ACS Chemical Biology最新文献

Autophagy inhibition represents a promising therapeutic approach for the management of various cancers including nonsmall cell lung cancer (NSCLC). We previously reported SBP-7455, a dual inhibitor of unc-51-like kinase 1 (ULK1) and its homologue ULK2 and described its effects on triple-negative breast cancer (TNBC) cells. Herein we report the design, synthesis, and characterization of SBP-5147 and SBP-7501, two new dual ULK1/2 inhibitors that are cytotoxic against NSCLC cells, inhibit autophagic flux in A549 cells, and present greater oral exposure than SBP-7455 at a lower dose. In addition, SBP-5147 effectively modulates autophagy and increases the expression of major histocompatibility complex (MHC) class I in NSCLC cells, which may support the rationale for ULK1/2 inhibition as a strategy to overcome resistance to immunotherapy. Together these data support the use of ULK inhibitors as part of a cancer treatment strategy, either as a single agent or in combination with current therapies.

A mechanistic basis for luciferase bioluminescence provides a glimpse into its evolutionary role for organism survival, as it provides a blueprint to engineer luciferase enzymes for advanced technological applications. Gaussia Luciferase is among the brightest natural luciferases, but (1) the evolutionary development of its luminescence behavior remains unclear, (2) recent fundamental studies utilized Escherichia coli expression systems instead of eukaryotic expression systems, and (3) notable mutants have been discovered but not integrated into a comprehensive mechanistic analysis. We describe new mechanistic observations from GLuc by addressing these gaps. We monitored the fluorescent coelenterazine-to-coelenteramide conversion to study turnover kinetics of mammalian-derived GLuc; this assay characterized the positive cooperativity kinetics of GLuc. The nonluminescent mutants, R76A and R147A, still turn over the substrate with high efficiency, each demonstrating sustained positive cooperativity. Through mass spectrometry, mutational analysis, and analytical liquid chromatography, we demonstrate that GLuc undergoes methionine oxidation during substrate turnover and that this impacts the luciferase's flash-type luminescence; we did not observe indications of covalent attachment with the substrate, product, or their intermediates. Chromatography of luciferases derived from ancestral sequence reconstruction highlighted that the extent of methionine-induced surface changes was greater for earlier ancestral luciferases. Ancestral sequence reconstruction also revealed that earlier ancestral copepod luciferases produced less light when compared to GLuc.

Poly-ADP-ribosylation (PARylation) is a reversible post-translational modification that occurs in higher eukaryotes. While thousands of PARylated substrates have been identified, the specific biological functions of most PARylated proteins remain elusive. PARylation stoichiometry is a critical parameter to assess the potential functions of a PARylated protein. Here, we developed a large-scale strategy to measure the stoichiometries of protein PARylation. By integrating chemically mild cell lysis conditions, boronate enrichment, and carefully designed titration experiments, we were able to determine the PARylation stoichiometries for a total of 235 proteins. Importantly, this approach enables the capture of all PARylation events, regardless of their amino acid acceptor linkages. We revealed that PARylation occupancy spans over 3 orders of magnitude. However, most PARylation events occur at low stoichiometric values (median 0.58%). Notably, we observed that high-stoichiometry PARylation (>1%) predominantly targets proteins involved in transcription regulation and chromatin remodeling. Thus, our study provides a system-scale, quantitative view of PARylation stoichiometries under genotoxic conditions, which serves as an invaluable resource for future functional studies of this important protein post-translational modification.

ADP-ribosyl ubiquitination is a unique form of crosstalk between post-translational modifications, where recently RNF114 has been identified as the first dedicated reader of this dual modification. New findings reveal that RNF114 extends the initial hybrid ADPr-Ub modification with K11-linked poly-Ub chains. This Letter highlights the recent advances made in methods and tools to study this hybrid signal and addresses some key questions that remain.

Type II aromatic polyketides represent a structurally diverse class of natural products with medicinally relevant properties, and their biosynthesis usually involves biosynthetic intermediates with terminal carboxyl groups. In certain instances, terminal decarboxylation occurs, which can significantly impact the structural complexity. However, the enzymes and their involved mechanisms of terminal decarboxylation in type II aromatic polyketide biosynthesis have rarely been studied. This study has now shown that MrqO5, a member of the antibiotic biosynthesis monooxygenase (ABM) family, unexpectedly functions as a terminal decarboxylase involved in the biosynthesis of murayaquinone. Furthermore, an in vitro biochemical study demonstrated that two homologous proteins of MrqO5 exhibited similar decarboxylase activity. Therefore, the functional assignment and mechanistic investigation of this polyketide terminal decarboxylase elucidated an overlooked step in type II polyketide biosynthesis. Also, the discovery of this new family of decarboxylases expands the functions of the ABM superfamily proteins. Our structural characterizations, combined with site-directed mutagenesis studies, have unveiled the key residues involved in the decarboxylation and allowed an enzymatic decarboxylation mechanism to be proposed. Our studies advance the currently incomplete understanding of type II aromatic polyketide biosynthesis and gain the insight necessary for future engineering of these enzymes.

