Precise control of protein-specific O-GlcNAcylation in cells remains a major challenge. Chemically induced proximity (CIP) offers a promising path forward, but its application to targeted protein O-GlcNAcylation has been limited by the lack of ligands that can bind the O-GlcNAc transferase (OGT) without inhibiting its catalytic function. Here, we repurpose a potent OGT inhibitor into a noninhibitory covalent probe using ligand-directed release chemistry (LDR). The resulting ligands covalently label OGT while preserving its enzymatic activity. Building on this scaffold, we developed a self-assembling O-GlcNAcylation Targeting Chimera (OGTAC) that recruits OGT to its native substrate casein kinase IIα (CK2α) in living cells, selectively elevating CK2α O-GlcNAcylation without affecting global modification levels. This new class of self-assembling chimeras covalently engages OGT to induce protein-specific O-GlcNAcylation, offering a versatile platform for dissecting and controlling this essential modification in living cells. Our findings open the door to next-generation OGTACs and related therapeutic strategies for the targeted modulation of the O-GlcNAc signaling.

Ganfeborole (GSK3036656) inhibits the leucyl-tRNA-synthetase (mtLeuRS) and is in Phase 2a clinical trials for the treatment of tuberculosis. Here we show that ganfeborole is a time-dependent inhibitor of mtLeuRS (IC 1 nM) and generates a postantibiotic effect of 77 h at 50xMIC (MIC 0.058 μM) with H37Rv, indicating that mtLeuRS is a highly vulnerable drug target and supporting the excellent in vivo efficacy of the drug. Ganfeborole is also a potent time-dependent inhibitor of LeuRS (ecLeuRS, IC 2 nM), however no antibacterial activity is observed toward up to 1 mM ganfeborole despite the observation that less potent ganfeborole analogs have antibacterial activity. To rationalize this observation, we propose that ganfeborole forms a complex with AMP that binds to the ecLeuRS editing site but does not impact aminoacylation. In support, addition of 12.5 μM norvaline generates a ganfeborole MIC of 0.4 μM since ecLeuRS is unable to hydrolyze norvaline-tRNA. Additionally, mutations that reduce the affinity and residence time of ganfeborole-AMP on ecLeuRS result in antibacterial activity. We propose that the activity of ganfeborole toward is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNA complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.

The Watson-Crick-Franklin (WCF) rules describing nucleobase pairing in antiparallel strands of DNA and RNA can be exploited to create artificially expanded genetic information systems (AEGIS) with as many as 12 independently replicable nucleotides joined by six hydrogen bond pairing schemes. One of these additional pairs joins two nucleotides trivially designated as (6-amino-5-nitro-(1)-pyridin-2-one) and (2-amino-imidazo-[1,2-]-1,3,5-triazin-(8)-4-one). The : pair has supported 6-nucleotide PCR to give diagnostics products, in environmental surveillance kits, and for laboratory evolution (LIVE) that has generated, , molecules that inactivate toxins, antibody analogs that bind cancer cells, therapeutic candidates that deliver drugs to those cells, reagents to identify targets on those cells' surfaces, reagents to move cargoes across the blood-brain barrier, and catalysts with ribonuclease activity. However, the nucleoside is acidic, with a p of ∼7.8. In its deprotonated form, forms a WCF pair with G. This leads to the slow replacement of pairs by C:G pairs during PCR or, in the reverse process, their introduction. Here, we examine analogs of that retain the same donor:donor:acceptor hydrogen bonding pattern as earlier generations of the heterocycle, still form a WCF pair with , but have a higher p. Experiments with Taq polymerase show that the rate of loss of pairs decreases markedly as the p of the heterocycle increases. This provides direct support for the hypothesis that : pairs are in fact lost via deprotonated : mismatches. Further, it provides a : system that can be replicated with very high fidelity, with >97% retention of the : pairs over 10,000-fold amplification.

