Cell Chemical Biology

Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole-core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the {Omega}perX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 g mL1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.

The SARS-CoV-2 pandemic has underscored the urgent need for broad-spectrum antivirals in pandemic preparedness efforts. Nucleoside analogs targeting viral polymerases are often considered in this context. Here, we employ ensemble biochemical assays and single-molecule magnetic tweezers to characterize the detailed mechanism of action of the adenosine analog CMX521 (developed through Phase 1 clinical studies), a broad-spectrum antiviral against caliciviruses and coronaviruses, against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The triphosphate form of CMX521 is efficiently incorporated by RdRp, even against saturating ATP concentrations. Analog incorporation induces only a brief pause in nascent RNA synthesis. When embedded in the template strand, CMX521 causes the polymerase to stall ~9 s on average due to impaired uridine opposite incorporation. Multiple CMX521 residues in the template strand completely inhibit polymerase elongation. When the coronavirus polymerase is associated with the viral helicase, CMX521 strongly promotes copy-back RNA synthesis suggesting a second inhibitory mechanism for CMX521. Collectively, our findings establish a two-pronged mode of coronavirus polymerase inhibition by CMX521.

The cellular uptake and propagation of tau are central features of tauopathies, including Alzheimers disease, and are mediated by the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1). While prior studies have implicated LRP1 in tau binding and internalization, the biochemical features of this interaction and its suitability for therapeutic targeting remain incompletely defined. Here, we establish a quantitative and scalable framework to interrogate the tau-LRP1 interaction and identify small-molecule modulators. We engineered and purified the LRP1 ligand-binding domain 4 (BD4), a key region mediating tau interaction, and developed multiple orthogonal assays, including fluorescence polarization, split luciferase complementation, and time-resolved FRET, to measure LRP1-BD4 interactions with tau and a known peptide ligand. Across assay formats, we observe consistent binding affinities in the nanomolar range and demonstrate competitive displacement by tau, receptor-associated protein (RAP), and a peptide ligand, supporting overlapping binding interfaces. Leveraging these platforms, we performed small molecule high-throughput screening and identified a set of candidate inhibitors of the LRP1-BD4-tau interaction. Selected compounds reduced tau uptake in a cellular assay, phenocopying competitive inhibition by tau and a peptide ligand. Together, these studies define the LRP1-BD4-tau interaction as a biochemically tractable and druggable interface and establish an integrated discovery pipeline linking mechanistic characterization to functional cellular outcomes. This work provides a foundation for the development of therapeutic strategies targeting LRP1-mediated tau uptake.

Prion diseases are fatal neurodegenerative disorders driven by prion protein (PrP) misfolding, and lowering cellular PrP represents a promising therapeutic strategy. Here we report a small-molecule approach that reduces PrP by modulating pre-mRNA splicing of the PRNP gene. Through chemical modification of the clinically approved splicing modulator risdiplam, we generated CP3, the first class of compounds that selectively activate a cryptic exon in PRNP and routes its mRNA product for degradation, reducing PrP by ~70% in cells. We demonstrate that CP3 activity critically depends on alternative splicing factor Luc7L, revealing a novel requirement for alternative splicing factors in small molecule splicing modulation. Strikingly, co-administration of CP3 with a Luc7L activator PTC258 significantly lowers PrP levels in the brains of transgenic mice. These results establish splicing modulation as a powerful strategy for PrP reduction and highlight the potential of small-molecule cooperativity for therapeutic RNA targeting.

Despite the success of direct-acting antivirals, preventing hepatitis C virus (HCV) reinfection remains a critical global challenge. To address this, we leveraged deep learning-based de novo protein design to engineer mini-proteins targeting the large extracellular loop (LEL) of the HCV co-receptor CD81. These mini-proteins are predicted to precisely dock into CD81-LEL, occluding the critical binding interface required for the HCV E2 glycoprotein. Through biophysical screening, we identified mini-19, a lead candidate with sub-nanomolar affinity (KD = 0.5 nM) and high thermostability up to 95{degrees}C. Flow cytometry and super-resolution imaging confirmed that mini-19 specifically recognizes the native topology of CD81 on cells and extracellular vesicles. Functionally, mini-19 neutralized HCVcc infection (IC50 = 1.2 nM). Molecular dynamics simulations demonstrated that mini-19 acts as a structural clamp, restricting the fusogenic conformational plasticity of the receptor. By targeting a conserved host factor rather than the rapidly mutating viral envelope, mini-19 provides a high genetic barrier to resistance. Beyond offering a prophylactic strategy against HCV, our findings establish a stable biologic platform for targeting tetraspanin-enriched microdomains and modulating host-pathogen interactions.

