ACS Chemical Biology最新文献

The nonribosomal cyclic peptides (NRcPs) rufomycins, produced by Streptomyces atratus, have attracted attention as antimycobacterials. Thus, there has been interest in engineering the corresponding biosynthetic pathway to produce novel derivatives. We have thus investigated the type I thioesterase (TE) of the NRPS RufT that catalyzes rufomycin peptide macrocyclization to understand its tolerance to changes in substrate peptide sequence. In contrast to our previously reported efficient cyclization chemistry, the recombinant RufT-TE domain and RufT-PCP-TE didomain, while tolerating some substrate structural changes, both produce high levels of hydrolyzed peptide. Closer analysis led to the identification of the natural product diketopiperazine rufomyazine in assays. The data indicate, with significant implications for rufomycin production, that RufT produces both cyclic and linear peptides. We propose that rufomyazine forms non-enzymatically from the linear peptide. In addition, it provides evidence for TE domains as gatekeepers in NRPS biosynthesis.

Iron is essential for bacterial growth, and Pseudomonas aeruginosa synthesizes the siderophores pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression of femA, a gene encoding the ferri-mycobactin outer membrane transporter in P. aeruginosa. Notably, the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting their weaker iron-chelating ability compared to that of pyoverdine. ⁵⁵Fe uptake assays demonstrated that both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind AA₂-Fe or DHA₂-Fe complexes. Key interactions are conserved between FemA and these two complexes, with specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These findings show that under moderate iron deficiency, when only PCH is produced by P. aeruginosa, degradation products AA and DHA enhance iron uptake by inducing femA expression and facilitating iron transport through FemA. This provides new insights into the pathogen’s strategies for iron homeostasis.

Spurred by the authors’ own recent discovery of reactive metabolite-regulated nexuses involving lipid droplets (LDs), this perspective discusses the latest knowledge and multifaceted approaches toward deconstructing the function of these dynamic organelles, LD-associated localized signaling networks, and protein players. Despite accumulating knowledge surrounding protein families and pathways of conserved importance for LD homeostasis surveillance and maintenance across taxa, much remains to be understood at the molecular level. In particular, metabolic stress-triggered contextual changes in LD-proteins’ localized functions, crosstalk with other organelles, and feedback signaling loops and how these are specifically rewired in disease states remain to be illuminated with spatiotemporal precision. We hope this perspective promotes an increased interest in these essential organelles and innovations of new tools and strategies to better understand context-specific LD regulation critical for organismal health.

The AMP transferase, FICD, is an emerging drug target fine-tuning stress signaling in the endoplasmic reticulum (ER). FICD is a bifunctional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER-resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules that inhibit pathogenic FICD variants are lacking. Using an in vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also inhibit pathogenic FICD variants and improve proinsulin processing in β cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.

RNA guanine (G)-quadruplexes (rG4) are unique noncanonical structures composed of stacked guanine quadruplexes that play diverse roles in regulating gene expression, from transcription to protein synthesis. This study proposes a new splice-switching therapy using G-quadruplex-inducing antisense oligonucleotides (G-ASOs) to reinstate dystrophin expression in Duchenne muscular dystrophy (DMD) models. G-ASOs consist of two functionally independent domains that enable the formation of RNA/DNA hetero-G-quadruplex (hG4) structures. The antisense domain binds to complementary sequences within the target RNA, while the G-rich domain, which contains a sequence of continuous guanines (G-tract), interacts with the G-rich region of target RNA to form an hG4 structure. This precise binding forms an hG4 structure that effectively interrupts alternative splicing. In contrast to the traditional methods that block sterically, this technique employs steric hindrance by forming hG4 structures. Significantly, our findings show that hG4 structures can still form effectively even when the G-rich region of the target RNA and the antisense sequence are as much as 70 nucleotides apart. To address the challenges associated with G-quadruplex formation via G-ASO self-assembly, we developed bulge-containing G-ASOs. This enhancement improves both the efficiency of hG4 formation and the induction of exon-skipping therapy. In summary, this study highlights the potential of G-ASOs in gene therapy, specifically DMD, and marks significant progress in the development of novel therapeutic strategies. These findings highlight the effectiveness of G-ASOs in exon-skipping therapy and demonstrate the advancements in RNA structural manipulation.

Ferritin degradation pathways, particularly NCOA4-mediated ferritinophagy, are crucial for maintaining iron homeostasis. Here, we demonstrate the coexistence of two NCOA4 isoforms, one iron–sulfur cluster-free and one iron–sulfur cluster-bound, in oxygenated cell cultures. Using a combination of spectroscopic and analytical techniques, in vitro characterization of the NCOA4 fragment (383–522), denoted NCOA4-D, revealed a predominance of monomeric species with a relatively stable [2Fe-2S] cluster under normoxic conditions. The results demonstrate distinct interactions between NCOA4-D isoforms and ferritin, underscoring the influence of cellular oxygen and iron concentrations on NCOA4’s regulatory functions, pathways, and ferritin’s fate. Our findings suggest that different NCOA4-initiated degradation pathways may concurrently occur in cells and highlight the necessity of further exploring the role of the Fe–S cluster in NCOA4 as an iron-sensing mechanism for maintaining cellular iron homeostasis.

