ACS Chemical Biology最新文献

Secondary metabolite profiling of a recently described new cyanobacterial species Floridanema aerugineum identified abundant amounts of the polyketide-peptide tychonamide A. Investigation of the biosynthetic gene clusters in the F. aerugineum genome identified a putative tychonamide biosynthetic gene cluster with module and domain architecture consistent with the tychonamide A structure. High resolution liquid chromatography–mass spectrometry/MS (LC-MS/MS) identified new tychonamide analogs C-E (1–3), and bioactivity predictions led us to test the protease inhibition activities of the tychonamides. The results of this analysis illustrated the biosynthesis of the 3-amino-2,5,7-trihydroxy-8-phenyloctanoic acid moiety (Atpoa) in tychonamide A, further deduced the absolute configuration of tychonamide A, and uncovered new biological activities (human neutrophil elastase inhibition) that may be relevant in environmental science and pharmaceutical development.

Chemokines are secreted blood proteins that steer leukocyte migration in the inflammatory response. Neutralization of chemokines is believed to be a beneficial therapeutic strategy for the treatment of inflammation-associated diseases. Proteolytically stable chemokine-binding peptides could be suitable candidates for the development of chemokine-neutralizing agents. Here, we report the mirror-image phage display selection of cyclic all-D-peptides against the C–X–C motif chemokine ligand 8 (CXCL8). Selection yielded structurally diverse all-D-peptides with submicromolar affinity to the target CXCL8 chemokine and different selectivity to related chemokines. Binding of these all-D-peptides caused dissociation of the native CXCL8 dimer and disruption of its binding to GAGs, without an effect on in vitro cell migration. This work demonstrates the example of mirror-image phage display selection of cyclized all-D-peptides and its utility for the development of chemokine-binding agents.

The growth of bacterial pathogens is limited by nutritional immunity, where the infected host deploys transition metal scavenging proteins including calprotectin (CP) to starve the bacterium of essential transition metals. Prior work reveals that CP induces a significant Zn- and Fe-starvation response in the Gram-negative opportunistic pathogen Acinetobacter baumannii in liquid culture. Here, we employ a quantitative chemoproteomics platform to pinpoint changes in abundance-corrected cysteine reactivity─and by extension cellular metal occupancy in metalloenzymes─that occur when A. baumannii is challenged with physiological CP in liquid culture relative to an untreated WT control. Changes in protein abundance with CP stress reveal a pronounced Zn-limitation and Fe-starvation response and reciprocal regulation of three enzymes of central carbon metabolism, including aconitase. A majority of the 2645 quantifiable Cys-containing peptides that show an increase in abundance-corrected Cys reactivity (150) are derived from known Zn-, Fe-, and Fe–S-cluster proteins, revealing a significant decrease in metal occupancy (undermetalation) across the proteome. Myriad cell processes are impacted by undermetalation, including enzymes that function in the TCA cycle and respiration, GTP metabolism, ribosome remodeling, tRNA charging, and proteostasis. In an effort to identify an undemetalated client enzyme for the candidate GTPase-powered metallochaperone ZigA, we performed this chemoproteomics experiment in a CP-stressed ΔzigA strain relative the CP-stressed wild-type strain. These findings reveal that the loss of ZigA is effectively silent in this assay. We conclude that CP induces a widespread, negative impact on the metalation status of the metalloproteome that results in a significant nutrient limitation response.

DMAT-type prenyltransferases (PTases) are essential for the biosynthesis of structurally diverse hybrid terpenoids. Here, we report the discovery and functional characterization of DiaB, a previously uncharacterized fungal PTase identified through genome mining. Heterologous expression of the dia gene cluster in Aspergillus oryzae NSAR1 led to the production of five new meroterpenoids. In vivo and in vitro studies revealed that DiaB is a PTase that catalyzes the regiospecific O-farnesylation of free l-threonine and l-serine, a previously unknown activity for this enzyme class in fungi. The functions of all enzymes in the cluster were elucidated, revealing a novel pathway to prenylated amino acids. This work expands the substrate scope of DMAT-type PTases and provides a new biocatalytic tool for meroterpenoid diversification.

Hydropersulfides (RSSH) are increasingly recognized for their potent redox-modulating and cytoprotective properties, yet their therapeutic potential remains underexplored due to their chemical instability. Here, we report the design and optimization of azoreductase (AzoR)-responsive RSSH donors for programmed release. We explore azo-linked precursors that undergo AzoR-mediated reduction to form phenylamino intermediates, which are designed to trigger RSSH release via spontaneous 1,6-elimination. A series of precursors was synthesized to evaluate the structure–activity relationships governing elimination efficiency. Direct attachment of aliphatic or aromatic RSSH moieties at the benzylic position of the azobenzene core (azodisulfides, AzDS-1, AzDS-2, AzDS-3) resulted in stable intermediates that failed to eliminate RSSH under physiological conditions. Even an electron-rich substituent such as a methoxy group on the azobenzene core was not sufficient to drive the elimination. To improve leaving group ability, we synthesized an azoperthiocarbonate donor (AzPTC) that enabled AzoR-triggered RSSH release but also underwent undesired nonenzymatic hydrolysis. Finally, incorporation of a hydrolytically stable perthiocarbamate yielded the precursor azoperthiocarbamate (AzPTB) that released RSSH selectively upon AzoR activation via 1,6-elimination, decarboxylation, and an intramolecular cyclization cascade. AzPTB demonstrates high enzymatic turnover and excellent stability under neutral aqueous conditions. These results provide key structural parameters that govern RSSH release via 1,6-elimination and establish AzPTB as a robust platform for site-selective delivery. This work expands the chemical biology toolkit for probing RSSH signaling and supports future efforts in redox-based therapeutic development.