Our ability to respond to emerging pandemics and pathogen resistance relies critically on our ability to build vaccines quickly and efficiently. In this report we used an efficient enzymatic oxidative coupling reaction to create a viral capsid-based vaccine platform that is modular and quickly adaptable for many different pathogens. Tyrosinase-mediated oxidative coupling was used to conjugate C-terminal tyrosine residues on peptide antigens to cysteine residues installed inside MS2 viral capsids. This strategy is particularly promising because the capsids protect the internally conjugated peptides from protease degradation before they are delivered into cells. The vaccine constructs were tested for MHC presentation followed by T-cell activation. Mutants of the MS2 capsid itself activated DC2.4 cells, serving as an adjuvant to help induce the immune response to delivered antigens. The MS2-peptide constructs were shown to be stable in serum, activate DC2.4 cells, and lead to MHC presentation of peptide antigens with subsequent activation of antigen-specific T-cell hybridomas. Taken together, these results demonstrate effective activation of the adaptive immune system . This synthetic platform can be used to build new vaccines for many different diseases for which immunodominant peptide antigens are known because the antigens can be quickly interchanged while the MS2 scaffold remains the same. Additionally, this platform allows for multiple peptide antigens to be delivered simultaneously in each capsid, which could provide enhanced immunity against resistant pathogen strains and be useful for cancer vaccine development.

The specific incorporation of lanthanide ions is a promising strategy to equip biomolecules with a new function. Their long-lived luminescence, strong anomalous X-ray scattering, paramagnetism, Lewis acidity, and photoredox activity are attractive features for protein-based probes, materials, and catalysts. However, natural lanthanide-binding proteins are rare, and de novo design is often complicated by unspecific binding to negatively charged patches on protein surfaces. We thus aimed to develop an efficient workflow to screen libraries of protein scaffolds for their ability to coordinate lanthanides. Here, we introduce a microtiter plate-based assay, which employs commercial filter plates and a dual readout based on sensitized Tb luminescence. We first benchmarked our procedure using control proteins with and without lanthanide-binding sites, demonstrating that site-specific coordination and surface binding can be distinguished. The stringency of this protocol also allowed screening for small lanthanide-binding peptides in the presence of a large expression tag. We then designed a de novo scaffold library derived from a helical bundle protein and applied our screening platform. We could identify lanthanide-binding variants with nanomolar affinity, distinct lanthanide specificity, and increased thermostability in response to metal binding. Our approach will support the discovery and evolution of lanthanide-binding peptides and proteins for various applications in vitro and in living cells.

Capnine-like sulfonolipids are sulfonate-containing analogs of sphingolipids found in many Bacteroidetes bacteria, where they govern essential functions such as gliding motility, outer membrane polysaccharide assembly, and antibiotic susceptibility. In gut-associated anaerobic Bacteroidetes, these sulfonolipids also modulate host-microbe interactions. In aerobic bacteria, the capnine precursor cysteate is produced by a pyridoxal phosphate (PLP)-dependent cysteate synthase (CapA1), a close homologue of cystathionine β-synthase (CBS). By contrast, the mechanism of cysteate production in anaerobic Bacteroidetes bacteria has not been biochemically studied. Herein, we report the characterizations of archaeal cysteate synthase homologue from the anaerobic bacteria (CapA2). Biochemical assays confirm its ability to catalyze the conversion of -phosphoserine (OPS) to cysteate. Crystal structures of CapA2 in complex with PLP and OPS-PLP identify essential catalytic residues and reveal a structural similarity to threonine synthase, unlike CapA1, which is more similar to CBS. Comparative analysis of CapA1 and this nonorthologous CapA2, including structural differences, catalytic versatility, and phylogenetic distribution across Bacteroidetes, suggests convergent evolution of cysteate synthase activity. Our work clarifies the details of sulfonolipid synthesis in anaerobic bacteria and the biochemical origins of this structurally distinctive lipid in the gut microbiome.

Hexokinase domain containing protein 1 (HKDC1) is a recently discovered fifth human hexokinase isozyme that is significantly upregulated in several disease states, including lung and liver cancers. Cellular studies suggest that HKDC1 is a low activity hexokinase; however, its functional characteristics have remained enigmatic. Here, we describe the kinetic and regulatory features of recombinant human HKDC1, demonstrating it to be a robust hexokinase (/ = 1.5 × 10 M s) with a unique glucose value (0.49 ± 0.07 mM) that differs markedly from all other human hexokinase isozymes. The isolated C-terminal domain of HKDC1 displays kinetic characteristics nearly identical to the full-length enzyme, whereas the N-terminal domain is inactive. Unlike all other 100 kDa vertebrate hexokinases characterized to date, HKDC1 is insensitive to product inhibition by physiological concentrations of glucose 6-phosphate, with apparent inhibition constants above 1 mM. The hexokinase activity of HKDC1 is also insensitive to Dinaciclib, a pan cyclin-dependent kinase inhibitor that reportedly disrupts the ability of nuclear localized HKDC1 to phosphorylate retinoblastoma-binding protein 5. Conversely, the hexokinase activity of HKDC1 is potently inhibited by a synthetic glucosamine derivative previously developed for hexokinase 1 and 2, with an IC value of 103 ± 6 nM. An HKDC1 variant associated with retinitis pigmentosa, T58M, displays a modest, but statistically significant 2-fold decrease in catalytic efficiency (/) compared to the wild-type enzyme. Together, our results provide a detailed functional characterization of recombinant HKDC1 and set the stage for investigating the link between HKDC1 catalysis and human disease.

