ACS Chemical Biology最新文献

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is widely valued for its speed and sensitivity in biomolecular analysis, yet the inherently nonquantitative nature hampers its use in many applications including high-throughput screening. Here, we introduce an iodo-based labeling strategy that enables accurate quantification of peptides and peptide libraries using high-resolution MALDI FT-ICR MS. The peptides are coupled at the N-terminus with benzoic acid (BA) or 4-iodobenzoic acid (IBA) to generate the analyte and its internal standard, respectively, differing only by a single iodine substitution. This new labeling strategy was first validated using a simple four-peptide mixture, and subsequently applied to quantitatively evaluate glycine-zipper peptide libraries containing up to 125 members for the discovery of bacterial-binding peptides. Screening of these libraries against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis revealed peptides with strong and selective interactions with the bacteria. This universally applicable, cost-effective, and straightforward approach for peptide quantification significantly enhances the reliability and accuracy of high-throughput peptide screening via MALDI FT-ICR MS.

Hydrogen sulfide (H₂S) fluorescent probes are important tools for imaging and understanding H₂S in biology. One significant requirement for such probes is that they are highly selective for H₂S over competing analytes, which are often present at much higher levels than endogenous H₂S. Different approaches have been used to generate selective H₂S probes, and recently, highly selective probes using 2-thiophene esters have been reported. We report here that in contrast to prior reports, thiophene ester probes are not selective for H₂S but rather report on both biothiols and esterase activity. We do demonstrate, however, that the rate of reactivity toward H₂S can be enhanced by incorporating an ortho aldehyde, leading to an 85-fold rate enhancement. We anticipate that this work will further clarify effective approaches for selective H₂S detection and also advance strategies for improving the selectivity of electrophilic probes for H₂S and other related nucleophiles.

N–H/N interactions, between an amide N–H on one residue (i + 1) and the amide N lone pair on the prior (i) residue, have been observed in key structures in proteins, including turns, loops, and α-helices. However, there remains an incomplete understanding about the inherent nature of N–H/N interactions and how they can impact protein structure and dynamics. The crystal structure of a molecule (Boc-hyp(4-I-Ph)-NHCy) with an N–H_i+1/N_i interaction was obtained. This structure had a close H_i+1···N_i distance of 2.30 Å, well below the 2.75 Å sum of the van der Waals radii of H and N. This structure exhibited substantial (12°; 0.17 Å) pyramidalization (partial sp³ character) of the proline N_i nitrogen. This pyramidalization reduces the planarity and electron delocalization inherent to an amide bond, as a result of the nitrogen N_i lone pair engaging directly with the hydrogen on the subsequent amide. DFT calculations on Ac-Pro-NHMe indicate that nitrogen pyramidalization and partial loss of amide planarity are inherent features of an N–H/N interaction. In addition, calculations demonstrate that the δ conformation, which has an N–H/N interaction, exhibits a substantially lower barrier to proline cis-trans isomerism than the PPII conformation, which lacks an N–H/N interaction, and that a closer N–H/N interaction and greater N pyramidalization are observed in the transition state than in the ground states. Analysis of small-molecule crystal structures indicates that close H···N distances and substantial N_i pyramidalization are inherent to N–H/N interactions. N–H/N interactions are ubiquitous in the PDB at all 20 canonical amino acids when they are in the δ/α_R or δ’/α_L conformations, consistent with N–H/N interactions being central local structural elements in proteins, as well as in early folding transitions in proteins (single residue δ/α_R → type I β-turn → 3₁₀-helix → α-helix).

Caspase-1 is a key mediator of the inflammasome pathway, which is associated with several inflammatory disorders including obesity, diabetes mellitus, cardiovascular diseases, cancers, and chronic respiratory diseases. Although substrate-based probes can be used to visualize the activity of caspase-1, none are selective enough for use as imaging agents. Here, we report the design and synthesis of an AND-gate substrate probe (Cas1-Cat-Cy7) that requires processing by both caspase-1 and cathepsins to produce a signal. Because both enzymes are found together and active in tissue locations where cells are undergoing caspase-1-mediated pyroptosis, the resulting probe can be used to image sites of caspase-1-mediated inflammation. We demonstrate that the probe produces selective signals in ex vivo biochemical and cellular assays and in a mouse model of acute inflammation.

Developing potent and selective protease inhibitors remains a grueling, iterative, and often unsuccessful endeavor. Although macromolecular inhibitors can achieve single-enzyme specificity, platforms used for macromolecular inhibitor discovery are optimized for high-affinity binders, requiring extensive downstream biochemical characterization to isolate rare inhibitors. Here, we developed the High-throughput Activity Reprogramming of Proteases (HARP) platform. HARP is a yeast-based functional screen that isolates protease-inhibitory macromolecules from large libraries by coupling their inhibition of endoplasmic reticulum-resident proteases to a selectable phenotype on the cell surface. Endowed with high dynamic range and resolution, HARP enabled the isolation of low-nanomolar-range inhibitory nanobodies against tobacco etch virus protease and human kallikrein 6, including a rare 10.5 nM K_I TEVp uncompetitive inhibitor. Structural modeling and deep sequencing all provide insights into the molecular determinants of inhibitors and reinforce HARP’s foundational findings. Overall, HARP is a premier platform for discovering modulatory macromolecules from various synthetic scaffolds against enzyme targets.