Rifamycins inhibit bacterial transcription by targeting RNA polymerase (RNAP), but their clinical effectiveness is limited by the rapid emergence of resistance caused by mutations within the rifamycin-binding pocket. Rifamycin B (Rif B), one of the earliest discovered members of this antibiotic family and a precursor of clinically used derivatives, has remained poorly characterized because of its chemical instability and relatively weak antibacterial activity. Here, we revisit Rif B using biochemical and structural approaches. We show that Rif B remains sufficiently stable under assay conditions and retains inhibitory activity against RNAP variants carrying clinically relevant rifampicin-resistance mutations. We report the first crystal structure of Rif B and determine the structure of Rif B bound to bacterial RNAP. The structures reveal that the distinctive C-4 O-carboxymethyl substituent of Rif B forms an intramolecular interaction in the free molecule but establishes a salt bridge with fork loop 2 of the RNAP {beta}-subunit upon binding. This additional interaction explains the reduced sensitivity of Rif B to resistance-associated substitutions and identifies the C-4 position as an underexplored site for rational rifamycin modification. These findings redefine Rif B as a mechanistically distinct rifamycin scaffold and provide new insights for developing inhibitors targeting rifampicin-resistant RNAP.

DNA mismatch repair (MMR) deficiency has been widely utilized in forward genetic screens to identify drug resistance mutations and elucidate the mechanisms of action of cytotoxic small molecules. However, MMR deficiency generates a characteristic mutational signature enriched for C>T transitions that precludes saturation of mutagenesis. We hypothesized that expression of proofreading deficient DNA polymerase epsilon, as has been observed in patients with biallelic MMR deficiency, would enhance mutagenesis and enable identification of new resistance mutations and cytotoxin mechanisms. Here, we combine auxin-inducible degradation of Msh2 with doxycycline-inducible expression of the proofreading-deficient Pol{varepsilon}P286R mutant in murine small cell lung cancer (SCLC) cells. Simultaneous MMR deficiency and mutant Pol{varepsilon} expression markedly increased the emergence of bortezomib-resistant clones relative to either perturbation alone and targeted sequencing of Psmb5 in bortezomib-resistant clones identified recurrent mutations previously reported in chemical mutagenesis-based resistance screens. We next applied this platform to investigate resistance to lurbinectedin, a clinically relevant therapy for SCLC. Whole-exome sequencing revealed recurrent loss-of-function alterations in nucleotide excision repair (NER) genes, most notably Ercc5 and Ercc4, implicating NER deficiency as a major mechanism of lurbinectedin resistance and supporting a central role for NER in mediating lurbinectedin-induced cytotoxicity. Collectively, these findings establish inducible ultramutagenesis as a powerful and versatile platform for the unbiased discovery of drug resistance mechanisms and therapeutic vulnerabilities.

