Identification of a small molecule CAPON binder using affinity selection-mass spectrometry screening.

The discovery of bioactive small molecules is dominated by iterative design-make-purify-test cycles focused on specific protein targets. In contrast, phenotype-driven discovery can yield bioactive molecules with unexpected mechanisms of action, and open paths to first-in-class drugs. Here, we present a fully closed-loop, algorithm-driven workflow for phenotypic-driven molecular discovery. Initially, a large virtual reaction space is constructed from pairs of potential substrates and co-substrates. Batches of reactions are then algorithmically-designed and automatically executed, and the products screened in a phenotypic assay; based on observed hits, the algorithm then directs subsequent round(s) of discovery and optimisation until a user-defined end-point is reached. The approach was exemplified using Rh-catalysed annulations of hydoxamate esters with alkene/alkyne co-substrates, coupled with the cell painting assay, and enabled the discovery and structural evolution of a series of tubulin modulators. Because the workflow is agnostic to both the chemistry and the assay modality, the approach may be generalisable for the automated function-directed exploration of synthetically-accessible chemical space. The approach has the potential to accelerate the discovery of chemical probes and to unlock opportunities for drug discovery.

Disease-associated RNAs are increasingly recognized as promising therapeutic targets for small-molecule intervention. While DNA-encoded libraries (DELs) have long been established for protein ligand discovery, recent studies have demonstrated their feasibility for identifying RNA-binding small molecules. To further advance RNA-targeted ligand discovery, a diverse, solid-phase DEL enriched in privileged RNA-binding scaffolds was constructed and applied to identify ligands of r(G4C2)exp, a toxic RNA repeat expansion implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). DEL selection outcomes were analyzed through large-scale molecular docking integrated with physicochemical and structure-activity relationship (SAR) analyses. Correlations were observed between docking predictions and experimental enrichment trends, supporting lead identification. The lead compound was subsequently optimized based on rational design, resulting in analogues with enhanced binding affinity and bioactivity. These findings demonstrate that RNA ligand identification can be effectively achieved by combining DNA-encoded library technology with computational approaches for rational design and analysis and highlight a broadly adaptable platform for RNA-targeted small-molecule discovery.

Cellular Casitas B-lineage lymphoma (c-Cbl), a RING-type E3 ligase, regulates the degradation of diverse proteins, whose dysregulation is implicated in solid tumors and hematological malignancies. The conformational change of c-Cbl with substrate binding plays a critical role in the activation of c-Cbl, which facilitates the opening of the RING domain and exposes c-Cbl's ubiquitin-conjugating enzyme (E2) recognition sites to promote E2 binding and following ubiquitin transfer. However, the molecular mechanism of this conformational transition that is essential for c-Cbl-targeted drug discovery remains unclear. Here, by performing NEB (nudged elastic band) calculations, molecular dynamics (MD) simulations, and Markov state model (MSM), we revealed the molecular mechanism of c-Cbl transformation from autoinhibited to partially open conformation upon substrate binding at the molecular level and identified the key metastable states of c-Cbl during this process, which are beneficial for discovery and development of small molecules targeting c-Cbl.

DNA-encoded library (DEL) technology has emerged as a transformative platform for the discovery of bioactive small molecules against challenging therapeutic targets including G protein-coupled receptors (GPCRs). As a clinically pivotal class of membrane-bound targets, GPCRs pose inherent challenges in the discovery of novel ligands. This Microperspective highlights recent methodological advances (2015-2026) that enable DEL selections against GPCRs, thereby facilitating the identification of diverse ligand modalities, including agonists, antagonists, allosteric modulators, and biased ligands. Furthermore, we discuss current challenges and future directions in the application of DEL technology to GPCR drug discovery, with a specific emphasis on opportunities in receptor stabilization, selection strategy design, and computational method development.