Citrullination and homocitrullination of arginine and lysine can significantly impact protein structure and function. Citrullination of arginine is an enzymatic modification catalyzed by Protein Arginine Deiminases (PADs). Homocitrullination of lysine is a nonenzymatic modification that occurs in the presence of high concentrations of cyanate. Both post-translational modifications are elevated in Rheumatoid Arthritis (RA) and other inflammatory diseases. Moreover, autoantibodies targeting these PTMs are associated with the development of RA. Identifying arginine and lysine residues that are hypersensitive to these modifications is critical for deepening our understanding of the functional effects of (homo)citrullination. Current methods use a phenylglyoxal-biotin probe for the protein-level identification of citrullinated proteins, however, this platform does not inform on the exact site of citrullination. Herein we describe the development of a desthiobiotin-phenylglyoxal (DB-PG) probe, which can be used to selectively enrich and subsequently release (homo)citrullinated peptides for the site-specific identification of citrullinated arginines and homocitrullinated lysines. (Homo)citrullinated peptides enriched using DB-PG were subjected to quantitative mass-spectrometry analysis to (1) identify PAD2 and PAD4-selective citrullination sites across ∼800 arginine residues and (2) evaluate ∼1400 lysine residues for sensitivity to homocitrullination by cyanate. Projecting forward, this platform will enable the comprehensive analysis of (homo)citrullination in complex proteomes.

Inspired by the uncommon furanose configuration of 3′-deoxyapio-containing nucleic acids (apioNAs), we developed a facile and convenient synthesis of 24 building blocks of this modified nucleic acid monomer, including phosphoramidites, H-phosphonates, solid supports, and nucleoside triphosphates. The building blocks included those containing the four canonical RNA bases A, G, U, and C as well as T and 5-methyl-C and were synthesized starting from a single common sugar intermediate derived from d-(+)-xylose. DNA and RNA duplexes with a single apioNA modification in one strand were less thermodynamically stable than unmodified DNA or RNA. The crystal structure of apioNA-modified RNA octamer showed that the apioNA residue adopts an RNA-like structure but local reorientation of the apioNA sugar and 2′-phosphate and the difference in helical rise on the 5′ side of the apioNA T relative to RNA likely contribute to the destabilizing effect of apioNA residues. At the terminus of a DNA strand, this modification provides extremely high resistance against both 3′- and 5′-exonucleases even when linked to the adjacent residue by a phosphodiester moiety. Molecular modeling of a DNA duplex containing apioNA was used to rationalize the DNA duplex destabilization and the exonuclease resistance resulting from incorporation of the apioNA residue. Use of apioNA NTPs as substrates for previously engineered α-l-threofuranosyl polymerases depends on both the enzyme and the nucleobase. These data indicate that apioNAs warrant further evaluation, and the building blocks synthesized will allow incorporation of apioNA into therapeutic oligonucleotides.

Aggregation during solid-phase peptide synthesis (SPPS) remains a key limitation, often leading to low coupling efficiencies and poor crude purities. Our previously introduced synthesis tag (“SynTag”) for chemical protein synthesis combines six C-terminal Arg(Pbf) residues with a MeDbz linker to suppress aggregation via helical structure induction and serves as a handle for native chemical ligation (NCL). To apply the concept to short, yet aggregation-prone sequences, some practical limitations need to be addressed: Tag removal needs to be simplified, its utility demonstrated on more commonly used resin types and loadings, and the method must be effective on larger scale. To this end, we developed a simplified C-terminal hexaarginine tag (“ArgTag”) and refined an enzymatic method for efficient removal with Carboxypeptidase B, enabling selective cleavage under mild, linker-free conditions. We evaluated the ArgTag across six solid supports (resins) of varying polarity and loading. Using automated fast-flow SPPS (AFPS), we observed consistent aggregation suppression and improved crude purities across all resin types. We finally demonstrated the efficiency of our ArgTag on larger scale using more economical synthesis parameters. This work broadens the applicability of the SynTag strategy to short, yet difficult peptide sequences and offers a more scalable solution to improve SPPS efficiency for challenging targets.

Adenosine Deaminases Acting on RNA (ADARs) are an important class of RNA editing enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Since inosine is typically read as guanosine (G) during translation, ADARs can produce A to G transitions in dsRNA. Site-directed RNA editing (SDRE) is a promising therapeutic tool wherein guide RNAs can be used to direct endogenous human ADARs to reverse disease-causing mutations in specific RNA transcripts. Guide RNA (gRNA) modifications at locations that contact the ADAR active site are often used to improve editing efficiency. However, little is known about rate-enhancing chemical modifications in the gRNA at the dsRNA binding domain (dsRBD)-RNA interface. Analysis of published crystal structures of ADAR2 bound to dsRNA suggested positions at this interface would be sensitive to gRNA modification. In this work, gRNAs bearing 2′-modifications in the dsRBD binding site were synthesized and subsequently tested to determine their effects on the editing rate of therapeutically relevant ADAR targets. We found that replacing a single 2′-OH at specific positions on the gRNA with a 2′-F substantially increased the rate of in vitro ADAR2-catalyzed adenosine deamination for two different sequences, whereas 2′-OMe at these positions was inhibitory. This effect was also validated in cellulo. The rate of ADAR1-catalyzed deamination is not stimulated by these 2′-F modifications. A crystal structure of an ADAR2 fragment bound to duplex RNA bearing a single 2′-F at guide position +13 suggested a favorable interaction between the side chain of N241 of the auxiliary ADAR2 monomer and the 2′-F modification. Furthermore, electrophoretic mobility shift assays and mass photometry indicate 2′-F at position +13 facilitates ADAR2 dimerization on the RNA substrate. This work advances our understanding of the RNA features that define superior ADAR substrates and inform the design of gRNAs for therapeutic RNA editing.