Glioblastoma remains a highly aggressive and therapy-resistant brain tumor, with limited benefit from the current standard-of-care regimen combining surgery, radiotherapy, and temozolomide. Overcoming chemoresistance therefore represents a critical unmet clinical need. Here, we investigate the anticancer potential of Succisa pratensis and its ability to enhance TMZ efficacy in GBM models. Treatment with S. pratensis markedly reduced cell proliferation and migration while significantly increasing sensitivity to TMZ. Integrated multi-omics analyses revealed extensive metabolic rewiring, characterized by suppression of central carbon metabolism and activation of stress-adaptive pathways. Mechanistically, we identify the Pregnane X Receptor, a key regulator of drug metabolism and chemoresistance, as a central node affected by treatment. Although S. pratensis increased PXR expression, this was not accompanied by induction of canonical downstream targets, including MDR1 and ALDH1A1, indicating a functional impairment of PXR transcriptional activity. Consistently, pharmacological inhibition of PXR using the antagonist SPA70 further potentiated the cytotoxic effects of S. pratensis and TMZ. Docking analyses suggest that specific secondary metabolites, including apigenin-derived compounds, may interact with the PXR ligand-binding domain, providing a potential molecular basis for this effect. Collectively, our findings indicate that S. pratensis enhances TMZ efficacy by inducing metabolic vulnerability and functionally impairing PXR signaling. These results highlight the therapeutic potential of plant-derived metabolites as adjuvant strategies to overcome chemoresistance in glioblastoma. Article HighlightsO_LISuccisa pratensis enhances temozolomide efficacy in glioblastoma by reducing proliferation, migration, and clonogenic growth. C_LIO_LIIntegrated proteomic and metabolomic analyses reveal extensive metabolic rewiring, with suppression of central carbon metabolism and induction of stress-adaptive pathways. C_LIO_LIPregnane X Receptor (PXR), a key regulator of chemoresistance, is functionally impaired despite increased expression, resulting in reduced activation of drug-resistance genes. C_LIO_LIPharmacological inhibition of PXR further potentiates the antitumor effects of Succisa pratensis and temozolomide, promoting apoptotic cell death. C_LIO_LIApigenin-derived metabolites show high affinity for the PXR ligand-binding domain and emerge as promising candidates to overcome temozolomide resistance in glioblastoma. C_LI

Protein kinases are critical regulators of cellular signaling, but precise modulation of their activity remains challenging due to their high structural conservation. Here, we present de novo designed genetically encoded miniproteins capable of activating or inhibiting focal adhesion kinase (FAK) by directly targeting the kinase domain itself. Among 96 binders designed to stabilize distinct conformational states of FAK, 33 modulated kinase activity. Biochemical characterization of the four most potent modulators revealed that two designs inhibit FAK with low-nanomolar IC50 values while the remaining two potentiated FAK activity by more than two-fold. When expressed in cells, the modulators preserved the same inhibitory and activating effects observed in vitro, establishing that designed conformational binders can directly tune FAK signaling in living cells. Taking advantage of the high similarity between kinases, we redesigned the FAK inhibitors to inhibit Src kinase. Our approach establishes a versatile platform for selective and genetically encoded kinase control as a way to rewire cell signaling and as a starting point for the discovery of novel modulatory sites of kinases.

Camptothecin derivatives are first-line anticancer drugs used worldwide for the treatment of diverse malignant tumors. However, the biosynthetic pathway of camptothecin has remained elusive for five decades. Here, we fully map its entire biosynthetic route. We discovered five key missing enzymes (OpCAR, OpSDR11, OpCS, OpGH1, and OpSTR) via the combination of MALDI mass spectrometry imaging, single-cell RNA sequencing and co-expression analysis. Meanwhile, we demonstrated a free flavin mononucleotide triggered the non-enzymatic 6-5-6 to 6-6-5 fused-ring skeleton rearrangement, filling the last gap in camptothecin biosynthesis. Finally, we validated this identified pathway and achieved the de novo biosynthesis of camptothecin in Saccharomyces cerevisiae. These discoveries uncover the long-standing mystery underlying camptothecin and pave the way for manufacturing camptothecin and its derivatives through synthetic biology approaches.

Scrub typhus is a potentially fatal infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. While antibiotic treatment is generally effective, it requires extended treatment, and drug resistance and treatment failures have emerged. O. tsutsugamushi encodes a deubiquitylating enzyme, OtDUB, which interferes with host ubiquitin-dependent pathways. OtDUB cleaves ubiquitin from various substrates, but whether this activity can be selectively targeted by small molecules is unknown. Here we have screened a chemically diverse small-molecule library using a fluorescence-based deubiquitylation assay to identify potential inhibitors of OtDUB. Two compounds, gentisic acid and amiloride hydrochloride, inhibited OtDUB activity at low dosage, with little effect on the related Wolbachia CidB or yeast Ulp1 enzymes. Computational docking predicted the compounds engage regions near the OtDUB catalytic pocket, suggesting a competitive mode of inhibition; this was supported by enzyme kinetic analyses. Neither compound caused detectable cytotoxicity in mammalian cells. Amiloride hydrochloride treatment reduced both total cellular deubiquitylating activity and the O. tsutsugamushi bacterial load in infected cells. While the identified compounds are not optimized inhibitors, they establish that bacterial pathogen-encoded deubiquitylating enzymes can be targeted by small molecules. Overall, our results provide a framework for using selective inhibitors as tools to study DUB function in genetically intractable intracellular bacteria and as potential treatments for scrub typhus.

