ACS Chemical Biology最新文献

Histone deacetylase (HDAC) enzymes remove acetyl groups from acetyllysine-containing proteins, including nucleosomal histones to control gene expression. Beyond fundamental cell biology, HDAC activity is linked to various cancers, with many HDAC inhibitors developed as anticancer therapeutics. Among the 11 metal-dependent HDAC proteins, the four class IIa isoforms (HDAC4, 5, 7, and 9) are “pseudodeacetylases” without measurable enzymatic activity due to mutation of a catalytic tyrosine. Deacetylase-related activities of class IIa HDAC proteins are attributed to scaffolding functions, where recruitment of an active HDAC isoform leads to bound substrate deacetylation. Scaffolding of class IIa proteins beyond simple recruitment of an active HDAC is only starting to emerge. This review explores the various scaffolding roles of HDAC7, including recently reported acetylation-mediated reversible scaffolding, which is a form of acetyllysine-binding reader function. Studying the functional roles of HDAC7 will provide molecular insight into normal and pathological conditions, which could facilitate drug design.

Second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2) were developed by hybridizing a 3′-FAM-labeled 2′-OMe RNA strand with a 3′-Cy5-labeled 25mer RNA strand. These duplexes demonstrated the silencing of cytoplasmic mRNA targets in HeLa cells as measured by RT-qPCR and supported by western blot analysis. Fluorescence intensity and lifetime measurements revealed that a single guanosine (G) positioned adjacent to FAM achieves substantial static quenching at pH 5, with additional collisional quenching rendering the dye almost nonemissive. A FAM-G π–π stacking interaction was evidenced by a red-shifted absorbance spectrum for FAM. Decreased quenching at near-neutral pH enhances the FAM dynamic range in the physiologic pH window and improves the differentiation in cells between endocytic entrapment and cytoplasmic release. Flow cytometric analysis of intracellular pH and uptake using sMiRpH-2 was corroborated by live cell confocal microscopy and found to be predictive of knockdown efficacy. A sMiRpH-2 probe successfully predicted the relative efficacy of two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of nonviral siRNA delivery vectors.

The National Institutes of Health molecular probe ML283 was synthesized as a potent, selective inhibitor of the helicase encoded by the hepatitis C virus. Because modeling with AutoDock Vina predicted that ML283 might bind the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 13 (nsp13) helicase, the effects of a collection of ML283 analogs and other hepatitis C virus (HCV) helicase inhibitors on the SARS-CoV-2 helicase were analyzed. Only modest impacts on nsp13-catalyzed ATP hydrolyses were observed with some compounds, most of which were analogs of the drug ebselen, not ML283. In contrast, a new molecular-beacon-based helicase assay revealed that ML283 and many ML283 analogs are potent SARS-CoV-2 helicase inhibitors. Analog potencies correlate with the binding energies predicted by modeling, which suggests that a pocket surrounded by the carboxy-terminal nsp13 RecA-like helicase motor domain might be exploitable for antiviral drug development.

8-Oxoguanine (8-oxoG) is not only a biomarker of oxidative DNA damage but also an epigenetic-like regulator in mammalian cells. The identification and characterization of 8-oxoG-binding proteins would be crucial for further understanding the biological consequences of 8-oxoG. Here, we identified human Y-box-binding protein 1 (YBX1) as a novel binding protein for 8-oxoG modification in DNA by using a quantitative proteomic approach. Moreover, we found that the deficiency of YBX1 can substantially decrease the cellular sensitivity to oxidative stress and facilitate the repair of 8-oxoG embedded in DNA. These findings provided new insight into the biological significance of the functional interplay between YBX1 and 8-oxoG modification in DNA.

Ligand-conjugated small interfering RNAs (siRNAs) have emerged as a powerful approach to developing nucleic acid-based medicines. To achieve efficient mRNA knockdown, it is important to select targeting receptors with high expression and ligands that exhibit rapid internalization. However, the key characteristics of ligand–receptor sets involved in the postinternalization process remain largely unclear. In this study, we investigated the effect of ligand–receptor binding dissociation under low pH conditions, known as a postendocytic environment. Specifically, we chemically synthesized several modified epidermal growth factor (EGF) ligands that showed a variety of binding activities to the EGF receptor (EGFR) at low pH. Among these modified ligands, the siRNA conjugate with chemically synthesized EGF H10Y/H16Y, which is a less pH-responsive variant, exhibited reduced internalization and mRNA knockdown activity at high concentrations in EGFR-expressing cells. Additionally, we explored the use of antibody-related molecules (anti-EGFR IgG and Fab) as targeting moieties for siRNA conjugates. The anti-EGFR Fab-siRNA, which showed dissociation of EGF under low pH conditions, demonstrated stronger internalization and mRNA knockdown activity compared to the anti-EGFR IgG-siRNA, which strongly binds EGF at low pH. These data emphasize the importance of intracellular ligand–receptor dissociation and provide insights for future advancements in the field.

