Franz L, Rubil T, Balázs A
… +5 more, Overtus M, Kemnitz-Hassanin K, Govaerts C, Mall MA, Hackenberger CPR
Nat Chem Biol
· 2026 Jul · PMID 41998105
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Nanobodies are emerging as attractive biopharmaceuticals due to their small size, stability and target specificity. However, their therapeutic use has largely been restricted to extracellular targets because of a lack of...Nanobodies are emerging as attractive biopharmaceuticals due to their small size, stability and target specificity. However, their therapeutic use has largely been restricted to extracellular targets because of a lack of efficient delivery methods. This limitation is particularly relevant for diseases caused by dysfunctional intracellular proteins, such as cystic fibrosis. Here we show that cell-permeable nanobodies can modulate an intracellular disease-relevant target: the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel carrying the common F508del mutation. By combining a CFTR-binding nanobody with cell-penetrating peptides, we achieved intracellular delivery in cystic fibrosis bronchial epithelial cells. The delivered nanobody stabilizes misfolded F508del-CFTR, promotes its maturation and trafficking to the apical membrane and restores chloride channel activity. Moreover, the cell-permeable nanobody enhances the efficacy of approved CFTR modulator drug combination in primary airway epithelial cultures from patients with cystic fibrosis. These findings establish cell-permeable nanobodies as promising biopharmaceuticals for intracellular protein targeting and therapeutic modulation.
Calheiros de Carvalho A, Hurtado-Lopez N, Cano-Prieto C
… +20 more, von Bargen M, Damas-Ramos LC, Undabarrena A, Rago D, Chen L, Gadar Lopez AE, Jayachandran S, Trejo Alarcon LM, Li X, Arsovska D, Ahonen L, Kandasamy V, Sondt-Marcussenv L, Arango Saavedra M, Karyofyllis I, Exley KP, de Bekker C, Brogaard JS, Keasling JD, Cruz-Morales P
Pests cause up to 40% of global crops losses. Pesticide overuse drives resistance and poses notable risks to public health and the environment. Many hypocrealean fungi form symbiotic relationships with plants while antag...Pests cause up to 40% of global crops losses. Pesticide overuse drives resistance and poses notable risks to public health and the environment. Many hypocrealean fungi form symbiotic relationships with plants while antagonizing pests, making them valuable sources of biocontrol agents and biopesticides. However, little is known about their biosynthetic capabilities. Here we use phylogenomics, metabolomics and heterologous expression to catalog the biosynthetic repertoire of 82 plant-associated and insect-associated Hypocreales species. Annotation of 5,221 biosynthetic gene clusters reveals that ~80% of them encode unknown products. By linking biosynthetic gene clusters to molecules, we investigate the biosynthesis of several natural products, including pyridones, dethiosecoemestrin and efrapeptin. Additionally, by combining our metabologenomics workflow with synthetic biology, we characterize four nonribosomal peptide synthetase-like synthetases involved in the biosynthesis of hitherto unknown products. We believe that this work lays the groundwork for future efforts toward sustainable pest control in agriculture.
Tubulin-targeting agents such as paclitaxel have been a cornerstone of cancer treatment. However, the molecular basis by which prognosis-associated tubulin isotypes and mutations (that is, variants) affect drug efficacy...Tubulin-targeting agents such as paclitaxel have been a cornerstone of cancer treatment. However, the molecular basis by which prognosis-associated tubulin isotypes and mutations (that is, variants) affect drug efficacy remains unclear. Here we reveal that evolutionarily conserved tubulin residues modulate the allosteric network to determine paclitaxel efficacy. The paclitaxel resistance of human β3-tubulin depends on a residue distant from the taxane-binding pocket. The ~2.3 Å-resolution cryo-EM microtubule reconstructions demonstrate that the paclitaxel-sensitizing tubulin mutation induces allostery at the paclitaxel-binding site, intertubulin interactions and nucleotide-binding pockets. In particular, the reoriented guanine triphosphate (GTP)-hydrolyzing catalytic α-tubulin E254 residue enhances the GTP cap, reducing the catastrophe frequency of dynamic microtubules. Examining genome-edited cancer cells with the paclitaxel-sensitized mutant β3-tubulin indicates that the affinities of tubulin variants for paclitaxel determine drug efficacy. Our findings provide mechanistic insights into the development of new tubulin-targeting therapeutics not only for cancer but also for tubulinopathies associated with mutations in specific tubulin isotypes.