Discovering high-affinity ligands directly from protein structures remains a key challenge in drug discovery. BindCraft is a structure-guided generative modeling platform able to de novo design miniproteins with a high affinity for a large set of targets. While miniproteins are valuable research tools, short peptides offer substantially greater therapeutic potential. However, given their lack of stabilized tertiary structures, de novo generation of functional peptides is a remarkable challenge. Here, we show that BindCraft is able to generate high affinity peptides, solely based on target structure, with remarkable success rates. For the oncoprotein MDM2, BindCraft generated 70 unique peptides; 15 were synthesized, and 7 showed specific binding with nanomolar affinities. Competition assays confirmed site-specific binding for the intended target site. For another oncology target, WDR5, six out of nine candidates bound the MYC binding WBM site with submicromolar affinity. Bindcraft's high fidelity structure prediction enabled one shot peptide optimization via rational chemical modification, improving the potency of one WDR5 binder by 6-fold to a of 39 nM. BindCraft also generated candidate peptides for targeting PD-1 and PD-L1. However, none of the tested peptides showed detectable binding. Together, these results establish a first evaluation of BindCraft for peptide binder prediction. Despite remaining limitations, this tool shows the potential to rival display technologies in delivering high-affinity ligands for therapeutic development.

Live-cell activity-based protein profiling (ABPP) with mass spectrometry enables the proteome-wide quantification of compound reactivity, yet resulting datasets often suffer from low data completeness for high-priority targets and do not give users the option to measure compound-induced protein changes within the same screening assay. To address these limitations, we developed CysDig, an enrichment-free chemoproteomics platform for the targeted covalent drug discovery in live cells. Using the CysDig platform, we screened 288 cysteine-reactive electrophiles against 300 functionally annotated cysteine sites. From this screen, we identified covalent binders that liganded dozens of sites and identified multiple instances of acute compound-induced protein degradation of ACAT1. We validated a molecule that engaged with the active site of HECT E3 ligase HUWE1 and showed that chemical inhibition stabilized known substrates. Together, these findings establish CysDig as a powerful, targeted platform for live-cell covalent drug screening, expanding the current repertoire of available approaches for ligand discovery in live cells.

Tracking small-molecule distribution in heterogeneous cell samples at single-cell resolution remains a major analytical challenge. Here, we present a tellurophene-functionalized analogue of the proteasome inhibitor Carfilzomib (TeCar) whose distribution can be followed by mass cytometric (MC) quantification while preserving target engagement and cytotoxicity. Structural and biochemical analyses confirm that TeCar binds the proteasome in a mode comparable to the clinically approved parent compound. Using MC, we demonstrate selective TeCar accumulation in malignant over immune cells within mixed populations, with cancer cells exhibiting 15 to 30-fold higher uptake. Tellurium signal correlates with proteasomal activity, and differential labeling among immune subsets reveals functional heterogeneity not captured by transcriptomics alone. These findings establish tellurophene tagging as a minimally perturbing and broadly applicable strategy for functional distribution studies at single-cell resolution.

Histone acetylation, governed by histone deacetylase (HDAC) enzymes, plays a pivotal role in cell biology. Elevated HDAC expression is linked to a poor prognosis in various diseases, including cancer, making HDAC inhibitors clinically valuable. Among the 11 metal-dependent HDAC isoforms, the exceptional ability of HDAC11 to regulate both the deacetylation and defattyacylation of proteins suggests an expansive role in cellular processes. However, since HDAC11 is one of the least studied HDAC isoforms, the known roles for HDAC11 in cell biology are limited. In this study, proteomics-based mutant trapping was performed to identify nonhistone substrates of HDAC11 and link HDAC11 activity to specific cellular events. Proteomics revealed 64 putative substrates, with follow-up studies documenting that HDAC11 deacetylates the BRAF kinase on K680 to suppress kinase activity and cell proliferation. Given the established role of BRAF in cancer, HDAC11-mediated deacetylation likely influences signaling pathways in tumor progression, underscoring the diverse regulatory role of HDAC11 in cellular events.

