ACS Chemical Biology最新文献

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.

Macrophages remove apoptotic cells via phagocytosis, also known as efferocytosis, during inflammation to maintain tissue homeostasis. This process is accompanied by various metabolic changes in macrophages including the production of lipid metabolites by fatty acid oxygenases. Among these, highly reactive metabolites, called lipid-derived electrophiles (LDEs), modify cysteines and other nucleophilic amino acids in intracellular proteins. However, the landscape and functions of the modifications by these electrophilic metabolites have been poorly characterized. In this study, we used activity-based protein profiling to quantitatively profile the cysteine reactivity landscape and identify the potential targets of endogenous LDE modification during efferocytosis in mouse peritoneal macrophages. Using this methodology, we identified multiple cysteine sites that are highly likely to be modified by LDEs generated by 12/15-lipoxygenase (12/15-LOX), an efferocytosis-related fatty acid oxygenase that is highly expressed in peritoneal macrophages. Among these, actin-depolymerizing protein Cofilin-1 was found to be a target of 12/15-LOX-derived LDEs. In vitro Cofilin-1 activity was attenuated by 12/15-LOX-derived LDEs, and intracellular actin stabilization and efferocytosis were substantially enhanced by the LDE treatment of mouse peritoneal macrophages. These results highlighted the role of intracellular LDE modification during efferocytosis in macrophages.

RNA interference (RNAi) has rapidly matured as a novel therapeutic approach. In this field, chemical modifications have been critical to the clinical success of short interfering RNAs (siRNAs). Notwithstanding the significant advances, achieving robust durability and gene silencing in extrahepatic tissues, as well as reducing off-target effects of siRNA, are areas where chemical modifications can still improve siRNA performance. The present study developed the challenging synthesis of amide-linked guanosine dimers (G_AM1G and G_AM1A) and completed an “amide walk” one by one, systematically replacing every internucleoside phosphate with an amide linkage in a guide strand targeting the PIK3CB gene. Dual-luciferase and RT-qPCR assays in HeLa cells showed that, in a model system of unmodified siRNAs, the amide linkage at position 3 (between nucleosides 3 and 4) suppressed the cleavage of off-target YY1 and FADD mRNAs similarly to the industry gold standard modification glycol nucleic acid (GNA). These results suggest that amide linkages in the seed region have strong potential to improve the specificity of siRNAs by suppressing the microRNA-like off-target activity.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs primarily known for guiding chemical modifications of RNA, but their broader cellular roles and contributions to human diseases remain elusive. This In Focus article introduces the development of snoRNA-enriched kethoxal-assisted RNA–RNA sequencing (snoKARR-seq), a transcriptome-wide approach to uncover snoRNA targets with enhanced sensitivity and specificity. This method revealed an unexpected role for snoRNAs in protein translocation and secretion, expanding our understanding of their noncanonical functions.

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.

Hyperammonemia is characterized by the accumulation of ammonia within the bloodstream upon liver injury. Left untreated, hyperammonemia contributes to conditions such as hepatic encephalopathy that have high rates of patient morbidity and mortality. Previous studies have identified gut bacterial urease, an enzyme that converts urea into ammonia, as a major contributor to systemic ammonia levels. Here, we demonstrate use of benurestat, a clinical candidate used against ureolytic organisms in encrusted uropathy, to inhibit urease activity in gut bacteria. Benurestat inhibits ammonia production by urease-encoding gut bacteria and is effective against individual microbes and complex gut microbiota. When administered to conventional mice with liver injury induced by thioacetamide exposure, benurestat reduced gut and serum ammonia levels and rescued 100% of mice from lethal acute liver injury. Overall, this study provides an important proof-of-concept for modulating host ammonia levels and microbiota-driven risks for hyperammonemia with gut microbiota-targeted small-molecule inhibitors.

