In eukaryotic cells, the precise spatial localization of RNAs and proteins is essential for proper cellular function. Genetically encoded photocatalytic proximity labeling techniques have expanded our ability to map subcellular proteomes and transcriptomes, but their temporal resolution remains limited. Here we introduce Lantern, an engineered flavoprotein optimized via directed evolution, which enables sub‑minute, spatially resolved labeling of cellular biomolecules. Lantern is targetable to diverse subcellular compartments, including the endoplasmic reticulum (ER), mitochondria and stress granules (SGs), to map local transcriptomes (CAP-seq) and proteomes (CAP-MS). Using Lantern, we observed that N6-methyladenosine-rich RNAs are recruited to SGs within 10 minutes of stress induction, and ER‑proximal RNAs associate with G3BP1 during early SG assembly. Additionally, Lantern was adapted for cell surface tagging (CAP-CELL), enabling spatially resolved cell typing and identifying cell−cell interactions. Collectively, this study establishes Lantern as a powerful tool that offers unprecedented temporal resolution for investigating the dynamic organization of subcellular molecular networks. Fang, Ren, Zheng, Wang et al. developed Lantern, a variant of miniSOG, through directed evolution for subcellular transcriptomes and proteomes analyses inside living cells, enabling findings that RNA mobilization precedes protein recruitment in stress granule assembly.
By evolving a flavoprotein in mammalian cells, a super-fast, genetically encoded photocatalyst called Lantern was developed, allowing the mapping of dynamic RNA and protein organization at high spatiotemporal resolution during stress.
Pretomanid and delamanid are prodrugs, whose active derivatives have been reported to target decaprenylphosphoribose-2'-reductase, DprE2, while quabodepistat is a noncovalent inhibitor of decaprenylphosphoribose-2'-oxidase, DprE1. Both enzymes are involved in Mycobacterium tuberculosis cell wall synthesis, but the mechanism of the DprE1-DprE2 epimerase complex and its inhibition by these compounds remain unclear. We report cryo-EM structures of the M. tuberculosis DprE1-DprE2 complex bound with either substrate or quabodepistat in DprE1, and with either activated pretomanid or delamanid in DprE2, respectively. DprE1-DprE2 assembles as a membrane-associated tetramer of a DprE2 dimer flanked on each side by a DprE1 subunit. Both pretomanid and delamanid bind to DprE2 in an NADH-adduct form and within a conserved binding pocket that extends from the NADH-binding site to the substrate-binding site. Quabodepistat binds to DprE1 with a unique mode. Our data reveal the mode of action of these drugs, allowing rational design of new derivatives for improved tuberculosis treatments.
Cell-surface sialoglycans overexpressed on cancer cells suppress activation of tumor-infiltrating immune cells (TIICs) and thus represent promising targets for cancer immunotherapy. However, the functional roles of sialylation in TIICs remain incompletely understood. Here we developed a strategy for single-cell correlative analysis of α2,3- and α2,6-linked sialoglycans and transcriptomes to determine the correlations of cell-surface sialylation with gene expression of the same cells in a linkage-specific manner by single-cell RNA sequencing. We found that α2,3- and α2,6-sialoglycans were distinctively regulated across specific TIIC subpopulations in murine tumors. Notably, downregulation of α2,6-sialoglycans was identified as a cell-surface marker for intratumoral tumor-specific antigen-reactive CD8+ T cells with high avidity. Mechanistically, high-avidity (but not low-avidity) activation of murine CD8+ T cells within the tumor suppressed β-galactoside α2,6-sialyltransferase 1 expression, which downregulates α2,6-sialylation and exposes galectin-1 ligands. Functionally, galectin-1 binding promoted T cell apoptosis and immune evasion in cancer. This work demonstrates the functional significance and therapeutic potential of specific sialoglycans on TIICs.
The approvals of four CDK4/6 (cyclin-dependent kinases 4 and 6) inhibitors by the US Food and Drug Administration-palbociclib, abemaciclib, ribociclib and trilaciclib-as anticancer or supportive therapies establish the critical role of CDKs in regulating the cell cycle and cancer progression. However, the emergence of drug-resistant cancers underscores the adaptability and plasticity of this cellular pathway, highlighting the need for additional therapeutic strategies. CDK2, the key kinase activated downstream of CDK4/6, has emerged as a prominent pharmaceutical target, with numerous selective inhibitors reported in recent years. This Perspective discusses the adaptive plasticity of cell-cycle control in cancer cells and then highlights current strategies to target newly emerging CDK2 druggable pockets-including orthosteric, substrate-binding and allosteric sites-with an emphasis on chemical biology approaches, aiming to inspire the next generation of CDK2 inhibitors and kinase-targeted therapies more broadly.
