharmacology and Toxicology

DOI: 10.64898/2026.10.12.758423

Abstract

NOS1AP is an adaptor for neuronal nitric oxide synthase that engages several partner proteins and is implicated in Alzheimer's disease. Efforts to drug NOS1AP have sought to disrupt these; we instead report pharmacological stabilization of one interaction. Affinity selection of a 1.4-billion-member DNA-encoded library identified a benzimidazole-5-carboxamide chemotype, optimized by fragment-directed SAR to NOSTRA with submicromolar NOS1AP binding affinity. Of six interactions, NOSTRA enhanced only SCRIB binding, with a cooperativity factor of 30, predominantly through a 14-fold slowing of dissociation; native mass spectrometry resolved a NOS1AP:NOSTRA:SCRIB ternary complex. In healthy human iPSC-derived cortical neurons, NOSTRA engaged the endogenous complex and activated Rac1 without cytotoxicity; in Alzheimer's patient-derived neurons, it lowered the Aβ42/Aβ40 ratio and p-tau217, reduced neurofilament light and improved network activity. With an in vitro ADME profile consistent with CNS exposure, NOSTRA is the first molecular glue for a NOS1AP complex, establishing interaction stabilization as a viable NOS1AP pharmacology.

Malkeet Singh Bahia, Yifan Jiang, Arthur Gonse, Robert A. Batey, Michał Koziarski

Digital Discovery

DOI: 10.1039/d6dd00117c

Abstract

Over the past several decades, DNA-encoded libraries (DELs) have become an established and efficient approach for the synthesis and screening of large molecular libraries to facilitate drug discovery. Although DELs generate substantially larger amounts of protein–ligand binding data than traditional screening approaches, the quality of the resulting molecular enrichment counts is often poor. Consequently, a careful treatment of enrichment counts is often required to use DELs for further analysis. This review examines different sources of noise influencing DEL screening data quality, and strategies for de-noising enrichment counts, including ML frameworks. Furthermore, it summarizes successful DEL-ML case studies for hit identification and benchmarks for different ML methods.

Hee Wang , Seungyoon Jang , Sanggil Kim , Jungyeon Kim , Deokhee Kim , Yeongju Lee , Hyun Soo Lee , Min Hyeon Shin , Hyun-Suk Lim

RSC Chemical Biology

DOI: 10.1039/d6cb00273k

Abstract

Protein–protein interactions (PPIs) represent an attractive class of therapeutic targets, yet the discovery of PPI inhibitors remains challenging because PPI interfaces are typically shallow and lack well-defined binding pockets. Moreover, target binding alone does not necessarily translate into functional inhibition, as compounds can engage a target protein without disrupting its interaction with a partner, and in some cases may even stabilize the complex. Combinatorial library technologies such as one-bead–one-compound (OBOC) screening enable the exploration of vast chemical diversity, but because conventional OBOC screening selects hits based on target binding rather than biological function, it is susceptible to nonfunctional or false-positive hits. These include compounds that bind outside the functional PPI interface and therefore lack inhibitory activity, compounds that stabilize rather than disrupt the PPI, and artifacts arising from avidity, nonspecific adsorption, or physical entrapment of the target protein within the polymer bead matrix. Here, we report a function-based OBOC screening platform that establishes PPI disruption, rather than target binding, as the primary selection criterion. By integrating a FRET-based competition assay with OBOC combinatorial libraries, bead-displayed compounds are selected only when they competitively and potently displace a quencher-tagged binding partner from a preassembled target complex, ensuring that fluorescence-positive beads directly report potent, functional PPI disruption. To validate this strategy, we applied the platform to the SRC-1/STAT6 interaction, we screened a stapled peptide OBOC library, directly identifying highly potent functional inhibitors. Proof-of-concept experiments using DNA-encoded beads further confirmed the compatibility of this selection strategy with DNA-encoded library technologies, which enable access to substantially larger and more diverse combinatorial libraries. Together, this work establishes a broadly applicable framework for transforming affinity-based OBOC screening into function-based screening, enabling the direct discovery of biologically active PPI inhibitors.

Masaki Koshimizu, Masashi Suzuki, Saori Miya, Hiroyuki Miyachi

Organic Chemistry Frontiers

DOI: 10.1039/d6qo00903d

Abstract

DNA-encoded library (DEL) technology enables efficient exploration of chemical space, but its synthetic scope remains constrained by the limited repertoire of DNA-compatible transformations. Pyrazoles are privileged motifs in medicinal chemistry, yet they remain underrepresented in DEL synthesis because their preparation often requires conditions that are poorly compatible with DNA. Here we report a mild, DNA-compatible three-component reaction for on-DNA pyrazole construction from DNA-conjugated aldehydes, hydrazines, and nitroolefins. The reaction proceeds in aqueous media at room temperature without added oxidants or heating, while preserving DNA integrity and amplifiability under the tested conditions. Systematic optimisation identified buffer composition and pH as key parameters, enabling broad substrate tolerance across all three reaction partners. A streamlined preparation workflow further makes a practical subset of nitroolefins accessible as DEL building blocks. Mechanistic experiments are consistent with a hydrazone-controlled [3 + 2] cycloaddition pathway under on-DNA conditions. Multistep DEL synthesis and chemical-space analysis show that this chemistry enables access to drug-like pyrazole-centred libraries that complement regions of ChEMBL chemical space with relatively low compound density, including areas that are not efficiently populated by a corresponding amide-based DEL design. More broadly, this study illustrates how reaction design, combined with scaffold-focused chemical-space analysis, can guide the introduction of underrepresented heterocyclic motifs into DEL discovery collections.

Gaonan Wang, Xiaona Zhang, Zhuo Wen, Xiaodong Shi, Weiren Cui, Alexander L. Satz, Wenji Su, Qi Zhang

Organic Letters

DOI: 10.1021/acs.orglett.6c03765

Abstract

The Doebner reaction is a classical method for the synthesis of quinoline-4-carboxylic acids via the condensation of anilines, aldehydes, and pyruvic acid. However, its application in DNA-encoded library (DEL) technology has been limited due to harsh reaction conditions that degrade the DNA. Herein, we report a DNA-compatible Doebner reaction that proceeds under mild and catalyst-free conditions. This methodology enables the efficient construction of desirable quinoline scaffolds on-DNA with a broad substrate scope for electron-rich and electron-neutral aromatic amines, although electron-deficient aromatic amines generally afford lower conversions. The qPCR and NGS results demonstrate that the reaction conditions preserve DNA amplifiability and sequence fidelity.

Christodoulos Mitrogiannis, Sebastian Moes, Ottilia Andersson, S. Pauliina Turunen, Christian Sköld, Anders Karlén, Johan Wannberg, Oscar Verho

ACS Medicinal Chemistry Letters

DOI: 10.1021/acsmedchemlett.6c00385

Abstract

DNA-encoded chemical library technology offers a cost-effective method for screening billions of compounds in a single experiment. However, achieving broad coverage of lead-like chemical space remains challenging. Here, we describe the design and synthesis of a three-cycle, peptide-like DEL comprising 403,200 compounds, including an in silico-guided strategy for building block additions. A chemical space model based on 4,774 commercially available amino acid building blocks was used to identify amino acids underrepresented in the original design, in order to expand the library’s physicochemical diversity. Following split-and-pool synthesis, affinity selections against three targets were performed to assess library performance. Selections against carbonic anhydrase II successfully identified a known sulfonamide-containing pharmacophore. These results demonstrate that a computationally optimized, medium-sized DEL can effectively explore lead-like chemical space and identify specific binding motifs for purified protein targets.