Ketoreductases (KRs) are domains in the reductive loops of type I polyketide synthases (PKSs) and are responsible for the majority of stereocenters in reduced polyketides. Although the highly stereoselective reduction of ACP-bound β-ketothioester intermediates by KRs is crucial for the overall functioning of PKSs, the substrate-dependent stereoselectivity of KRs is a factor that is not yet fully understood, especially for KR domains in late PKS modules that act on biosynthetic precursors with complex polyketidic moieties. We present studies on the three KR domains FosKR7, PlmKR6, and EryKR6 from the biosynthetic pathways of fostriecin, phoslactomycin, and erythromycin by in vitro assays using close surrogates of the octaketidic FosKR7 biosynthetic precursor, complex derivatives and a diketide in the form of their biomimetic N-acetylcysteamine thioesters. Supported by molecular modeling, specific interactions of the studied KR domains with the extended polyketide moieties of their natural precursors were identified and correlated to the differences in stereoselectivity observed in the in vitro assays. These results reinforce the importance of the substrate-dependent stereoselectivity of KR domains in PKSs and suggest more detailed experimental and structural studies with isolated KRs and full PKS modules that could ultimately lead to improved results in PKS engineering.

Current methods for the macrocyclization of phage-displayed peptides often rely on small molecule linkers that nonspecifically react with targeted amino acid residues. To expand tool kits for more regioselective macrocyclization of phage-displayed peptides, this study explores the unique condensation reaction between an N-terminal cysteine and nitrile along with the reactivity of an internal cysteine. Five 2-cyanopyrimidine derivatives were synthesized for this purpose and evaluated for their selective macrocyclization of a protein-fused model peptide. Among these, two novel linkers, 2-chloro-N-(2-cyanopyrimidin-5-yl)acetamide (pCAmCP) and 2-chloro-N-(2-cyanopyrimidin-4-yl)acetamide (mCAmCP), emerged as efficient molecules and were demonstrated to macrocyclize phage-displayed peptide libraries flanked by an N-terminal and an internal cysteine. Using these linkers to generate macrocyclic peptide libraries displayed on phages, peptide ligands for the ZNRF3 extracellular domain were successfully identified. One of the identified peptides, Z27S1, exhibited potent binding to ZNRF3 with a K_D value of 360 nM. Notably, the selection results revealed distinct peptide enrichment patterns depending on whether mCAmCP or pCAmCP was used, underscoring the significant impact of linker choice on macrocyclic peptide identification. Overall, this study validates the development of two novel regioselective, small molecule linkers for phage display of macrocyclic peptides and highlights the benefits of employing multiple linkers during phage selections.

Bacterial peptidoglycan, the essential cell surface polymer that protects bacterial integrity, also serves as the molecular pattern recognized by the host’s innate immune system. Although the minimal motifs of bacterial peptidoglycan fragments (PGNs) that activate mammalian NOD1 and NOD2 sensors are well-known and often represented by small canonical ligands, the immunostimulatory effects of natural PGNs, which are structurally more complex and potentially can simultaneously activate both the NOD1 and NOD2 signaling pathways in hosts, have not been comprehensively investigated. In particular, many bacteria incorporate additional structural modifications in peptidoglycans to evade host immune surveillance, resulting in diverse structural variations among natural PGNs that may influence their biological effects in hosts. The focus of this study is on the amidation status of γ-d-glutamic acid and meso-diaminopimelic acid (mDAP) at the second and third positions of stem peptides in peptidoglycan, which represent key structural features that vary across different bacterial species. With four synthetic mDAP-containing disaccharide PGNs of different amidation states, we systematically investigated their structure–activity relationship in stimulating host innate immune responses in vitro. Our findings revealed that the amidation of disaccharide PGNs has distinct effects on NOD1 and NOD2 induction, along with their differential immunostimulatory activities in macrophage cells. Additionally, we found that, like the canonical NOD2 ligand, natural PGNs confer immune tolerance to LPS, and amidation states do not affect this outcome. Overall, our work highlights the potential immunological implications of these differentially amidated mDAP-type disaccharide PGNs in host–microbe crosstalk.

Small molecule degraders such as PROteolysis TArgeting Chimeras (PROTACs) and molecular glues are new modalities for drug development and important tools for target validation. When appropriately optimized, both modalities lead to proteasomal degradation of the protein of interest (POI). Due to the complexity of the induced multistep degradation process, controls for degrader evaluation are critical and commonly used in the literature. However, comparative studies and evaluations of cellular potencies of these control compounds have not been published so far. Here, we investigated a diverse set of ubiquitin pathway inhibitors and evaluated their potency and utility within the CRBN and VHL-mediated degradation pathway. We used the HiBiT system to measure the level of target rescue after treatment with the control compounds. In addition, the cell health was assessed using a multiplexed high-content assay. These assays allowed us to determine nontoxic effective concentrations for control experiments and to perform rescue experiments in the absence of cellular toxicity.