We have used small molecule chemical design and protein engineering to fashion an orthogonal ligand-channel pair that enables acute and selective inhibition of a genetically encoded voltage-gated sodium channel (NaV). A modified ligand based on the nerve toxin, saxitoxin (STX), binds potently and reversibly to a NaV bearing two unique amino acid mutations in the outer mouth of the channel (Site 1). The same ligand is ineffective at blocking wild-type NaVs and does not disrupt action potential signals in neuronal cells or brain tissue at working concentrations. The versatility of this tool derives from the highly conserved outer pore, as the same two amino acid replacements can be introduced in NaV1.1-1.4, 1.6, or 1.7, thus sensitizing each channel to inhibition by the designer STX ligand. This technology will empower studies of NaV-isoform physiology to better understand the role of NaVs in health and disease.
Chemically induced proximity has transformed targeted protein degradation but has been applied far less extensively to directly reprogram protein function through post-translational modification (PTM). Here, we develop an O-GlcNAcylation-targeting Chimera (OGTAC) that recruits O-GlcNAc transferase (OGT) to the oncogenic transcription factor c-Myc, enabling targeted O-GlcNAcylation in living cells without globally perturbing cellular O-GlcNAcylation. OGTAC suppresses HeLa cell proliferation, rewires c-Myc genomic occupancy, and reprograms expression of the downstream oncogene MALAT1 in an O-GlcNAcylation-dependent manner. By selectively modulating the regulatory state of c-Myc rather than its abundance, OGTAC establishes targeted O-GlcNAcylation as a chemically induced proximity strategy for functional rewiring of transcription factors. More broadly, this work expands proximity-induced protein regulation beyond degradation to programmable PTM.
Reactive cysteines serve important functions in proteins, and characterizing their engagement by different electrophiles facilitates biological discovery and covalent drug development. Here, we show that the common lysis buffer components phenylmethylsulfonyl fluoride (PMSF) and orthovanadate generate a lysis-derived oxidant that engages cysteines during cell lysis. This oxidant sulfonylates N-acetyl-D-glucosamine kinase (NAGK) C217, producing a mobility shift on SDS-PAGE. C217 lies within the ATP-binding pocket, and a C217S mutant exhibits reduced ATP affinity and enzymatic activity. Competitive iodoacetamide-alkyne activity-based protein profiling (IAA-ABPP) chemoproteomics further showed that the PMSF/orthovanadate oxidant defines a cysteine-engagement profile that partially differs from that of pervanadate. These findings reveal an unrecognized source of chemical reactivity during protein extraction that expands the toolkit for cysteine-engagement profiling and underscores how sample preparation chemistry shapes chemoproteomic measurements.
Protein degrader drugs such as PROTACs are being advanced as therapeutics targeted against oncogenic proteins. During tumorigenesis, oncogenic proteins can become constitutively activated via mechanisms including gene amplification, which increases protein production, and point mutations, which can extend protein half-life. Few experimental studies have addressed how disease-associated changes in target protein homeostasis influence PROTAC activity. We developed orthogonal methods to increase production or enhance stability of β-catenin, an important oncoprotein and target for degrader therapeutics, and used the dTAG system to evaluate the consequences for PROTAC activity. Stabilizing oncogenic missense mutations increase protein expression up to 5-fold but do not alter the PROTAC-imposed minimal steady-state level. In contrast, transcriptional upregulation increases both pre- and post-treatment target levels, revealing a synthesis-dependent ceiling on achievable depletion. Our results highlight distinct constraints on PROTAC activity arising from different mechanisms of oncogene activation, with potential implications for preclinical modeling, drug resistance and personalized medicine.
Renal ischemia-reperfusion injury (IRI), a leading cause of acute kidney injury, is driven by coordinated inflammatory signaling and ferroptotic cell death, yet effective therapies remain limited. Here, we show that H-151, a covalent stimulator of interferon genes (STING) inhibitor, also suppresses ferroptosis through a STING-independent mechanism. H-151 functions as a broad-spectrum radical-trapping antioxidant that directly scavenges radicals generated during the Fenton reaction, thereby blocking lipid peroxidation. In a murine renal IRI model, H-151 attenuated tissue damage and restored renal function through concurrent inhibition of STING signaling and ferroptosis. These findings establish radical-trapping antioxidant activity as an additional mechanism of H-151 and identify dual inhibition of inflammatory signaling and ferroptosis as a promising therapeutic strategy for IRI and related disorders.