Bcl-2-associated athanogene 3 (BAG3) is a multifunctional co-chaperone protein that regulates apoptosis, autophagy, and proteostasis through interactions with HSP70 and other partners. Overexpression of BAG3 contributes to tumor cell survival, metastasis, and chemotherapy resistance, making it an appealing but challenging anticancer target due to its intrinsic disorder and lack of structural data. Here, we report a fragment-based drug discovery (FBDD) approach to identify novel small molecules targeting human BAG3. A fragment library of 783 compounds was screened using a thermal shift assay (TSA) against recombinant BAG3 expressed in mammalian cells, followed by hit validation through ligand-observed NMR (WaterLOGSY). Eleven fragments stabilized the protein, and seven were confirmed as binders. Among them, a 6-chloro-2-oxindole fragment ( Fr1 ) exhibited the strongest interaction, with a dissociation constant (K D ) of 97.8 ± 11.1 μM. Structure–activity relationship (SAR) studies focused on maintaining the 6-chloro-2-oxindole core and optimizing substitutions at position 3, identified derivative 7 as a promising lead. Derivative 7 bound BAG3 with improved affinity (K D ≈ 22 μM), as confirmed by grating-coupled interferometry, and displaced Fr1 in competition NMR assays. This work demonstrates the feasibility of applying FBDD to intrinsically disordered and structurally unresolved proteins such as BAG3, providing a validated chemical starting point for the development of selective BAG3 inhibitors. These findings expand the druggability landscape of BAG3 and highlight fragment-based methodologies as powerful tools to explore protein–protein interaction targets previously considered intractable.

HECT-type ubiquitin ligases: Emerging principles in the era of full-length structures.

The blood–brain barrier (BBB) is crucial for neural homeostasis, tightly regulating molecular exchange between the circulation and brain. However, this selective protection also greatly limits drug delivery to the central nervous system, posing a major challenge for treating neurological disorders. Pharmacological strategies that transiently and safely increase BBB permeability could therefore transform brain drug delivery, yet systematic discovery of such modulators remains hampered by the limitations of current in vitro and in vivo approaches. Here we present FishNAP, a non-invasive, high-throughput zebrafish platform for real-time assessment of BBB permeability in vivo. FishNAP captures developmental changes in barrier function and detects dysfunction in genetic mutants. Using this platform, we screened 2,320 FDA-approved small molecules for compounds capable of opening an intact BBB and identified 11 that reproducibly increased permeability. Seven of these molecules allowed entry of a 1 kDa tracer into brain tissue, and five also permitted passage of a larger 10 kDa Dextran. Barrier integrity recovered within 24 hours for all seven compounds, indicating reversible modulation. Finally, testing three representative molecules (Calcitriol, Lovastatin, and Sunitinib) in adult mice revealed increased BBB permeability and reduced Claudin- 5 expression, demonstrating conserved mechanisms of BBB-regulation across vertebrates. FishNAP thus enables systematic discovery of BBB modulators with direct translational potential for brain drug delivery.

G protein-coupled receptors orchestrate numerous physiological processes and represent the largest class of drug targets, yet their intracellular regulators, the β-arrestins, remain largely underexplored. Despite their crucial roles in receptor desensitization, trafficking, and signaling, few modulators have been identified, with limited isoform selectivity. Therapeutic efforts have mainly focused on receptor-level biased ligands to indirectly influence arrestin pathways. However, advances in small-molecule discovery and peptide design are now expanding the feasibility of directly modulating β-arrestins using structurally tailored ligands, primarily as research tools and potential therapeutic leads. Along with the recent identification of disease-associated mutations and first-generation modulators, these developments create new opportunities for selective and mutation-specific targeting. In this review, we summarize β-arrestin biology and signaling, highlight recent discoveries of disease-associated mutations and β-arrestin modulators, and discuss emerging strategies for precision drug development of arrestin-targeting compounds, with a focus on peptides.

Discovery of potent bifunctional small molecules targeting DNA-PK and HDAC6 with desirable pharmacokinetic properties for acute myeloid leukemia treatment.

Neurolysin (Nln) is a peptidase recognized for its cerebroprotective function in acute ischemic stroke. This study aimed to identify small molecule activators of Nln as research tools to further explore the role of this enzyme in stroke and other neurological disorders. Building on our previous computational screen of ∼140,000 compounds from the National Cancer Institute Developmental Therapeutics Program database, we extended experimental testing to the top 100 candidates using an Nln enzymatic assay. A pyridine-piperazine derivative (Py-Pip) was identified as a hit molecule and was characterized in detail. Py-Pip concentration-dependently enhanced the hydrolysis of both synthetic and natural substrates (neurotensin, angiotensin I, and bradykinin) by rat Nln, and displayed comparable activating effects on human and mouse orthologs. Importantly, Py-Pip exhibited a favorable selectivity profile, showing no potentiation of homologous metallopeptidases or unrelated enzymes. Kinetic analysis revealed that Py-Pip increases the catalytic efficiency (Vmax/Km) of Nln via a nonessential activation mechanism, whereas competition assays with inhibitor dynorphin A(1-13) confirmed that Py-Pip acts at a distinct, nonoverlapping site. Direct binding was further validated by orthogonal biophysical techniques, including differential scanning fluorimetry, microscale thermophoresis, and biolayer interferometry, whereas circular dichroism spectroscopy indicated activator-induced secondary structural changes. These findings validate that Nln activity can be enhanced by small molecules and establish Py-Pip as a novel, nonpeptide scaffold for developing potent, "drug-like" activators to investigate Nln biology and therapeutic potential. SIGNIFICANCE STATEMENT: This study reports the discovery of a novel nonpeptide small molecule that selectively enhances the activity of neurolysin (Nln), a peptidase implicated in cerebroprotection. Unlike previous peptide-based activators, this molecule provides a stable, "drug-like" scaffold and a structural foundation for the development of potent Nln activators to probe Nln biology and therapeutic potential in ischemic stroke.