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@982d1forg.highwire.dtl.DTLVardef@ba86f2org.highwire.dtl.DTLVardef@1f19f2borg.highwire.dtl.DTLVardef@8e807_HPS_FORMAT_FIGEXP M_FIG C_FIG

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the reading frame and abolish expression of functional dystrophin protein. Antisense oligonucleotides (ASO) can restore production of partially functional dystrophins by inducing exon skipping to restore the reading frame of dystrophin transcripts. While exon skipping is an FDA approved therapeutic strategy, there are currently no approved therapies for patients amenable to exon 44 skipping (8% of DMD patients). Here, we carried out a discovery campaign to identify phosphorothioate (PS) ASOs that efficiently induce exon 44 skipping and to define key sequence and chemistry features associated with activity. A tiling and micro-tiling approach with 18mer fully PS and 2-O-methoxyethyl (2MOE) modified ASOs in patient-derived myotubes identified five exonic target regions that promote skipping. ASO activity was strongly correlated across skeletal muscle and iPSC-derived cardiomyocytes, indicating similar exon 44 splicing regulation across cell types. Optimization studies showed that for 2MOE PS ASOs, 16-20mers were generally most active, while longer ASOs often had reduced activity due in part to impaired productive uptake into cells. Swapping out 2MOE modifications at both terminal positions for locked nucleic acids (LNAs) rarely improved activity and could also reduce it. Finally, promising candidates were tested in a humanized mouse model with an exon 44 skippable deletion, where one ASO outperformed others, inducing dose-dependent exon 44 skipping and dystrophin restoration in mouse models. These findings define practical design rules for exon 44-targeted ASOs and provide a foundation for therapeutic development.

Messenger RNA (mRNA) is canonically composed of ribonucleotides, with sporadic incorporation of deoxyribonucleotides into natural RNA transcripts being traditionally regarded as a rare, deleterious error arising from transcriptional infidelity. Here, we challenge this paradigm by demonstrating controlled partial substitution of ribonucleotides with deoxyribonucleotides during in vitro transcription (IVT) generates intact, stable and fully translationally competent IVT-mRNA. Unexpectedly, chimeric DNA-RNA backbone modification exhibits markedly enhanced IVT-mRNA translation several fold across multiple cell types and in vivo via diverse dosing routes relative to their ribonucleotide-based counterparts. 25% substitution of cytidine triphosphate with deoxycytidine triphosphate achieved best-performing translational output, surpassing the current gold-standard N1-methylpseudouridine (m1{Psi})-modified IVT-mRNA in a B16-OVA tumor vaccination model. These findings identify nucleotide class composition as a previously unrecognized parameter governing IVT-mRNA function and establish hybrid ribonucleotide-deoxyribonucleotide backbone engineering as a versatile strategy to expand the chemical space for next-generation mRNA therapeutics.