Spirochetes are especially invasive bacteria that are responsible for several human diseases, including Lyme disease, periodontal disease, syphilis, and leptospirosis. Spirochetes rely on an unusual form of motility based on periplasmic flagella (PFs) to infect hosts and evade the immune system. The flexible hook of these PFs contains a post-translational modification in the form of a lysinoalanine (Lal) cross-link between adjacent subunits of FlgE, which primarily comprise the hook. Lal cross-linking has since been found in key species across the phylum and involves residues that are highly conserved. The requirement of the Lal cross-link for motility of the pathogens Treponema denticola (Td) and Borreliella burgdorferi (Bb) establish Lal as a potential therapeutic target for the development of antimicrobials. Herein, we present the design, development, and application of a NanoLuc-based high-throughput screen that was used to successfully identify two structurally related Lal cross-link inhibitors (hexachlorophene and triclosan) from a library of clinically approved small molecules. A structure–activity relationship study further expanded the inhibitor set to a third compound (dichlorophene), and each inhibitor was demonstrated to biochemically block autocatalytic cross-linking of FlgE from several pathogenic spirochetes with varied mechanisms and degrees of specificity. The most potent inhibitor, hexachlorophene, alters Lal cross-linking in cultured cells of Td and reduces bacterial motility in swimming plate assays. Overall, these results provide a proof-of-concept for the discovery and development of Lal-cross-link inhibitors to combat spirochete-derived illnesses.

With the growing number and diversity of known genome sequences, there is an increasing opportunity to regulate gene expression through synthetic, cell-permeable small molecules. Enhancing the DNA sequence recognition abilities of minor groove compounds has the potential to broaden their therapeutic applications with significant implications for areas such as modulating transcription factor activity. While various classes of minor groove binding agents can selectively identify pure AT and mixed AT and GC base pair(s) containing sequences, there remains a lack of compounds capable of distinguishing between different AT sequences. In this work, we report on the design compounds that exhibit selective binding to -TTAA- or -TATA- containing DNA minor groove sequences compared with other AT ones. Several studies have shown that the -AATT- and -TTAA- sequences have distinct physical and interaction properties, especially in terms of their different requirements for recognition in the minor groove. Achieving strong, selective minor groove binding at -TTAA- sequences has been challenging, but DB1003, a benzimidazole-furan-furan diamidine, has demonstrated cooperative dimeric binding activity at -TTAA-. It has significantly less binding preference for AATT. To better understand and modify the selectivity, we synthesized a set of rationally designed analogs of DB1003 by altering the position of the five-membered heterocyclic structure. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1992, a benzimidazolefuran-thiophene diamidine, binds strongly to -TTAA- as a positive cooperative dimer with high cooperativity. The high-resolution crystal structure of the TTAA-DNA-DB1992 complex reveals that DB1992 binds as an antiparallel π-stacked dimer with numerous diverse contacts to the DNA minor groove. This distinctive binding arrangement and the properties of diamidines at the -TTAA- minor groove demonstrate that benzimidazole-furan-thiophene is a unique DNA binding pharmacophore. Competition mass spectroscopy and circular dichroism studies confirmed the binding stoichiometry and selectivity preference of the compounds for the -TTAA- sequence.

Chronic kidney fibrosis poses a significant global health challenge with effective therapeutic strategies remaining elusive. While cell-extracellular matrix (ECM) interactions are known to drive fibrosis progression, the specific role of focal adhesions (FAs) in kidney fibrosis is not fully understood. In this study, we investigated the role of FAs in kidney tubular epithelial cell fibrosis by employing precise nanogold patterning to modulate integrin distribution. We demonstrate that increasing ligand spacing disrupts integrin clustering, thereby inhibiting FA formation and attenuating fibrosis. Importantly, enhanced FA activity is associated with kidney fibrosis in both human disease specimens and murine models. Mechanistically, FAs regulate fibrosis through mechanotransduction pathways, and our in vivo experiments show that suppressing mechanotransduction significantly mitigates kidney fibrosis in mice. These findings highlight the potential of targeting FAs as a therapeutic strategy, offering new insights into clinical intervention in kidney fibrosis.

Rho-associated protein kinase (ROCK) inhibitors are promising therapeutic agents for reducing elevated intraocular pressure in patients with glaucoma. We explored new ROCK inhibitors derived from bioactive metabolites produced by microbes, specifically cryptic metabolites from Nocardiopsis sp. MCY7, using a liquid chromatography-mass spectrometry-based chemical analysis approach integrated with metal stress-driven isolation. This strategy led to the identification of two previously undescribed linear peptides, nocarnickelamides A and B (1 and 2), and an unreported cittilin derivative, cittilin C (3). The planar structures of 1-3 were elucidated using UV spectroscopy, high-resolution mass spectrometry, and nuclear magnetic resonance. The absolute configurations of 1 and 2 were assigned using the advanced Marfey's method. Biological assays demonstrated that nocarnickelamides (1 and 2) exhibited dual inhibitory activity against ROCK1 (IC₅₀ 29.8 and 14.9 μM, respectively) and ROCK2 (IC₅₀ 27.0 and 21.9 μM, respectively), with molecular simulations suggesting binding to the ATP-binding site. In human trabecular meshwork cells, 2 significantly inhibited the activation of ROCK-regulated cytoskeletal contraction markers such as the myosin light chain. Nocarnickelamide B (2) is a novel dual ROCK1/2 inhibitor and a potential pharmacophore for designing new therapeutic agents to reduce intraocular pressure in glaucoma.