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.

Powered by dramatic advances in computer hardware, the advent of ultralarge make-on-demand virtual libraries, and a shift in small-molecule discovery toward more challenging targets with limited known actives, there has been a growing interest in the development of performant virtual screening methods that can reliably deliver novel hits. We report on a new method called Glide WS, that builds on our earlier efforts (WScore) to introduce an explicit representation of water structure and dynamics to an empirical scoring function suitable for high-throughput docking. This scoring function has been carefully tuned using absolute binding free energy perturbation calculations (ABFEP). Compared with Glide SP, Glide WS offers significant gains in the two primary tasks for molecular docking in drug discovery, pose prediction and virtual screening enrichment. For docking accuracy, Glide WS achieves a self-docking accuracy of 92% on a diverse set of 1477 protein ligand complexes as compared to 85% for Glide SP, using a criterion of 2.5 Å. We also demonstrate significantly improved virtual screening enrichment using a diverse data set covering of 38 targets together with three different computationally generated libraries of decoys, combined with standard known ChEMBL actives. We focus on ligands ranked in the top few percent of the database (the subset that is relevant to practical virtual screening efforts) and demonstrate that, along with improved enrichment of ChEMBL actives, Glide WS achieves a remarkable reduction in the number of poorly scoring decoys (as calibrated by ABFEP calculations), across a high percentage of targets, as compared to Glide SP. These results suggest that considerably higher hit rates will be observed, as compared to conventional rigid receptor docking, in practical virtual screening applications.

Refractory hepatocellular carcinoma (HCC) perpetuates metastasis or recurrence through anti-cancer drug resistance, necessitating more effective and reliable therapeutic strategies. We propose a new therapeutic approach involving the discovery of novel small molecules through target identification and validation in a patient-derived metastatic HCC model. We showed that calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α)-mediated enhancement of sarco/endoplasmic reticulum (ER) calcium ATPase 1 (SERCA1) expression level was pivotal events under anti-cancer drug treated conditions in patient-derived metastatic HCC cells. Increased SERCA1 was regulates to overloaded free calcium. SERCA is widely recognized as a key regulator of cytosolic free calcium under severe ER stress conditions. Though a cardiac dysfunction was unavoidable in vivo because of non-specific inhibition of SERCA isoforms by standard SERCA inhibitors. Based on the molecular structure of SERCA1, we discovered and synthesized two SERCA1-specific inhibitors, candidate 56 and 62. These compounds significantly reduced tumor size in the metastatic HCC xenograft tumor model without cardiac contractile dysfunction. This study first showed survival mechanism of patient-derived metastatic HCC cell, and propose a new therapeutic approach by the new small molecules, candidate 56 and 62, which are SERCA1 isoform-specific inhibitors without cardiac dysfunction by SERCA1 selectively inhibition.

BackgroundAlzheimer’s disease (AD) remains a devastating neurodegenerative disorder with complex pathophysiology, necessitating innovative therapeutic strategies. Among the emerging targets in AD research, INPP5D, a gene that encodes the SHIP1 phosphatase, stands out as a critical regulator of microglial function and a promising avenue for intervention. Microglia, the brain's resident immune cells, play a dual role in neuroprotection and neurodegeneration, contingent on various activation states. Recent findings by Oblak et al. have illuminated INPP5D’s central role in maintaining microglial homeostasis, positioning it as a key molecular regulator influencing the balance between beneficial and detrimental microglial responses.MethodSHIP1 exerts its effects as a negative regulator of intracellular signaling cascades, which in turn govern microglial processes such as phagocytosis, inflammatory cytokine release, and synaptic pruning. Dysregulation of these pathways has been linked to exacerbated neuroinflammation and impaired clearance of amyloid‐beta and other pathological substrates in AD. Leveraging insights from high‐throughput assays, advanced bioinformatics, and in vivo models, Oblak et al. have elucidated the mechanistic underpinnings of SHIP1’s influence on microglial activity and its downstream impact on neuronal health. These studies also underscore the potential of targeting SHIP1 to recalibrate microglial phenotypes, offering a novel therapeutic strategy for slowing or preventing AD progression.ResultIn this presentation, we will discuss how the integration of functional genomics, structural biology, and pharmacological screening within the TREAT‐AD consortium has advanced our understanding of INPP5D/SHIP1. Focus will be placed on the discovery and characterization of small molecules that modulate SHIP1 activity and siRNAs that downregulate INPP5D expression. These innovative tools not only validate the therapeutic potential of INPP5D/SHIP1 but also pave the way for combination therapies and precision medicine approaches tailored to individual patients.ConclusionThe session aims to catalyze collaboration by presenting INPP5D/SHIP1 research as a cornerstone of TREAT‐AD’s mission to accelerate the discovery and development of AD therapies.

High-throughput screening (HTS) remains a central pillar of small molecule discovery yet routinely fails for immune receptors and protein-protein interaction-driven targets. Here, we introduce HTS-Oracle, an experimentally validated AI system for prospective hit discovery that integrates molecular language modeling with cheminformatics to prioritize bioactive compounds at scale. We deploy HTS-Oracle across three clinically validated yet historically intractable immune targets, TREM2, CHI3L1, and CD28, representing cryptic binding pockets, intrinsically disordered proteins, and protein-protein interaction-driven immune checkpoint, respectively. Across the tested targets, HTS-Oracle reduces experimental screening requirements by up to >99% while increasing hit rates by up to 176-fold relative to traditional HTS. Notably, the platform remains predictive under extreme data sparsity, achieving an eightfold improvement for CD28 despite fewer than 2% actives in training. By consistently enriching for experimentally validated hits, HTS-Oracle establishes a new performance benchmark for hit discovery and unlocks small molecule access to immune targets long regarded as chemically inaccessible.

Targeted protein degradation (TPD) is a promising therapeutic strategy that requires the discovery of small molecules that induce the proximity between E3 ubiquitin ligases and proteins of interest. FBXO22 is an E3 ligase that is overexpressed in many cancers and implicated in tumorigenesis. While FBXO22 was previously identified as capable of recognizing ligands bearing a primary amine degron, further investigation and development of recruitment ligands are required to enable its broader utility for TPD. Here, we describe the discovery of chemical probes that can either selectively degrade FBXO22 or recruit this ligase for TPD applications. First, we described AHPC(Me)-C6-NH2 as a potent and selective FBXO22 degrader (DC50 = 77 nM, Dmax = 99%) that is suitable for interrogating the effects of FBXO22 loss of function. Further, we discovered that the simple hexane-1,6-diamine acts as a minimal FBXO22 self-degrader, whereas shorter C4 (putrescine) to C5 (cadaverine) analogs, found in mammalian cells, do not induce degradation. Finally, we found that 2-pyridinecarboxaldehyde (2-PCA) functions as a novel electrophilic degron capable of forming a reversible thioacetal with cysteine 326 for recruiting FBXO22. Conjugating 2-PCA to various ligands successfully induced the FBXO22-dependent degradation of BRD4 and CDK12. Collectively, these chemical probes will facilitate the study of FBXO22 biology and broaden its applicability in the TPD.