No therapies directly block apoptosis in tissue injury or the many diseases driven by cell loss. The BCL-2 family protein BAX is a central mediator of this pathway and C126 resides within a key regulatory region where physiologic or pharmacologic ligands can activate or inhibit its function. Here, we report enantioselective covalent BAX inhibitors that site-specifically react with C126 and confer cytoprotection across multiple cell types. These ligands constrain BAX conformation and suppress apoptosis in a strictly BAX-dependent manner. Medicinal chemistry optimization yielded covalent BAX inhibitor 3 (CBI-3), an analog with pharmacokinetics suitable for in vivo studies. In a murine model of Fas-induced fulminant hepatic failure, CBI-3 reduced hepatocyte apoptosis and preserved liver histology and survival. CBI-3 also conferred cytoprotection of motor neurons derived from human induced pluripotent stem cells of healthy and amyotrophic lateral sclerosis donors. These findings establish covalent BAX inhibition as a therapeutic strategy to directly block pathologic cell death.
BAX is a pro-death pore-forming BCL-2 family protein essential for the commitment to apoptotic programmed cell death of mammalian cells under stress. A BAX-selective covalent inhibitor acts as a safety pin, blocking BAX-dependent mitochondrial outer membrane permeabilization and apoptosis.
The multicellular forms and functions seen in biology are controlled by cellular communication and collective computation. Reprogramming natural systems is difficult because they comprise many signaling molecules connecting a web of regulatory networks within cells. Here we apply principles from pass transistor logic (PTL) to design bacteria that can be easily reconfigured to perform computations on a solid surface. Strains of Pantoea agglomerans were built to encode two transistors (N-type and P-type) whose inputs and outputs are small molecules. They are connected by three relay strains that convert molecular diffusion to unidirectional flow. To build circuits, an acoustic liquid handler prints patterns of these five strains on a surface. By changing the pattern, not requiring any genetic changes, different operations are implemented, including multi-input multioutput logic, demultiplexor, half-adder and full-adder. This work demonstrates that only five cell types, each encoding a simple operation, can be scaled to create complex computational operations.
Transcription factors (TFs) are aspirational therapeutic targets, as their dysregulation drives altered cell states. Yet many disease-relevant TFs are disordered and lack canonical binding pockets, frustrating direct small-molecule inhibition. Indirectly targeting the effector molecules that modulate TF function is a promising, underexplored alternative. Here we report a strategy for capturing cancer-specific protein-protein interactions using context-dependent µMap photoproximity labeling. With an intein-based method for catalyst conjugation in biochemically intact nuclei, we capture unique c-Myc interactomes in healthy and cancerous prostate cells and mine them for druggable vulnerabilities. We identify STE20-like kinase (SLK), a cancer-specific interactor that stabilizes c-Myc, drives epithelial morphology and is essential for tumorigenesis. Mechanistically, SLK phosphorylates c-Myc at serine 329, antagonizing GSK3β-dependent phosphodegron phosphorylation. This interaction is associated with a splicing change promoting nuclear localization of the long SLK isoform. Patient data link this isoform to c-Myc target expression across tumor types; the interaction validates across diverse tissues.
KRAS(Q61) mutant oncoproteins drive cancer through defective GTP hydrolysis. A study now shows that small molecules can restore catalytic activity while simultaneously shifting KRAS toward its inactive state, identifying an unexpected strategy to target previously untreatable KRAS mutants.
The RAS family of oncogenes (KRAS, HRAS, NRAS) is among the most frequently mutated genes in human cancer. Therapeutic development has largely focused on inhibitors for KRAS codon 12 mutations, while mutant-selective inhibitors for Q61 variants remain elusive. A common mechanistic feature of G12 and Q61 mutants is the reduced efficiency of GTP hydrolysis, which enriches RAS in its active, signaling-competent state. Here we report small molecules that accelerate GTP hydrolysis in KRAS-Q61 mutants as an alternative therapeutic strategy. These compounds compensate for the loss of the catalytic residue Gln61 by introducing a general base into the active site, selectively enhancing hydrolysis of KRAS-Q61X (X = H, L, K, R) mutants by up to 20-fold. In mutant cancer cell lines, these compounds reduce GTP-bound RAS levels and suppress downstream signaling. This work establishes a mechanistic foundation for small-molecule 'GTPase activators' and offers a new paradigm for targeting RAS-driven cancers.
