ACS Chemical Biology最新文献

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro. In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to study this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

As an important receptor in a host’s immune and metabolic systems, NOD1 is usually activated by Gram-negative bacteria having meso-diaminopimelic acid (m-DAP) in their peptidoglycan (PGN). But some atypical Gram-positive bacteria also contain m-DAP in their PGN, giving them the potential to activate NOD1. The prevalence of m-DAP-type Gram-positive bacteria in the gut, however, remains largely unknown. Here, we report a stem-peptide-based m-DAP-containing tetrapeptide probe for labeling and identifying m-DAP-type Gram-positive microbiota. The probe was synthesized via a five-step convergent approach and demonstrated moderate selectivity toward m-DAP-type bacteria in vitro. In vivo labeling revealed that ∼13.7% of the mouse gut microbiota (mostly Gram-positive) was selectively labeled. We then identified Oscillibacter and several other Gram-positive genera in this population, most of which were previously unknown m-DAP-type bacteria. The following functional assay showed that Oscillibacter’s PGN could indeed activate NOD1, suggesting an overlooked NOD1-activating role for these Gram-positive bacteria. These findings deepen our understanding of the structural diversity of gut microbes and their interactions with the host’s immune system.

MicroRNAs (miRNAs) play a significant role in tumor progression, and regulating miRNA expression with small molecules may offer a new approach to cancer therapy. Among them, miRNA-20b has been found to be dysregulated in several cancers, including nonsmall cell lung cancer (NSCLC). Herein, an in silico high-throughput computer screen was conducted to identify small molecules that downregulate miR-20b using the three-dimensional structure of the Dicer binding site on pre-miR-20b. Among 1058 small molecule compounds, Methotrexate (MTX), was discovered to be a potential miR-20b-specific inhibitor, which has been found to suppress miR-20b by specifically blocking Dicer processing in p53 wild-type A549 NSCLC cells but not in H1299 cells with p53 depletion. MTX effectively inhibited the proliferation, survival, migration, and invasion of A549 cells in a dose-dependent manner. Furthermore, the treatment of MTX up-regulated the expression of miR-20b target genes PTEN, STAT3, and HIF1α. Notably, MTX also significantly inhibited tumor growth in a mouse xenograft tumor model of NSCLC, with no observed tissue toxicity. Our findings indicate that MTX may have a novel role as an established drug in p53 wild-type NSCLC tumor therapy by down-regulating miR-20b expression. These findings are expected to provide preclinical evidence for miR-20b-targeting NSCLC therapeutic strategies.

Click chemistry is an immensely powerful technique for the synthesis of reliable and efficient covalent linkages. When undertaken in living cells, the concept is thereby coined bioorthogonal chemistry. Used in conjunction with the photo-cross-linking methodology, it serves as a sound strategy in the exploration of biological processes and beyond. Its broad scope has led to widespread use in many disciplines; however, this Review focuses on the use of click and bioorthogonal chemistry within medicinal chemistry, specifically with regards to drug development applications, namely, the use of DNA-encoded libraries as a novel technique for lead compound discovery, as well as the synthesis of antisense oligonucleotides and protein–drug conjugates. This Review aims to provide a critical perspective and a future outlook of this methodology, such as potential widespread use in cancer therapy and personalized medicine.

Tudor domains are histone readers that can recognize various methylation marks on lysine and arginine. This recognition event plays a key role in the recruitment of other epigenetic effectors and the control of gene accessibility. The Tudor-containing protein family contains 42 members, many of which are involved in the development and progression of various diseases, especially cancer. The development of chemical tools for this family will not only lead to a deeper understanding of the biological functions of Tudor domains but also lay the foundation for therapeutic discoveries. In this review, we discuss the role of several Tudor domain-containing proteins in a range of relevant diseases and progress toward the development of chemical tools such as peptides, peptidomimetics, or small-molecules that bind Tudor domains. Overall, we highlight how Tudor domains are promising targets for therapeutic development and would benefit from the development of novel chemical tools.

Bioluminescence imaging (BLI) is a powerful, noninvasive imaging method for animal studies. NanoLuc luciferase and its derivatives are attractive bioluminescent reporters recognized for their efficient photon production and ATP independence. However, utilizing them for animal imaging poses notable challenges. Low substrate solubility has been a prominent problem, limiting in vivo brightness, while the susceptibility of luciferins to auto-oxidation by molecular oxygen in air increases handling complexity and poses an obstacle to obtaining consistent results. To address these issues, we developed a range of caged PEGylated luciferins with increased auto-oxidation resistance and water solubility of up to 25 mM, resulting in substantial in vivo bioluminescence increases in mouse models. This advancement has created the brightest and most sensitive luciferase-luciferin combination, enabling high-speed video-rate imaging of freely moving mice with brain-expressed luciferase. These innovative substrates offer new possibilities for investigating a wide range of biological processes and are poised to become invaluable resources for chemical, biological, and biomedical fields.