Small-molecule probes are transformative for cell biology, offering unprecedented insights into subcellular structures, including in systems without molecular genetic tools. Centrioles are fundamental for generating the...Small-molecule probes are transformative for cell biology, offering unprecedented insights into subcellular structures, including in systems without molecular genetic tools. Centrioles are fundamental for generating the axoneme of cilia and flagella, as well as centrosomes, but a generic small-molecule probe allowing selective visualization of centriolar and axonemal microtubules is lacking. We engineered CenSpark as a cell-permeable dual-ligand fluorescent probe exploiting the juxtaposition of inner and outer microtubule-binding sites of microtubule triplets and doublets present exclusively in centriolar and axonemal microtubules. This design endows CenSpark high selectivity in live and fixed specimen analysis of centrioles, cilia and flagella across systems. We deployed CenSpark to uncover the rate of primary cilium formation and track centrioles in chimeric antigen receptor T cells during polarization at the immunological synapse with unprecedented resolution. Overall, CenSpark is a novel versatile small-molecule fluorescent probe to monitor centrioles, cilia and flagella without the need for genetic manipulation.
Group I introns are catalytic RNAs capable of self-splicing and generating circular RNAs, processes central to RNA metabolism and biotechnology. Yet, full-length ribozyme structures containing entire exon sequences and t...Group I introns are catalytic RNAs capable of self-splicing and generating circular RNAs, processes central to RNA metabolism and biotechnology. Yet, full-length ribozyme structures containing entire exon sequences and the structural basis of postsplicing circularization have remained limited. Using cryo-electron microscopy, we resolved multiple conformational states of the full-length Anabaena tRNA(Leu) precursor, capturing key intermediates of splicing and cyclization. In the apo state, the exons preassemble into a mature tRNA-like conformation that promotes P1 helix formation. Transitions through the splicing states involve substantial rearrangements essential for catalysis. Unlike other group I introns, the Anabaena intron circularizes without sequence loss, using its guanosine-binding site as the catalytic center. Mutational analyses confirm that G37 reorientation and a conserved wobble receptor motif precisely position the circularization site, driving efficient cyclization even in engineered PIE systems. These findings uncover unique mechanisms of RNA catalysis and establish structure-based optimization for advancing RNA circularization technologies.
Biological sulfation reactions require 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfate donor. While the biosynthetic pathway of PAPS has been well characterized, the phosphatase degrading PAPS remai...Biological sulfation reactions require 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfate donor. While the biosynthetic pathway of PAPS has been well characterized, the phosphatase degrading PAPS remains unidentified. Here, we discover MESH1 as a PAPS phosphatase that hydrolyzes PAPS into adenosine-5'-phosphosulfate and phosphate. Our crystallographic analysis of the MESH1-PAPS complex confirms PAPS as a bona fide substrate of MESH1. We further show that MESH1 localizes to Golgi, where sulfotransferases consume PAPS to produce sulfated glycosaminoglycan (sGAG). We show that MESH1 (also known as HDDC3) knockdown enhances sGAG production in a chondrogenic cell line. Furthermore, in brachymorphic mice, Mesh1 knockout significantly elevates sGAG levels in joint cartilage and improves bone density. In Caenorhabditis elegans lacking bpnt-1, neurotoxic PAP accumulation is alleviated by MESH1 overexpression, reducing upstream PAPS levels. Our biochemical, structural and functional findings establish MESH1 as a key PAPS phosphatase and highlights its potential as a therapeutic target in disorders characterized by sulfation deficiency.
Chronic itch, particularly in cholestatic and uremic conditions, poses a notable clinical burden, yet treatment options remain inadequate. MRGPRX4 (hX4), a bile-acid-sensing G-protein-coupled receptor predominantly expre...Chronic itch, particularly in cholestatic and uremic conditions, poses a notable clinical burden, yet treatment options remain inadequate. MRGPRX4 (hX4), a bile-acid-sensing G-protein-coupled receptor predominantly expressed in human sensory neurons, has emerged as a critical mediator of cholestatic pruritus. Here we identified and characterized HEP-50768, a potent and selective small-molecule inverse agonist of hX4 through high-throughput screening and structure-activity optimization. Structural elucidation through cryo-electron microscopy of the hX4-inverse agonist complex structure revealed the unique binding mode and inhibitory mechanism of HEP-50768. In hX4-humanized rats, HEP-50768 robustly suppressed bile-acid-induced pruritic behaviors. Comprehensive preclinical absorption, distribution, metabolism, excretion and safety profiling was performed in both rats and monkeys, and these findings establish HEP-50768 as a promising therapeutic candidate for chronic itch, supporting its advancement to clinical evaluation.