Conserved biosynthetic gene clusters (BGCs) are often tied to the production of natural products that perform critical functions in an organism’s physiology and ecological interactions. Here, by phylogenetic analysis across the bacterial genus, we report the obligate conservation of a BGC in genomes of cosmopolitan marine Microbulbifer bacteria. This genus is a common member of marine microbiomes, and this BGC was conserved in Microbulbifer genomes regardless of phylogenetic or geographical dispersal. The post-translationally modified peptidic product encoded by this BGC─which was accessed via heterologous production and its structure elucidated using a combination of mass spectrometry and NMR spectroscopy─was found to be a copper chelator. Similar BGCs were then found in genomes of other marine bacterial genera coinhabiting the microbiomes of sponges and corals. The phylogenomic workflows described herein were implemented in a pedagogic setting at the Georgia Institute of Technology to provide hands-on instruction to undergraduate students in bacterial phylogeny, genome mining, and natural product chemistry.

Quorum sensing (QS) allows bacteria to respond to changes in cell density and participate in collective behaviors. Interfering with QS could provide a strategy to block pathogenicity, reduce biofouling, and support biotechnology. Many common Gram-negative bacteria use LuxR-type QS receptors that regulate gene transcription in response to N-acyl l-homoserine lactone (AHL) signals. The most-studied LuxR-type receptors operate via an associative mechanism, i.e., they dimerize and associate with DNA upon ligand binding. In contrast, members of the less-studied class of dissociative LuxR-type receptors bind DNA as dimers in the absence of a ligand and dissociate from DNA upon ligand binding. Few chemical tools to modulate dissociative receptor activity are known. Such probes could provide new entry into mechanistic studies of LuxI/LuxR-type QS in general. In this report, we describe the discovery of synthetic modulators of EsaR, a dissociative LuxR-type receptor present in the plant pathogen Pantoea stewartii, based on AHL scaffolds. Compound activity was evaluated using both cell-based EsaR reporters and a phenotypic assay. We identified compound features associated with agonistic activity in EsaR, some of which were comparable to those of synthetic ligands active in other LuxR-type receptors. However, in contrast to prior studies of AHL mimics, no antagonists were uncovered in EsaR. These results provide chemical strategies to start to investigate mechanisms of ligand response in EsaR and define receptor features driving dissociative vs associative mechanisms in the LuxR-type receptor family. Our findings also suggest that alternate approaches may be required to develop competitive antagonists for dissociative LuxR-type receptors.

Most microorganisms produce far fewer secondary metabolites under laboratory culture conditions than would be expected based on the number of biosynthetic gene clusters (BGCs) present in their genomes. One strategy for inducing secondary metabolite production is to add chemical elicitors that disrupt bacterial metabolism. This one-strain-many-compounds (OSMAC) strategy has been used successfully to discover a broad range of natural products. However, traditional strategies for detecting changes in natural product production are not well suited to characterizing variations in the full secondary metabolome under elicitation conditions. One efficient tool to differentiate metabolites between experiments is IsoAnalyst, a parallel stable isotope labeling method that connects secondary metabolites to BGCs by determining the rates of incorporation for a set of isotopically labeled secondary metabolism building blocks. In this study three strains of Paraburkholderia were profiled under a range of OSMAC conditions and changes in secondary metabolism characterized using a combination of analytical tools including IsoAnalyst. Using these profiles, we assessed the degree of novel secondary metabolite production under different elicitation conditions. Prioritization of one compound class strongly induced in the presence of the antibiotic rifaximin led to the discovery of 2-hydroxyacyl putrescine compounds putrescinamides A (1) and B (2). The structures of these new metabolites were determined through a combination of multidimensional NMR experiments and total synthesis, which permitted the determination of their full absolute configurations. Together these stable isotope labeling experiments provide a unique perspective on system-wide variation in de novo secondary metabolite biosynthesis under elicitor conditions and highlight the impact of elicitor selection on metabolite induction in Burkholderiales strains.

Histone methylation depends on one-carbon metabolism, with methyl groups donated by methionine-, serine-, and glucose-derived intermediates. To dissect the metabolic origins of histone methylation, we developed Relative Quantitative Methyl Isotopomer Distribution Mass Spectrometry (RQMID-MS), a high-resolution mass spectrometry-based method that uses diagnostic low-mass fragment ions to quantify methyl group transfer from isotope-labeled precursors. Using this method, we mapped methylation sources to histone lysines in glioblastoma cells under nutrient and oxygen stress. Methionine was the dominant methyl donor under replete condition. Under combined serine and methionine depletion or prolonged methionine depletion alone, glucose emerged as a key compensatory source, particularly in U87 cells with elevated 3-phosphoglycerate dehydrogenase (PHGDH) expression. In contrast, U251 cells favored exogenous serine and glycine, correlating with higher levels of serine hydroxymethyltransferase 2 (SHMT2) expression. Hypoxia initially enhanced glucose-derived methylation but later suppressed it, likely due to impaired vitamin B₁₂-dependent remethylation of homocysteine. RQMID-MS enables precise tracking of methyl donor routing to histones and offers a robust platform for studying metabolic and epigenetic crosstalk in cancer and beyond.

Activity-based probes have been instrumental in the study of proteases, and quenched fluorescent versions can be utilized in real time imaging. Unfortunately, this application has not yet been reported for serine proteases, which make up the largest mechanistic class of proteases. Here, we describe quenched activity-based probes for detection of serine proteases, specifically the neutrophil serine proteases: neutrophil elastase, proteinase 3, and cathepsin G. We demonstrate that these reagents can selectively label serine proteases in complex proteomes and we illustrate their use in the live cell imaging of activation of primary human neutrophils. We expect that these reagents will find use in real-time imaging of active neutrophil serine proteases and may be further developed for imaging of other serine proteases.