ACS Chemical Biology最新文献

With the growing number and diversity of known genome sequences, there is an increasing opportunity to regulate gene expression through synthetic, cell-permeable small molecules. Enhancing the DNA sequence recognition abilities of minor groove compounds has the potential to broaden their therapeutic applications with significant implications for areas such as modulating transcription factor activity. While various classes of minor groove binding agents can selectively identify pure AT and mixed AT and GC base pair(s) containing sequences, there remains a lack of compounds capable of distinguishing between different AT sequences. In this work, we report on the design compounds that exhibit selective binding to -TTAA- or -TATA- containing DNA minor groove sequences compared with other AT ones. Several studies have shown that the -AATT- and -TTAA- sequences have distinct physical and interaction properties, especially in terms of their different requirements for recognition in the minor groove. Achieving strong, selective minor groove binding at -TTAA- sequences has been challenging, but DB1003, a benzimidazole-furan-furan diamidine, has demonstrated cooperative dimeric binding activity at -TTAA-. It has significantly less binding preference for AATT. To better understand and modify the selectivity, we synthesized a set of rationally designed analogs of DB1003 by altering the position of the five-membered heterocyclic structure. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1992, a benzimidazolefuran-thiophene diamidine, binds strongly to -TTAA- as a positive cooperative dimer with high cooperativity. The high-resolution crystal structure of the TTAA-DNA-DB1992 complex reveals that DB1992 binds as an antiparallel π-stacked dimer with numerous diverse contacts to the DNA minor groove. This distinctive binding arrangement and the properties of diamidines at the -TTAA- minor groove demonstrate that benzimidazole-furan-thiophene is a unique DNA binding pharmacophore. Competition mass spectroscopy and circular dichroism studies confirmed the binding stoichiometry and selectivity preference of the compounds for the -TTAA- sequence.

Chronic kidney fibrosis poses a significant global health challenge with effective therapeutic strategies remaining elusive. While cell-extracellular matrix (ECM) interactions are known to drive fibrosis progression, the specific role of focal adhesions (FAs) in kidney fibrosis is not fully understood. In this study, we investigated the role of FAs in kidney tubular epithelial cell fibrosis by employing precise nanogold patterning to modulate integrin distribution. We demonstrate that increasing ligand spacing disrupts integrin clustering, thereby inhibiting FA formation and attenuating fibrosis. Importantly, enhanced FA activity is associated with kidney fibrosis in both human disease specimens and murine models. Mechanistically, FAs regulate fibrosis through mechanotransduction pathways, and our in vivo experiments show that suppressing mechanotransduction significantly mitigates kidney fibrosis in mice. These findings highlight the potential of targeting FAs as a therapeutic strategy, offering new insights into clinical intervention in kidney fibrosis.

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.

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.

Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau ligase) E3 ligase ligands. However, the considerable size of the human E3 ligase family, newly developed E3 ligase ligands, and the favorable druggability of some E3 ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 ligase ligand efficiency for PROTAC development and the assessment of the corresponding "degradable" target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 ligase ligand negative controls, competition by kinase parent compounds, and neddylation and proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 ligase ligand systems for degrader development.

We report the discovery of small molecules that target the RNA tertiary structure of self-splicing group II introns and display potent antifungal activity against yeasts, including the major public health threat Candida parapsilosis. High-throughput screening efforts against a yeast group II intron resulted in an inhibitor class which was then synthetically optimized for enhanced inhibitory activity and antifungal efficacy. The most highly refined compounds in this series display strong, gene-specific antifungal activity against C. parapsilosis. This work demonstrates the utility of combining advanced RNA screening methodologies with medicinal chemistry pipelines to identify high-affinity ligands targeting RNA tertiary structures with important roles in human health and disease.

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.

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.

Multiple immune components in the complex and heterogeneous tumor-immune microenvironment (TIME) work cooperatively to promote or impede cancer immunotherapy. Synergistically co-managing multiple immune cells with single agents for advanced antitumor immunity remains desirable but challenging. This In Focus article introduces a triple orthogonal linker (T-Linker)-based multimodal targeting chimera (Multi-TAC) platform, enabling the single-agent-mediated tumor-targeted co-engagement of multiple immune cell types within TIME for potentiated immunotherapy.