Spatiotemporal control of neuronal activity has largely relied on genetically defined cell populations, limiting the ability to interrogate native biomolecules within intact circuits. Pharmacological approaches with high spatial precision are therefore needed to preserve physiological network architecture. Here, we demonstrate confined neuromodulation through two-photon molecular tattooing (sequential localized photocrosslinking). We introduce AL-701, an aryl azide derivatized photoreactive NMDA receptor antagonist that covalently binds receptors upon two-photon laser irradiation. In zebrafish larvae, molecular tattooing enabled spatially restricted inhibition of NMDA receptors in a hindbrain subregion regulating the acoustic startle reflex. This localized inhibition led to persistent suppression of NMDA receptor-dependent habituation, demonstrating that regionally restricted modulation of receptor function can alter behavior. Our results show the proof-of-concept for localized photocrosslinking within cell clusters in a vertebrate nervous system and thereby establish molecular tattooing as a robust chemical biology tool for circuit modulation and optically controlled reprogramming of behavior in vivo.
Neuroinflammation is a major secondary driver of Alzheimer's disease (AD). In this issue of Cell Chemical Biology, Carnevale et al.1 demonstrate that S-nitrosylation of the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway sustains pathological neuroinflammation in AD, identifying a promising therapeutic target for this devastating disease.
The ability to target oncogenic signals has transformed oncology. Targeted therapies typically inhibit oncogenic kinases and GTPases. Therapeutic augmentation of tumor-suppressive signaling could be a viable alternative but poses challenges. Specifically, designing compounds capable of stimulating kinase activity is more challenging than inhibitor design, and most kinases lack identified allosteric pockets that could be exploited for the development of allosteric activators. Inactivation of the tumor suppressor kinase liver kinase B1 (LKB1) is associated with poor prognosis and therapeutic resistance. Thus, augmented LKB1 function could be beneficial for cancer patients whose tumors retain intact copies of the gene. LKB1 signals as part of an obligate trimer including the scaffolding protein MO25 and the pseudokinase STE20-related kinase adapter protein (STRAD). As STRAD binds to but does not metabolize ATP, it provides a defined nucleotide binding pocket that may be targeted for an allosteric activation strategy. We have developed STRAD-binding compounds capable of activating LKB1 to augment tumor-suppressive signaling and reduce viability in cancer cell lines.
Lysine acylation has emerged as a rapidly expanding family of post-translational modifications that directly links cellular metabolism to protein regulation. Beyond lysine acetylation, advances in mass spectrometry and chemical biology have uncovered a diverse repertoire of acyl modifications spanning short-chain, branched, unsaturated, aromatic, and dicarboxylic groups. Together these modifications establish lysine acylation as a molecular interface through which metabolic state can shape protein function, chromatin regulation, and cell signaling. Yet fundamental questions remain regarding their biological significance, enzymatic regulation, site specificity, and whether many acylations function as bona fide regulatory signals or reflect metabolite-driven mechanisms. Here, we synthesize the current understanding of the metabolic origins, structural and biochemical properties, and their writer, reader, and eraser systems that govern lysine acylations. We further highlight emerging chemical biology approaches for detecting, manipulating, and functionally interrogating acyl marks and discuss the conceptual and technological advances needed to distinguish closely related modifications and establish their causal biological roles.
Deficiencies in glucocerebrosidase (GCase) are associated with Parkinson's disease (PD). The mechanistic basis of this association is being intensively investigated, and new treatments are being developed aiming to boost brain GCase activity in patients. Despite the importance of GCase in PD, studies are hampered by the continued reliance on weak and non-selective inhibitors like conduritol B epoxide. Here, we report easily prepared and selective irreversible inhibitors and complementary fluorescent activity-based probes (ABPs), along with straightforward methods to inhibit and/or monitor GCase activity. Robust characterization shows that compound CAz-5c selectively inactivates GCase in vitro, in live cells, and in vivo-including GCase in the brains of live mice, at low doses, overcoming a limitation of previously reported inhibitors. Collectively, these tools can be used to generate titratable chemical models of neuropathic GCase deficiency in animals with various genetic backgrounds with unrivaled molecular precision.
Therapeutic protein overexpression can overwhelm endoplasmic reticulum (ER) folding capacity, trigger unfolded protein response (UPR) signaling, and compromise the safety of gene and mRNA therapies. Here, we engineer stress-responsive RNA rheostats that couple transgene expression to endogenous ER stress sensing. Short RNA elements derived from X-box-binding protein 1 (XBP1) mRNA undergo inositol-requiring enzyme 1α (IRE1α)-dependent splicing under ER stress, inducing a frameshift that attenuates downstream protein expression. XBP1 switches function across DNA and mRNA delivery platforms and regulate the expression of fluorescent reporters, coagulation factor VIII, and Leronlimab, a therapeutic anti-CCR5 monoclonal antibody. Switch activation reduces ER stress markers while preserving expression under homeostatic conditions. We further demonstrate the regulation of Leronlimab expression in vivo using recombinant adeno-associated virus vectors. Together, these findings establish programmable RNA feedback control as a strategy for linking cellular proteostasis to therapeutic protein expression and improving the safety of gene and mRNA therapies.
