ACS Chemical Biology最新文献

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 Mycobacterium tuberculosis 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 (IC₅₀ 1 nM) and generates a postantibiotic effect of 77 h at 50xMIC (MIC 0.058 μM) with M. tuberculosis 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 Escherichia coli LeuRS (ecLeuRS, IC₅₀ 2 nM), however no antibacterial activity is observed toward E. coli 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^Leu. 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 M. tuberculosis is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNA^Leu complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.

Candida albicans (C. albicans) 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 Staphylococcus aureus (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 C. albicans. 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 C. albicans. Moreover, in vitro 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 C. albicans. 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.

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 (K_d ∼ 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.

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.

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.

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 K_D 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.

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.

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. RU-0610270, a sulfonamide derivative, is one of the most potent hits (IC₅₀ = 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 cpd 6 (IC₅₀ = 0.66 μM) as the most potent hit analog. We further profiled cpd 6 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.

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 Tb³⁺ 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.