Leveraging artificial intelligence in tissue regenerative engineering via small-molecule libraries.

Protein-protein interactions governed by conformationally heterogeneous domains remain difficult to drug because ligand-competent states are often absent from single static structures. Here, we present AtlasNMR, a statistical framework that transforms multi-model NMR ensembles into screening-ready conformational hypotheses for small molecule discovery. Using the neuronal nitric oxide synthase (nNOS) PDZ domain that engages the adaptor protein CAPON (NOS1AP) as a model system, AtlasNMR identified two representative conformational states capturing the dominant and minor populations of the NMR ensemble. Ensemble-based virtual screening followed by consensus ranking yielded MC-3 , a small molecule modulator that disrupts the NOS1-NOS1AP interaction in live cells and directly engages the nNOS PDZ domain. MC-3 produced convergent neuroprotective effects in disease-relevant neuronal models by reducing amyloid-β-induced cytotoxicity, suppressing NMDA-driven nitrosative stress, and attenuating pathological tau phosphorylation, while exhibiting a balanced early lead-like ADME and safety profile. Together, this work establishes a generalizable strategy for exploiting NMR ensemble heterogeneity to enable small molecule discovery against dynamic protein-protein interfaces.

ABSTRACTPrion propagation, in which the cellular prion protein (PrPC) is conformationally converted into an infectious structure (PrPSc), is now well understood. However, the molecular mechanism responsible for the neurotoxicity of prions remains unclear. Synaptic loss is one of the earliest events in bothin vivoandin vitromodels of prion disease. We previously developed a neuronal cell culture model to analyze the mechanisms of prion-induced synaptic degeneration in a physiologically relevant setting. Using this system, we showed that exposure of hippocampal neurons to PrPScengages a NMDAR/p38 mitogen-activated protein kinase (MAPK) signaling pathway that results in rapid, PrPC-dependent loss of synaptic transmission and retraction of dendritic spines. To comprehensively identify the components of this synaptotoxic signaling pathway, we measured changes in the phosphoproteome and transcriptome of hippocampal neurons exposed to PrPScwhile they were undergoing the process of dendritic spine retraction. We then used these data as input into the L1000 and P100 databases of transcriptomic and proteomic drug signatures, leading to the discovery of 17 compounds that were able to prevent PrPSc-induced spine retraction. These compounds converged on three protein kinase targets: Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and glycogen synthase kinase 3β (GSK3β). Using immunocytochemical staining, we confirmed that PrPSctreatment of hippocampal neurons induced phosphorylation of the three kinases and caused their rapid translocation to dendritic spines. Along with N-methyl-D-aspartate receptors (NMDARs) on the neuronal surface, which trigger an initial influx of Ca2+in response to PrPSc, these kinases constitute key nodes in a signaling network that mediates prion synaptotoxicity. Taken together, our results provide new insights into the mechanisms of prion neurotoxicity, and they identify novel molecular targets and inhibitory compounds that can be utilized for therapy of prion diseases.AUTHOR SUMMARYThe mechanism by which prions propagate is now well established, but how they cause neurodegenerative changes is still uncertain. The earliest effects of prion infection occur at the level of the synapse, and we previously established an experimental system using cultured hippocampal neurons to assay prion synaptotoxicity. To search comprehensively for components of the synaptotoxic signaling pathway, we employed a novel, small-molecule discovery pipeline based on the transcriptomic and phosphoproteomic profiles of prion-treated neurons. This approach converged on inhibitors of three different protein kinases (Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and glycogen synthase kinase 3β), which, along with N-methyl-D-aspartate receptors, constitute key nodes in a prion synaptotoxic signaling network that can be targeted for therapeutic benefit.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have emerged as promising candidates for clinical applications in vascular disease therapy and in the in vitro modeling of vascular regeneration. However, the translational potential of hUC-MSCs requires direct differentiation into functional vascular lineage cells, particularly vascular endothelial cells (VECs) and endothelial progenitor cells (EPCs). A critical challenge is the lack of reliable sources that yield sufficient quantities of mature VECs/EPCs for therapeutic purposes. To address this limitation, we established an efficient protocol for generating VECs from hUC-MSCs. Preconditioning hUC-MSCs using small molecules with cytoprotective properties can enhance their potential for use in cell-based therapeutics. Through systematic screening, we identified CPP as a novel small chemical molecule that effectively induces the endothelial differentiation of hUC-MSCs. Remarkably, our CPP-based induction protocol achieved > 90% conversion to functionally competent VECs within 5 days, as evidenced by both in vitro assays and in vivo functional validation. Single-cell RNA sequencing (scRNA-seq) analysis further delineated the differentiation trajectory and confirmed the acquisition of endothelial-specific molecular signatures during lineage commitment. These findings establish CPP as a potent inducer of rapid endothelial differentiation, and provide mechanistic insights into stem cell fate determination.Graphical Take home figure CPP drives the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into functionally competent vascular endothelial cells (VECs). Crucially, the long non-coding RNA MEG3 acts as a pivotal regulator within this differentiation pathway.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00278-2.