OptoGPCRs are light-activatable G protein-coupled receptors (GPCRs) used for optogenetic control of physiological processes. Most existing optoGPCRs are based on monostable opsins, which are limited by photobleaching and irreversibility. The bistable jumping spider rhodopsin 1 (JSR1) carrying the single point mutant S199F introduces a [~]150 nm spectral separation between the active and inactive states, enabling bidirectional control with distinct wavelengths of light. Here, we show that JSR1-S199F demonstrates robust, light-reversible arrestin recruitment and Gq/i protein activity. We then engineered JSR1-S199F-based optoGPCRs with Gs protein activity, expanding the limited repertoire of bistable Gs-coupled opsins. Specifically, we present optoDRD1, a chimeric optoGPCR that redirects the native Gq/i protein activity of JSR1 towards the Gs pathway of the dopamine D1 receptor (DRD1). Through systematic screening of intracellular domain combinations, we identified an optimal chimeric configuration comprising ICL2, ICL3, helix 8, and the C-terminus from DRD1. The resulting optoGPCR is activated by violet light ({lambda}max = 397 nm) and deactivated by green light ({lambda}max = 531 nm) at physiologically relevant light intensities. A single violet light pulse drives sustained Gs signalling for several hours, while green light deactivation enables precise signal termination at any timepoint. OptoDRD1 closely mimics wild-type DRD1 signalling kinetics and G protein selectivity. Compared to JellyOp, the only previously characterised natively Gs-coupled opsin, optoDRD1 shows higher signal amplitude and reversibility over multiple light cycles. We further demonstrate optoDRD1s utility for optogenetic control of Gs-regulated processes in vitro, including insulin secretion in human {beta}-cells and signalling modulation in a neuronal cell line, supporting its potential for in vivo applications. The biochemical stability and known structure of JSR1 make it a robust scaffold for this rational engineering and for future biophysical characterization. Together, optoDRD1 and JSR1-S199F expand the optoGPCR toolkit and open new opportunities for dissecting dopaminergic signalling, Gs-mediated physiology, and GPCR signalling pharmacology.

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer.

The cysteine to serine mutation at residue 481 of Bruton's tyrosine kinase (BTK) is the most common mechanism of clinical resistance against ibrutinib for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia. We report small molecule ligands containing chiral {beta}-lactone electrophiles to address this challenge. The asymmetric warhead enabled stereoselective covalent modification of wild-type and ibrutinib-resistant mutant BTK(C481S) through distinct sites of reactivity. Building on these findings, we developed kinase-directed {beta}-lactone probes and demonstrated that individual enantiomers preferentially engage distinct subsets of the kinome. These studies establish {beta}-lactones as stereochemically encodable covalent warheads whose stereochemistry can serve as a selectivity filter in covalent drug discovery.

Toxigenic bacterial infections in the gut are a significant contributor to the global burden of disease. Advanced tools for protein and live biotherapeutic engineering offer potentially transformative strategies for treating such diseases, while avoiding the collateral effects of traditional antibacterials. Here we used de novo protein design to identify inhibitors of the metzincin family protease Bacteroides fragilis toxin (BFT). These inhibitors, which bind distal to the active site, interfere with toxin-mediated E-cadherin cleavage and downstream proinflammatory signaling by blocking claudin-4 receptor binding. We tested the inhibitors as disulfide-stabilized variants administered directly to the cecum or in drinking water, as well as through in situ secretion by an engineered live biotherapeutic. Across these delivery modalities, the inhibitors successfully neutralized the toxin and effectively prevented BFT-associated gut pathology, including tumor formation. These results highlight the potential of de novo designed proteins as precise, non-antibiotic interventions to mitigate bacterial toxin-driven disease in the gut.

Immunoglobulins (e.g., IgGs) are critical effectors of the adaptive immune system that when overexpressed or dysregulated can result in autoimmune diseases. Thus, depletion of IgGs can be a promising therapeutic avenue. Here we developed genetically-encoded lysosome targeting chimeras (GELYTACs) that target circulating IgGs for clearance and degradation. The GELYTACs comprised two protein modules derived from insulin-like growth factor 2 (IGF2) and an IgG-binding nanobody, respectively, and mediated clearance of plasma IgG via the lysosomal trafficking receptor IGF2R. To achieve long-lasting IgG depletion, we encoded GELYTACs in an AAV gene therapy vector and established continuous expression in mice. We also developed conditional GELYACs that are activatable with disease-specific proteases or small molecule drugs. This work establishes GELYTACs as a possible therapeutic modality that is deliverable using genetic medicine approaches.

Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process -1,4- and -1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as -1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel -1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves -1,2-linkages via an anomer-inverting mechanism, with a distinct preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 [A]) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts a (/{beta})6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses -1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare -glucans.