ACS Chemical Biology最新文献

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.

Developing novel nonribosomal peptides (NRPs) requires a comprehensive understanding of the enzymes involved in their biosynthesis, particularly the substrate amino acid recognition mechanisms in the adenylation (A) domain. This study focused on the A domain responsible for adenylating l-2,4-diaminobutyric acid (l-Dab) within the synthetase of polymyxin, an NRP produced by Paenibacillus polymyxa NBRC3020. To date, investigations into recombinant proteins that selectively adenylate l-Dab─exploring substrate specificity and enzymatic activity parameters─have been limited to reports on A domains found in enzymes synthesizing l-Dab homopolymers (pldA from S. celluloflavus USE31 and pddA from S. hindustanus NBRC15115), which remain exceedingly rare. The polymyxin synthetase in NBRC3020 contains five A domains specific to l-Dab, distributed across five distinct modules (modules 1, 3, 4, 5, 8, and 9). In this study, we successfully obtained soluble A domain proteins from modules 1, 5, 8, and 9 by preparing module-specific recombinant proteins. These proteins were expressed in E. coli BAP-1, purified via Ni-affinity chromatography, and demonstrated high specificity for l-Dab. Through sequence homology analysis, three-dimensional structural modeling, docking simulations to estimate substrate-binding sites, and functional validation using alanine mutants, we identified Glu281 and Asp344 as critical residues for recognizing the side chain amino group of l-Dab, and Asp238 as essential for recognizing its main chain amino group in the A domain. Notably, these key residues were conserved not only across the A domains in modules 1, 5, 8, and 9 of P. polymyxa NBRC3020 but also in those of the P. polymyxa PKB1 strain, as confirmed by sequence homology analysis. Interestingly, in pldA and pddA, the key residues involved in recognizing the side-chain amino group of l-Dab, which are conserved among polymyxin synthetases of NBRC3020 and PKB1 strain, were not observed. This suggests a potentially different mechanism for l-Dab recognition.

The threat of multidrug-resistant bacteria has been increasing steadily in the past century, posing a major health risk (Organización Mundial de la Salud. Directrices Sobre Componentes Básicos Para Los Programas de Prevención y Control de Infecciones a Nivel Nacional y de Establecimientos de Atención de Salud Para Pacientes Agudos; Organización Mundial de la Salud: Ginebra, 2017). Even though every year, 226 million antibiotics are prescribed in the United States alone, 50% of these prescriptions are inappropriate for the patient's condition (CDC. Get Smart about Antibiotics Week; Centers for Disease Control and Prevention. 2016,https://www.cdc.gov/media/dpk/antibiotic-resistance/antibiotics-week-2016/dpk-antibiotics-week-2016.html). The increasing abuse of antibiotics in healthcare as well as agriculture has resulted in the rise of antibiotic resistance at an alarming rate. In a clinical setting, timely and accurate recognition of the pathogen allows for the most effective choice of treatment, highlighting the need for novel, fast, and reliable antibiotic susceptibility testing. Traditional susceptibility testing techniques require costly and complex experimental setups or extended cell incubation periods, delaying a timely treatment response to the infection. Herein, we report that a short-pulse fluorescent d-amino acid (FDAA)-based approach provides insight not only into bacterial antibiotic susceptibility but also into the mechanism of action of the antibiotic. Using the FDAA-labeling signal as a reflection of peptidoglycan (PG) integrity after antibiotic treatment, we observed that drugs targeting PG biosynthesis resulted in a significant decrease in fluorescence, while antimicrobials affecting other cellular targets resulted in no fluorescence changes. Our method was validated and optimized via fluorescence microscopy and spectrofluorometry, shortening the required procedure time to 15 min and providing reliably reproducible results. Significantly, we demonstrate that our protocol can be used to identify β-lactam-resistant bacterial strains, further demonstrating the utility of these valuable molecular tools.

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.

Class A G protein-coupled receptors (GPCRs) are key mediators in numerous signaling pathways and important drug targets for several diseases. A major shortcoming in GPCR ligand screening is the detection limit for weak binding molecules, which is especially critical for poorly druggable GPCRs. Here, we present a proximity-based screening system for class A GPCRs, which adopts the natural two-step activation mechanism of class B GPCRs. In this approach, class A/B chimeras with the extracellular domain of the class B receptor CRF₁R grafted to the transmembrane domain of target class A receptors are stimulated with hybrid ligands. These ligands contain a high-affinity peptide derived from CRF, which recruits the hybrid ligands to the engineered target GPCR, dramatically increasing the local concentration of the test substances. We exemplified this method for neurotensin receptor 1 (NTR₁) and endothelin receptor B (ET_B), two important class A GPCR drug targets for pulmonary arterial hypertension or psychological disorders and neurodegenerative diseases. We observed >20× activity enhancement by the directed proximity approach, enabling the detection of weakly activating sequences that would have otherwise remained undetected. Our approach allows to probe GPCR activation in the membrane of living cells and may be especially useful for GPCRs for which it has been difficult to generate small drug-like molecules.

Precise cell elimination within intricate cellular populations is hampered by issues arising from the multifaceted biological properties of cells and the expansive reactivity of chemical agents. Current chemical platforms are often limited by their complexity, toxicity, and poor physical/chemical properties. Here, we report on the synthesis of a structurally versatile library of chemically tunable bisacylphosphane oxides (BAPOs), which harnesses the spatiotemporal precision of light delivery, thereby establishing a universal strategy for on-demand, precise cellular ablation in vitro and in vivo.

Lysosomal storage disorders (LSDs) and adult neurodegenerative disorders like Alzheimer's disease (AD) share various clinical and pathophysiological features. LSDs are characterized by impaired lysosomal activity caused by mutations in key proteins and enzymes. While lysosomal dysfunction is also linked to AD pathogenesis, its precise role in disease onset or progression remains unclear. Lysosomal ionic homeostasis is recognized as a key feature of many LSDs, but it has not been clinically linked with AD pathology. Thus, investigating whether this regulation is disrupted in AD is important, as it could lead to new therapeutic targets and biomarkers for this multifactorial disease. Here, using two-ion mapping (2-IM) technology, we quantitatively profiled lysosomal pH and Ca²⁺ in blood-derived monocytes from AD patients and age-matched controls and correlated lysosome ionicity with age and key markers of AD pathology, namely, amyloid deposits, tauopathy, neurodegeneration, and inflammation. Together, the data show that the ionic milieu of lysosomes is dysregulated in monocytes of AD patients and correlates with key plasma biomarkers of AD. Using a machine learning model based on the above parameters, we describe a proof-of-concept combinatorial biomarker platform that accurately distinguishes between patients with AD and control participants with an area under the curve of >96%. Our study introduces a convenient, noninvasive platform with the potential to diagnose Alzheimer's disease based on fluid, cellular, and molecular biomarkers. Further, these findings highlight the potential for investigating therapeutic mechanisms capable of restoring lysosome ionic homeostasis to ameliorate AD.

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.

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.