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma. Fusion-positive (FP) RMS typically results in lower survival rates compared to fusion-negative RMS. FP-RMS is characterized by the presence of tumor-specific fusion oncogenes, most commonly PAX3::FOXO1 or PAX7::FOXO1, created by chromosomal translocations joining the DNA binding domains of PAX3 or PAX7 with the transcriptional activation domain of FOXO1. PAX3::FOXO1 plays a critical role in FP-RMS oncogenesis in both tumor initiation and maintenance. Thus, PAX3::FOXO1 represents an excellent target for therapeutic intervention in FP-RMS for which no new treatment option has been introduced in over 40 years. Following our discovery of small molecules that can directly bind to the fusion protein, we designed PAX3::FOXO1-targeting Proteolysis Targeting Chimeras (PROTACs) using the E3 ligase cereblon (CRBN) ligand via different linkers. This first generation of 15 PAX3::FOXO1-PROTACs demonstrated that endogenous PAX3::FOXO1 can be degraded in FP-RMS cell lines with protein degradation reaching over 50%. To further optimize our PROTACs, we identified SKP1 as the E3 ligase with the highest expression in both FP-RMS patient tumors and cell lines and as a dependency in FP-RMS cell lines which would limit emerging resistance towards SKP1-based PROTACs in contrast to CRBN-based PROTACs. The newly designed SKP1-based PAX3::FOXO1-PROTACs and their CRBN-based counterparts demonstrated on-mechanism PAX3::FOXO1 protein degradation via the proteasome as PAX3::FOXO1 protein levels could be rescued with proteasome inhibitor MG132 and NEDD8-activating enzyme inhibitor MLN4924. Moreover, we confirmed PROTACs-mediated ubiquitination of PAX3::FOXO1 protein. As expected with the loss of PAX3::FOXO1 function, we observe signs of myodifferentiation in FP-RMS cells shown in a spindle-like cell morphology and decreased expression of PAX3::FOXO1 target genes such as MYOD1 and B7-H3. Importantly, treatment of FP-RMS cells with PAX3::FOXO1-PROTACs resulted in impairment in anchorage-independent growth in soft agar which was not observed in non-FP-RMS cells. Taken together, we demonstrate proof of principle of PROTACs targeting the oncogenic fusion protein PAX3::FOXO1. Our designed PROTACs will not only be useful tools in studying PAX3::FOXO1 biology but could also serve as scaffolds for designing clinical-grade molecules to assess the therapeutic potential of PAX3::FOXO1-targeting PROTACs. Citation Format: Nikola Knoll, Kayra Somay, Foram Shah, Purushottam Tiwari, Isabel Frye, Jeffrey Toretsky, Aykut Üren. PAX3::FOXO1-targeting PROTACs in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr LT006.