Noncanonical base pairs play an important role in enabling the structural and functional complexity of RNA. Molecular recognition of such motifs is challenging because of their diversity, significant deviation from the Watson–Crick structures, and dynamic behavior, resulting in alternative conformations of similar stability. Triplex-forming peptide nucleic acids (PNAs) have emerged as excellent ligands for the recognition of Watson–Crick base-paired double helical RNA. The present study extends the recognition potential of PNA to RNA helices having noncanonical GoU, AoC, and tandem GoA/AoG base pairs. The purines of the noncanonical base pairs formed M⁺·GoU, T·AoC, M⁺·GoA, and T·AoG Hoogsteen triples of similar or slightly reduced stability compared to the canonical M⁺·G–C and T·A–U triples. Recognition of pyrimidines was more challenging. While the P·CoA triple was only slightly less stable than P·C–G, the E nucleobase did not form a stable triple with U of the UoG wobble pair. Molecular dynamics simulations suggested the formation of expected Hoogsteen hydrogen bonds for all of the stable triples. Collectively, these results expand the scope of triple helical recognition to noncanonical structures and sequence motifs common in biologically relevant RNAs.

Flavin-dependent azoreductases have been applied to a wide range of tasks from decolorizing numerous azo dyes to releasing azo-conjugated prodrugs. A general narrative reiterated in much of the literature suggests that this enzyme promotes sequential reduction of both the azo-containing substrate and its corresponding hydrazo product to release the aryl amine components while consuming two equivalents of NAD(P)H. Indeed, such aryl amines can be formed by incubation of certain azo compounds with azoreductases, but the nature of the substrates capable of this apparent azo bond lysis remained unknown. We have now prepared a set of azobenzene derivatives and characterized their turnover and products after treatment with azoreductase from Escherichia coli to discover the structural basis regulating aryl amine formation. Without resonance donation by aryl substituents, reduction ceases at the hydrazo product. This indicates that azoreductases do not act on the hydrazo bond. Instead, aryl amine formation depends on a spontaneous hydrazo bond lysis that is promoted by resonance stabilization and subsequent reduction of the quinone-like intermediate by azoreductase. Experimental and computational approaches confirm the substituent dependence of this process. With knowledge of this requirement, full release of aryl amines from azo-conjugates can now be designed and applied with confidence.

Although methods for Cys-specific bioconjugation and functionalization of proteins have been developed and widely utilized in biomolecule engineering and therapeutic development, reagents for this purpose are generally designed to accomplish bioconjugation only. Consequently, additional clickable groups must be attached to these reagents to accomplish functionalization. Herein, we describe a new, simple, dual-performing bioconjugation–functionalization reagent, VMeTz, which possesses an electron-withdrawing tetrazine (Tz) substituted vinyl (V) moiety to serve as both a Michael receptor for selective conjugation with Cys and a site for click with TCO derivatives to introduce functionality. Critically, VMeTz contains a methyl group that prevents the formation of multiple Tz-containing Cys-adducts. Reactions of VMeTz with Cys-containing peptides and proteins both in vitro and in live cells produce single stable Michael adducts with high selectivity. Moreover, the Cys-VMeTz peptide and protein conjugates undergo facile click reactions with TCO-functionalized reagents for labeling and protein profiling. Furthermore, VMeTz selectively activates and delivers the TCO-caged toxic substances Dox and PROTAC ARV-771 to cancer cells to produce therapeutic effects that are comparable to those of the parent drugs. Collectively, the studies demonstrate that VMeTz is a useful reagent for therapeutically significant Cys-specific protein bioconjugation and functionalization.

Target protein degradation (TPD) is a promising strategy for catalytic downregulation of target proteins through various cellular proteolytic pathways. Despite numerous reports on novel TPD mechanisms, the discovery of target-specific ligands remains a major challenge. Unlike small-molecule ligands, aptamers offer significant advantages, owing to their SELEX-based systematic screening method. To fully utilize aptamers for TPD, we designed an aptamer and N-degron ensemble system (AptaGron) that circumvents the need for synthetic conjugations between aptamers and proteolysis-recruiting units. In our AptaGron system, a peptide nucleic acid containing an N-degron peptide and a sequence complementary to the aptamer was designed. Using this system, we successfully degraded three target proteins, tau, nucleolin, and eukaryotic initiation factor 4E (eIF4E), which lack specific small-molecule ligands. Our results highlight the potential of the AptaGron approach as a robust platform for targeted protein degradation.