Loss of functional β cells is a hallmark of diabetes, and restoring β cell mass remains a critical goal in the quest for a specific therapy. One potential strategy is to convert non-β cells in the islet, such as α cells, into insulin-producing cells. Although several compounds have been identified to induce β cell-like features in α cells, none have been successfully translated into clinical applications. In this study, we identify PRC2 inhibitors as potent inducers of β cell-enriched gene expression in α cells, acting through modulation of the AR-ETV1 complex. AR inhibition suppresses glycogen synthesis and enhances the pentose phosphate pathway. Direct metabolic reprogramming with methyl esterified 6-phosphogluconate, an intermediate metabolite of the pentose phosphate pathway, induces β cell-like features in α cells, stimulates β cell regeneration and ameliorates diabetes. Our findings demonstrate that metabolic reprogramming drives β cell regeneration and highlight a promising therapeutic strategy for diabetes.
Glycolysis fuels vital cellular functions, and its dysregulation has been implicated in cancer, neurodegeneration, antibiotic resistance and diabetes. The glycolytic dependency of cancer, known as the Warburg effect, represents a key vulnerability for development of targeted anticancer agents; however, the development of such agents remains challenging owing to metabolic heterogeneity and resistance. Here we developed a covalent phosphofructokinase-1 liver type (PFKL) activator that couples glycolytic activation with delivery of a cytotoxic carnitine palmitoyltransferase 2 (CPT2)-targeting payload to cancer cells in vitro and in vivo. The electrophile-drug conjugate site-specifically and proteome-wide selectively modifies K677 in the allosteric effector site to stabilize the R-state tetramer of PFKL, while concomitantly releasing a CPT2-selective inhibitor to destabilize cell metabolism. The delivery mechanism of electrophile-drug conjugates is analogous to that of antibody-drug conjugates, but differentiated by their selective covalent targeting of intracellular proteins.
Selenium has a paradoxical role in biology, being essential at trace levels yet toxic at slightly higher doses. To operate within this narrow range, organisms have developed specialized pathways for selenium uptake, transport and usage/storage, primarily through selenoprotein biosynthesis. This Review summarizes current understanding of selenium handling and selenoprotein production, and discusses how selenium metabolism shapes susceptibility to ferroptotic cell death. We highlight both the role of selenoproteins in cellular defense and the unexpected roles of small-molecule metabolites, such as hydrogen selenide. We further discuss recent progress in analytical and chemical approaches, including mass spectrometry, activity-based probes and synthetic selenium donors, which are beginning to enable direct interrogation of these transient species. Together, these developments position selenium metabolism as a dynamic, chemically tractable regulator of redox biology and a promising therapeutic target.
Antibiotic resistance is a global health threat, driving the need for new molecules that kill bacteria via nontraditional mechanisms. Here, we present a computational de novo design strategy for α-helical peptides that self-assemble into large, stable and membrane-spanning nanopores with antimicrobial activity, including in vivo efficacy against drug-resistant pathogens. Molecular dynamics simulations guided the selection of sequences for transmembrane barrel-stave pore formation, which were validated by microscopy, electrophysiology and fluorescence assays. Using computational and experimental analyses, including negative design controls, we developed general design guidelines and 52 modular sequence templates with tunable antimicrobial, pore-forming properties. Mechanistic studies confirmed bacterial cytoplasmic membrane disruption via designed nanopore formation. A tuned lead peptide selectively killed drug-resistant ESKAPEE bacteria, including Acinetobacter baumannii, without harming human cells, and showed anti-infective efficacy in preclinical mouse infection models. The framework presented here enables the design of synthetic peptide nanopores for precision antimicrobials, anticancer agents, molecular sensors and delivery systems.
Fungal polyketides are a rich source of bioactive molecules. Their biosynthesis is often activated by environmental conditions that are hard to reproduce under laboratory conditions. Heterologous expression bypasses native regulation, enabling systematic polyketide discovery. The most widely used fungal hosts are the highly tractable Saccharomycotina yeasts with a narrow product scope and the metabolically robust but less tractable filamentous Eurotiomycetes. Here we established yeasts of the genera Exophiala and Knufia as hosts for polyketide production. These hosts are genetically tractable and robust, allowing us to heterologously produce six fungal polyketides with different domain architectures: 6-methylsalicylic acid, YWA1, monocillin II, farinosone B and monacolin J. Our findings demonstrate that these yeast hosts can efficiently produce complex polyketides allowing systematic polyketide synthase expression.
Fungal polyketides represent a treasure trove of industrially relevant compounds, yet sustainable high-level production can be challenging. Black yeasts have emerged as promising heterologous hosts for commandeering these complex biosynthetic pathways.