Bacteria manufacture a diversity of natural products with pharmaceutical value, many from modular polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS), or hybrid pathways. In these pathways, each module extends a biosynthetic intermediate by an acyl unit (PKS) or amino acid (NRPS), employing a carrier domain (CP) to deliver the pathway intermediate to successive active sites and to the subsequent module. Docking domains (DD) at polypeptide termini ensure pathway fidelity by specific noncovalent association of sequential modules. The vatiamide biosynthetic gene cluster encodes a rare trifurcated pathway, enabled by a short linear motif (SLiM) at the C-terminus of VatM that docks with identical β-hairpin domains (βHDs) at the N-termini of VatN, VatQ, and VatS. Taking inspiration from Nature, we examined the utility of DDs for engineering by quantitating affinity and catalytic throughput in the Vat system and an unrelated SLiM-βHD dock from the carmabin pathway. The SLiM-βHD dock was the sole determinant of affinity of natural and engineered module partners ( ∼ 1 μM). The effectiveness of engineered DDs was evaluated relative to natural partners and docks. DD affinity was predictive of catalytic success in most, but not all, of the dozen cases tested. Thus, while the DD determines affinity and selectivity, other factors also affect catalytic throughput when a DD is engineered into a non-native environment. This study enhances our understanding of the interactions that enforce PKS/NRPS pathway fidelity and highlights the challenges of engineering these systems to diversify the repertoire of natural products.

() is a conditionally pathogenic fungus in humans, with its virulence significantly modulated by alterations in the composition of commensal bacteria and the surrounding microecological environment, particularly during cohabitation with methicillin-resistant (MRSA). Despite this, the molecular mechanisms underlying these interactions remain inadequately elucidated. In this study, we utilized an integrative multiomics approach, including proteomics and proteomics of post-translational modifications (PTMs), to systematically examine the impact of MRSA on protein expression and PTM patterns in . Our findings indicate that the presence of MRSA markedly influenced the expression of virulence-associated proteins and modified the phosphorylation and acetylation levels of key proteins involved in essential signaling and metabolic pathways. These modifications were predominantly associated with biological processes such as energy metabolism, metabolic reprogramming, and stress response. Functional enrichment analyses further indicated that these PTMs may play crucial roles in regulating the pathogenicity and environmental adaptability of . Moreover, enzyme activity assays revealed that lysine acetylation induced by MRSA modulated the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and homoisocitrate dehydrogenase (HIcDH). This suggests that such modifications are involved in the metabolic adaptation and functional reprogramming of . In conclusion, this study provides novel insights into the regulation of fungal physiology mediated by MRSA through PTMs, thereby offering a new theoretical framework for understanding fungal pathogenesis and for the development of enhanced anti-infective strategies within the context of bacterial-fungal interactions.

Therapies that stimulate DLX5-driven osteogenesis in bone-marrow-derived mesenchymal stem cells (BMSCs) with bone morphogenetic proteins (BMPs) potently accelerate the healing of delayed union and nonunion fractures, but their superior osteoinductive activity is often offset by severe adverse effects. To provide a safer and more effective alternative, we engineered a circular aptamer-antisense oligonucleotide chimera (CircApt-ASO) to activate DLX5-regulated osteogenesis by silencing STAT5A, a key negative regulator of DLX5. CircApt-ASO utilizes a transferrin receptor 1 (TFR1)-binding aptamer, enabling both specific nanoaffinity targeting of BMSCs and efficient intracellular delivery of the anti-STAT5A ASO. Compared with chemically modified linear aptamer-ASO chimeras, CircApt-ASO chimeras exhibit superior biostability and more robust STAT5A gene silencing but negligible cytotoxicity relative to liposome-based ASO delivery methods. Importantly, we demonstrated that CircApt-ASO drastically activated the expression of DLX5 and its downstream osteogenesis-related genes in dose- and time-dependent manners, leading to markedly enhanced osteogenic differentiation. The high stability, potent osteoinductive activity, and minimal cytotoxicity of CircApt-ASO highlight its strong therapeutic potential for promoting bone regeneration in conditions such as delayed union and nonunion fractures.

A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in and translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.