Molecular glues promote protein-protein interactions by enhancing the surface complementarity between proteins. Those that recruit an E3 ubiquitin ligase to a target can elicit ubiquitination and subsequent destruction o...Molecular glues promote protein-protein interactions by enhancing the surface complementarity between proteins. Those that recruit an E3 ubiquitin ligase to a target can elicit ubiquitination and subsequent destruction of the target protein-a mechanism that underpins the field of targeted protein degradation (TPD). Here we explored whether small-molecule binders to the CTLH E3 ligase subunit GID4 could act as molecular glues. We discovered that CLEO4-88 functions as a molecular glue (EC = 12.5 nM) to promote the interaction of GID4 with the peroxisomal thiolase ACAA1 in vitro and in cellulo. An atomic structure of the ternary complex revealed an allosteric mechanism whereby CLEO4-88 binds solely to GID4 and induces a conformational change conducive to binding ACAA1. Biochemical analysis demonstrated that, while ACAA1 cannot be recruited by GID4 to a CTLH holoenzyme for ubiquitination, ternary complex formation inhibits ACAA1 thiolase activity, thus demonstrating potential utility beyond TPD.
Endosomal function is essential for pattern recognition receptor signaling, through endosomal Toll-like receptor (TLR) sensing of nonself RNA and DNA. The specific interaction of the calcium sensor Munc13-4 with syntaxin...Endosomal function is essential for pattern recognition receptor signaling, through endosomal Toll-like receptor (TLR) sensing of nonself RNA and DNA. The specific interaction of the calcium sensor Munc13-4 with syntaxin 7 (STX7) regulates endosomal flux and Munc13-4 depletion decreases the systemic inflammatory response to unmethylated DNA. Using high-throughput screening and orthogonal cell-based validation, we identified small-molecule inhibitors of the Munc13-4-STX7 interaction, ENDOtollins (ENDOs). ENDOs inhibit extracellular signal-regulated kinase signaling in neutrophils and interferon (IFN) regulatory factor signaling in plasmacytoid dendritic cells (DCs) in response to endosomal TLR ligands but not to plasma membrane agonists, highlighting specificity for the endocytic pathway. Mechanistically, ENDOs inhibit endolysosomal flux and decrease endolysosomal cargo degradation. Chemical optimization identified ENDO12 as the most potent inhibitor. ENDO12 inhibited primary DC responses to TLR3, TLR7 and TLR9 and reduced CpG-induced systemic inflammation, manifested as decreased levels of the proinflammatory mediators myeloperoxidase, interleukin 6 and IFNγ. Our findings have significant implications for immunodeficiency, inflammation and innate immunity.
Small molecules that induce protein interactions hold tremendous potential as new medicines, probes for molecular pathways and tools for agriculture. Explosive growth of targeted protein degradation drug development has...Small molecules that induce protein interactions hold tremendous potential as new medicines, probes for molecular pathways and tools for agriculture. Explosive growth of targeted protein degradation drug development has spurred renewed interest in proximity-inducing molecules, especially molecular glue degraders (MGDs). These compounds catalyze the destruction of disease-causing proteins by reshaping protein surfaces and promoting cooperative binding between ubiquitylating enzymes and target proteins. MGD discovery for predefined targets is a major challenge in contemporary drug discovery. Here, we solve this important chemical challenge through 'chemocentric' MGD discovery of ZZ1, a BET-family protein degrader and a prodrug of a negatively charged glue. ZZ1 activation unmasks a sulfinic acid that binds the modular CTLH ubiquitin ligase complex through a basic pocket in its YPEL5 subunit. These findings demonstrate a previously unrecognized capacity of YPEL5 to recruit CTLH substrates and enable the discovery of MGDs for exceedingly common acidic and basic degrons.
Bacteriophages use diverse genomic modifications, including substitutions on the cytosine pyrimidine ring through C-C and C-N bonds. Here, we report that coliphage HY126 encodes an Afh system with enzymatic activities no...Bacteriophages use diverse genomic modifications, including substitutions on the cytosine pyrimidine ring through C-C and C-N bonds. Here, we report that coliphage HY126 encodes an Afh system with enzymatic activities not previously implicated in DNA modification. This multienzyme cascade comprises the following: AfhB, a reduced flavin-dependent hydroxylase that catalyzes deoxycytidine monophosphate (dCMP) hydroxylation through C-O bond formation to yield 5-hydroxy-dCMP; AfhE and AfhF, which synthesize the sugar donor uridine diphospho-ᴅ-arabinose for AfhC; AfhC, a phage-encoded nucleotide arabinosyltransferase that arabinofuranosylates 5-hydroxy-dCMP, priming it for DNA incorporation; AfhG, a DNA arabinosyltransferase that adds a second arabinofuranose moiety through a β-1,3 linkage to 5-hydroxyarabinofuranosylated cytosine embedded in DNA, forming diarabinofuranosyl-5-hydroxycytosine. This work unveils a unique two-step arabinofuranosylation mechanism-involving distinct nucleotide priming followed by DNA modification-thereby elucidating an enzymatic strategy in the phage-host evolutionary arms race.