Cytotoxic chemotherapy is intended to eliminate transformed cells but can also provoke therapy-induced senescence, a persistent and pro-inflammatory cell state that can promote tumor progression. The molecular mechanisms that govern the apoptosis-senescence fate decision remain incompletely understood. Here, we show that the mitochondrial pore-forming proteins BAX and BAK function as a critical checkpoint that restricts entry to therapy-induced senescence. Genetic ablation of BAX and BAK markedly enhanced entry to senescence in response to multiple DNA-damaging agents, whereas loss of the BAX/BAK antagonists Bcl-xL or Mcl-1 suppressed entry to senescence and promoted cell death. Mechanistically, genotoxic stress induced BH3-only proteins, including Noxa, Bid, and Puma, creating a dependence on Bcl-xL and Mcl-1 to restrain BAX/BAK activation and maintain survival. These findings identify the Bcl-2 family network as a central regulator of entry to therapy-induced senescence, a pro-inflammatory cell state that goes beyond the mere avoidance of apoptosis.
Mitochondrial tricarboxylic acid (TCA) cycle metabolites have emerged as critical regulators of immunity and inflammation beyond their canonical metabolic functions. During inflammatory responses, these metabolites accumulate to millimolar concentrations in immune cells and act as endogenous damage-associated molecular patterns (DAMPs), linking metabolic state to immune regulation through receptor-dependent and receptor-independent mechanisms. Here, we characterize the inflammatory roles of TCA cycle metabolites as immunometabolites in infections, inflammatory and autoimmune diseases, and cancers. We discuss how their anti-microbial functions must be balanced against their capacity to drive and sustain inflammation. To capture the pleiotropic functions of immunometabolites, we introduce the concept of metabolic DAMPs (metaDAMPs), a class of metabolically derived danger signals that orchestrate immune responses. We highlight key metaDAMPs, including itaconate, succinate, and fumarate, and emerging immunometabolites such as malate and oxaloacetate. Finally, we highlight technological advances redefining our understanding of metabolite signaling and consider how targeting immunometabolite signaling may enable therapeutic intervention.
Proper subcellular protein localization is fundamental for protein function, yet cancer cells often exploit this machinery for survival. While the nuclear import receptor KPNB1 (importin β1) is overexpressed in various cancers, its oncogenic mechanisms remain incompletely understood and its therapeutic targeting remains in infancy. Through screening of an FDA-approved compound library, we uncovered simeprevir (Sim) and lusutrombopag (Lus) as direct KPNB1 inhibitors that disrupt its import complex assembly. These compounds blocked KPNB1-mediated nuclear import of p65, thereby suppressing Bcl-2 transcription and activating mitochondrial apoptosis. Overexpression of KPNB1, p65, or Bcl-2, but not nuclear localization signal (NLS)-deleted p65, reversed the effect of Sim/Lus. Sim induced apoptosis in multiple cancer cell lines by downregulating Bcl-2 expression and suppressed A549 xenografts growth without observable toxicity. Analysis of The Cancer Genome Atlas (TCGA) datasets substantiated the clinical relevance of the KPNB1-p65-Bcl-2 axis. Our study elucidates a therapeutically targetable KPNB1-p65-Bcl-2 survival axis in multiple cancers and establishes a foundation for developing next-generation KPNB1 inhibitors.
Damage-associated molecular patterns (DAMPs) are key mediators of inflammatory disease, among which HMGB1 is a prototypical extracellular alarmin and an attractive therapeutic target. Here, we report ZH-1a, a high-affinity DNA aptamer (Kd = 2.1 nM) identified through SELEX and sequence optimization, that preferentially recognizes the proinflammatory B-box region of HMGB1. ZH-1a functions as an extracellular HMGB1-neutralizing aptamer and suppresses HMGB1-induced inflammatory signaling, including cytokine secretion and NF-κB activation in macrophages. In vivo, ZH-1a reduced late-phase systemic inflammation and multiorgan injury in LPS-induced endotoxemia, improved survival in polymicrobial sepsis, and attenuated inflammatory responses and organ damage in an HMGB1-challenge model. In addition, ZH-1a alleviated joint inflammation and structural damage in collagen-induced arthritis, and further enhanced the therapeutic efficacy of methotrexate. Together, these findings establish ZH-1a as a promising anti-inflammatory aptamer targeting HMGB1 and support aptamer-based neutralization of pathogenic extracellular HMGB1 as a therapeutic strategy for inflammatory disease.
In an interview with Samantha Nelson, a scientific editor of Cell Chemical Biology, the authors of the minireview entitled "Pyroptosis across species and its potential impact on host defense against zoonotic pathogens" share their perspectives on their field and life as scientists.
Regulated cell death pathways play fundamental roles in development, tissue homeostasis, immunity, and disease, and have emerged as promising targets for therapeutic intervention. In this Cell Chemical Biology special issue on cell death mechanisms, this Voices article brings together researchers from across the field to address the following questions: which recent advances in cell death research excite you most, what key challenges remain, and what are the next big questions in the field?