Cyclic GMP-AMP synthase (cGAS) has emerged as a promising therapeutic target of several human diseases, including Alzheimer's disease (AD) and related disorders. As a cytosolic DNA sensor, cGAS generates an innate immune response to promote neuroinflammation by producing an endogenous agonist of the stimulator of interferon genes (STING), 2'3'-cyclic GMP-AMP (cGAMP), which activates the cGAS-STING pathway. We have performed a high-throughput screening of a chemical library containing over 300 K small molecules at the Fisher Drug Discovery Resource Center (DDRC), Rockefeller University (RU), to identify multiple hit inhibitors of human (h)-cGAS. We used a modified Kinase Glo Luminescent Kinase assay, which was earlier developed at RU and later used by multiple groups, including ours, to perform primary screening of the library using h-cGAS. The hit candidates bearing novel scaffolds are structurally diverse and exhibited in vitro activity in the low micromolar range. , a sulfonamide derivative, is one of the most potent hits (IC = 1.88 μM), selected for hit expansion and structure-activity relationship (SAR) analysis. We synthesized RU-0610270 (listed as cpd 1) and new analogs and evaluated them in vitro against h-cGAS to identify (IC = 0.66 μM) as the most potent hit analog. We further profiled and found that it modestly inhibited cGAMP levels by 29% at 30 μM in THP1 cells without detectable toxicity and by 76% at 100 μM, albeit with a moderate decrease (∼20%) in cell viability. These results highlight a novel chemical series with promising in vitro activity, providing a starting point for the development of selective and potent human cGAS inhibitors for clinical use.

Immunomodulatory imide drugs (IMiDs), including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short degron motif, which often comprises a zinc finger (ZF). These IMiDs, however, also induce the degradation of endogenous ZF-containing neosubstrates, including IKZF1, IKZF3, and SALL4. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogues against 8380 ZF mutants. This yielded a bumped IMiD analogue that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1, IKZF3, and SALL4. In proof-of-concept studies, this system was applied to induce degradation of the optimum degron fused to CDK9, HPRT1, NanoLuc, or TRIM28. We anticipate that this system will be a valuable addition to the current arsenal of degron systems for use in target validation.

DNA interstrand cross-links (ICLs) covalently link complementary DNA strands and represent one of the most cytotoxic forms of DNA damage. While their impact on free DNA has been extensively characterized, how ICLs influence nucleosomes─the fundamental units of chromatin─remains largely unexplored. Here, we generated nucleosomes containing site-specific ICLs using click chemistry and systematically examined their effects on nucleosome structure, dynamics, and transcription. Biophysical assays revealed that ICLs did not impair nucleosome assembly, DNA accessibility, or ATP-dependent sliding, and only slightly reduced nucleosome stability. However, in vitro transcription assays demonstrated that ICLs function as absolute barriers to RNA polymerase elongation, producing truncated transcripts that terminate precisely at the cross-linking site in both free DNA and nucleosomal contexts. Restriction enzyme protection assays further showed that transcription-induced nucleosome translocation was unaffected, indicating that arrest results from the inability of the elongation complex to separate cross-linked strands rather than impaired nucleosome mobility. These findings provide mechanistic insight into ICL-induced cytotoxicity at the chromatin level and establish site-specific cross-linked nucleosomes as valuable tools for probing DNA damage responses.

Chemokines are secreted blood proteins that steer leukocyte migration in the inflammatory response. Neutralization of chemokines is believed to be a beneficial therapeutic strategy for the treatment of inflammation-associated diseases. Proteolytically stable chemokine-binding peptides could be suitable candidates for the development of chemokine-neutralizing agents. Here, we report the mirror-image phage display selection of cyclic all-D-peptides against the C-X-C motif chemokine ligand 8 (CXCL8). Selection yielded structurally diverse all-D-peptides with submicromolar affinity to the target CXCL8 chemokine and different selectivity to related chemokines. Binding of these all-D-peptides caused dissociation of the native CXCL8 dimer and disruption of its binding to GAGs, without an effect on in vitro cell migration. This work demonstrates the example of mirror-image phage display selection of cyclized all-D-peptides and its utility for the development of chemokine-binding agents.

Noncoding RNAs account for up to 98% of the human transcriptome. It has become increasingly clear that noncoding RNAs play diverse and critical roles in many important cellular functions. Although modulation of noncoding RNAs using small molecules is a promising therapeutic strategy, there are relatively few well-characterized RNA-ligand structures. Therefore, the structure-interaction relationships of RNA-targeting small molecules remain underexplored. Here, we present a fragment-based screening approach using biophysical assays to identify and evaluate fragments that bind to the theophylline-binding RNA aptamer, which we use as a model system. We were able to identify high affinity fragment hits and generate models of RNA-ligand complexes using a combination of biophysical data and computational docking. Together, these findings provided insights into the RNA-fragment interactions that underpin binding. This approach demonstrates the feasibility of identifying high-affinity RNA-targeting small molecules with limited structural information.