Cross-link containing products from ribosomally synthesized and post-translationally modified peptides (RiPPs) are generated by radical SAM enzymes (rSAM). Here, we bioinformatically expanded rSAM enzymes based on the known families StrB, NxxcB, WgkB, RrrB, TqqB and GggB. Through in vivo functional studies in E. coli, the newly identified enzyme WprB from Xenorhabdus sp. psl was found to catalyze formation of a cross-link between Trp-C5 and Arg-Cγ at three WPR motifs on the precursor peptide WprA. This represents the first report of this type of cross-link by rSAM enzymes.

Rho-associated protein kinase (ROCK) inhibitors are promising therapeutic agents for reducing elevated intraocular pressure in patients with glaucoma. We explored new ROCK inhibitors derived from bioactive metabolites produced by microbes, specifically cryptic metabolites from Nocardiopsis sp. MCY7, using a liquid chromatography–mass spectrometry-based chemical analysis approach integrated with metal stress-driven isolation. This strategy led to the identification of two previously undescribed linear peptides, nocarnickelamides A and B (1 and 2), and an unreported cittilin derivative, cittilin C (3). The planar structures of 1–3 were elucidated using UV spectroscopy, high-resolution mass spectrometry, and nuclear magnetic resonance. The absolute configurations of 1 and 2 were assigned using the advanced Marfey’s method. Biological assays demonstrated that nocarnickelamides (1 and 2) exhibited dual inhibitory activity against ROCK1 (IC₅₀ 29.8 and 14.9 μM, respectively) and ROCK2 (IC₅₀ 27.0 and 21.9 μM, respectively), with molecular simulations suggesting binding to the ATP-binding site. In human trabecular meshwork cells, 2 significantly inhibited the activation of ROCK-regulated cytoskeletal contraction markers such as the myosin light chain. Nocarnickelamide B (2) is a novel dual ROCK1/2 inhibitor and a potential pharmacophore for designing new therapeutic agents to reduce intraocular pressure in glaucoma.

Conventional small-molecule drugs primarily operate by inhibiting protein function, but this approach is limited when proteins lack well-defined ligand-binding pockets. Targeted protein degradation (TPD) offers an alternative approach by harnessing cellular degradation pathways to eliminate specific proteins. Recent studies have expanded the potential of TPD by identifying additional E3 ligases, with DCAF16 emerging as a promising candidate for facilitating protein degradation through both proteolysis-targeting chimera (PROTAC) and molecular glue mechanisms. In this study, we revisited a previously reported compound and discovered that it covalently binds to DCAF16. We further optimized it into a FKBP12-targeting PROTAC, MC-25B. This PROTAC engages DCAF16 at cysteines C177–179, leading to the degradation of nuclear-localized FKBP12. We further demonstrated the versatility of this DCAF16 recruiter by degrading additional endogenous proteins. Compared to the first-generation DCAF16-based PROTAC, which was derived from a fragment electrophile, this DCAF16 recruiter-based PROTAC exhibits improved proteome-wide selectivity.

Dimethyl fumarate (DMF) is an established oral therapy for multiple sclerosis worldwide. Although the clinical efficacy of these fumarate esters has been extensively investigated, the mode of action and pharmacokinetics of fumarates have not been fully elucidated due to their broad-spectrum reactivity and complex metabolism in vivo. To better understand the mechanism of action of DMF and its active metabolite, monomethyl fumarate (MMF), we designed and utilized clickable probes to visualize and enrich probe-modified proteins. We further perform quantitative chemoproteomics analysis for proteome-wide target identification and validate several unique and shared targets of DMF and MMF, which provide insight into the reactivity, selectivity, and target engagement of fumarates.

Protein interactions play a crucial role in regulating cellular mechanisms, highlighting the need for effective methods to control these processes. In this regard, chemical inducers of proximity (CIPs) offer a promising approach to precisely manipulate protein–protein interactions in live cells and in vivo. In this study, we introduce pMandi, a photocaged version of the plant hormone-based CIP mandipropamid (Mandi), which allows the use of light as an external trigger to induce protein proximity in live mammalian cells. Furthermore, we present opabactin (OP) as a new plant hormone-based CIP that is effective in live mammalian cells at low nanomolar concentration and in live medaka embryos at submicromolar concentration. Its photocaged derivative, pOP, enables the induction of protein proximity upon light exposure in individual cells, enhancing spatiotemporal control to the level of single-cell resolution. Additionally, we explored the use of both photocaged CIPs to promote protein proximity in live medaka embryos.