Chitinase-3-like protein 1 (CHI3L1, also known as YKL-40) has emerged as a central effector of astrocyte-mediated neuroinflammation and a promising biomarker for Alzheimer's disease (AD). However, small molecule CHI3L1 inhibitors that modulate neuroinflammation are limited. Here, we report the discovery of a CHI3L1-targeted small molecule, DEL-C1, identified through DNA-encoded library (DEL) screening and validated using orthogonal biophysical, computational, and cellular approaches. DEL-C1 demonstrated direct CHI3L1 binding in microscale thermophoresis (MST) and surface plasmon resonance (SPR) assays, with reversible and concentration-dependent association. Molecular docking and 100 ns molecular dynamics simulations revealed a stable binding mode within the CHI3L1 substrate groove, anchored by Tyr206 and flanked by Trp99 and Trp352, supporting a thermodynamically favorable interaction. In vitro ADME profiling indicated a balanced physicochemical profile, permeability, and metabolic stability, consistent with CNS drug-like properties. Functionally, DEL-C1 reversed CHI3L1-induced astrocyte dysfunction by restoring Aβ uptake, lysosomal acidification, and proteolytic activity, while reducing CHI3L1 and IL-6 secretion. DEL-C1 also suppressed CHI3L1-driven NF-κB transcriptional activation, highlighting its anti-inflammatory potential. Collectively, this study establishes DEL-C1 as a promising small molecule modulator of CHI3L1 and a chemical tool to interrogate astrocyte-driven neuroinflammation in AD.

Chemical aggregates span multiple scales, from single molecules to nanoscale assemblies and macroscopic systems, exhibiting diverse functionalities towards biomedical purposes. The development of multiscale aggregation materials provides a powerful strategy to enable innovative solutions for treating diseases and performing bioanalysis. In this review, we summarize our recent progress in leveraging aggregation materials for antimicrobial therapy, bioanalysis, and drug delivery from a multiscale perspective. We highlight the discovery of small molecules, microfluidics-assembled or chemically synthesized nanoscale aggregation materials, and mesoscale biological aggregation interfaces for a broad range of biomedical applications. We also discuss current challenges in developing aggregation materials and explore directions for practical translations.

ΔFOSB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs), mediates long-term neuroadaptations underlying drug addiction, seizure-related cognitive decline, dyskinesias, and several other chronic conditions. AP-1 TFs are notoriously difficult to modulate pharmacologically because of the absence of well-defined binding pockets. Here, we identify a novel site on ΔFOSB, located outside the DNA-binding cleft, which accommodates small molecules. We show that sulfonic acid–containing compounds bind to this site via an induced-fit mechanism, reorienting side chains critical for DNA binding, and that they may hinder the ΔFOSB basic leucine zipper (bZIP) α-helix from binding to the major groove of DNA. In vivo, direct administration of one such compound, JPC0661, into the brain reduces ΔFOSB occupancy at genomic AP-1 consensus sites by approximately 60% as determined by CUT&RUN sequencing. These findings suggest that DNA binding and release by AP-1 TFs can be controlled via small molecules that dock into a novel site that falls outside the DNA-binding cleft. Minimal sequence conservation across 29 bZIP domain–containing TFs in this druggable groove suggests that it can be exploited to develop AP-1 subunit–selective compounds. Our studies thus reveal a novel strategy to design small-molecule inhibitors of ΔFOSB and other members of the bZIP TF family.

The prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties remains a central bottleneck in small-molecule discovery. We present the third-place solution from the PolarisHub Antiviral Competition, covering five end points broadly relevant to small-molecule design: human and mouse liver microsomal stability (HLM, MLM), MDR1-MDCKII permeability, kinetic solubility, and lipophilicity (LogD). Rather than pursuing complex machine learning architectures, we adopted a descriptor-first strategy. We systematically curated descriptors and models from ADMET Predictor as meta-features and then applied high-capacity tabular learners. A pretrained foundation model for tabular data (TabPFN), used in single-task regression, consistently outperformed or matched a strong gradient boosting baseline (CatBoost), yielding up to 44% mean absolute error (MAE) reduction across end points while simplifying deployment by eliminating an extensive hyperparameter search and producing compact models. Additionally, we engineered two feature sets that delivered modest gains in randomized cross-validation runs: (i) tuned fragment representations and (ii) site-of-metabolism pattern features. Overall, we used four groups of features: mechanistic, physicochemical, fragment, and metabolic. These results indicate that in practical ADMET modeling scenarios, where rich, validated descriptors are available, the competitive advantages often arise from principled feature engineering combined with robust, rather than overly complex, modeling approaches.