The growing threat of multidrug-resistant bacterial infections highlights the urgent need for antibiotics with novel mechanisms of action. Gromomycins, a newly identified class of triterpene antibiotics, exhibit potent activity against Gram-positive bacteria, including drug-resistant species, through a previously uncharacterized mode of action. Here, we report the discovery of a gromomycin-like biosynthetic gene cluster in the Actinoplanes genus through a genome mining approach, leading to the isolation and characterization of new bioactive derivatives that overcome resistance to clinically used drugs in vancomycin-resistant enterococci. Mechanistic studies revealed that gromomycins induce rapid potassium ion leakage and depolarization of the bacterial membrane, resulting in bactericidal activity against Staphylococcus aureus. Gromomycins disrupt the integrity of the cytoplasmic membrane, as evidenced by large pore formation, leakage of intracellular contents, and subsequent cell lysis. Supplementation with membrane lipids and fatty acids neutralized their antibacterial activity, suggesting a direct membrane-targeting mechanism, further supported by the inability to raise gromomycin resistance and their toxic effects on eukaryotic cells. Collectively, these findings deepen our understanding of gromomycin activity and demonstrate the utility of genome mining to uncover structurally novel and biologically active natural products.

Post-transcriptional RNA modifications are ubiquitous in biology, but the fate of epigenetic ribonucleotides after RNA turnover and the consequences of their metabolism and misincorporation into nucleic acids are largely unknown. Here, we explore epigenetic ribonucleoside metabolism in human cells by studying effects on cell growth, quantifying RNA misincorporation and identifying metabolic regulators, and exploring phenotypes associated with cytotoxicity. We find that bulky N⁶-modified adenosines (i.e., i⁶A) exhibit high levels of cytotoxicity and RNA misincorporation, whereas cells dramatically restrict the misincorporation of small N⁶-modified adenosines (i.e., m⁶A), partly through sanitization by enzymatic deamination, consistent with a recent report. Epigenetic ribopyrimidines also exhibit cytotoxicity, dependent on nucleoside kinase UCK2, but only at much higher concentrations than ribopurines. We further characterize the effects of cytotoxic ribonucleoside metabolism on nucleolar morphology and protein translation. Taken together, our work provides new insights into the metabolism of epigenetic ribonucleosides and mechanisms underlying their cytotoxicity to cells.

The plasma membrane is an important interface that integrates extracellular biochemical input with biophysical organization to regulate cell behavior. Galectin-3, a multivalent glycan-binding protein, can influence both events through the formation of extracellular glycan lattices on the surfaces of glycosylated cells. Although such lattices have been proposed to reshape membrane organization, their impact on nanoscale membrane phase behavior has remained difficult to quantify. Here, we establish a link between Galectin-3 lattice formation and the remodeling of plasma membranes by using imaging fluorescence correlation spectroscopy (ImFCS) to measure diffusion coefficients of a series of fluorescently labeled probes that partition into ordered or disordered regions of the cell membrane. Across several human cell types (BeWo, BxPC3, THP-1, and HEK293T), we observed that Galectin-3 induced significant changes in the lateral mobility of membranes in a manner dependent on the capacity of Galectin-3 to oligomerize and bind glycans, and that specific glycoproteins can play outsized contributing roles. Membrane regions enriched in Galectin-3 exhibited reduced diffusion, suggesting glycan lattices can serve as nucleation sites for ordered, raft-like microdomains. Finally, we also reveal that these Galectin-3-induced changes to membrane dynamics significantly amplifies Ca²⁺ triggered scrambling of phosphatidylserine exposure. Together, these findings identify Galectin-3 as an extracellular phase organizer that translates glycan recognition into nanoscale mechanical remodeling of the plasma membrane, potentially serving as a generalizable mechanism for fine-tuning cell behavior.

Fungal secondary metabolites have historically provided important applications in a variety of industries. Penicillium camemberti, a fungus with a role in cheese production, was domesticated to food use partly due to its metabolically depleted characteristic, minimizing the risk of toxic compound formation. However, antiSMASH analysis of the genome reveals that strains of the species do contain various cryptic biosynthetic gene clusters and, thus, have the potential capability of producing multiple secondary metabolites despite its limited compound production under normal laboratory conditions. Here, we genetically engineered Penicillium camemberti strain IMV00769, which is genetically similar to cheese-making isolates, by deleting negative global regulator, mcrA. This deletion resulted in the production of secondary metabolites not previously produced by this strain, including fumigermin, a compound patented for cosmetic applications for the reduction of skin wrinkles, enhancement of skin elasticity, and skin whitening. Our findings highlight the power of global regulator manipulation to activate cryptic biosynthetic pathways and expand the range of natural products accessible from domesticated fungal strains.

The Gram-positive pathogen Streptococcus pneumoniae, like the majority of bacteria, contains a peptidoglycan-based cell wall whose structure is highly dependent on the action of penicillin-binding proteins (PBPs). While the β-lactam antibiotics have been employed as an antimicrobial strategy for nearly a century, much remains unclear about how inhibitor structure informs potency and PBP isoform selectivity. Here, we obtained high-resolution structures (<2Å) of S. pneumoniae PBP1b cocrystallized with 6 β-lactams. Surprisingly, 2 structures feature a noncanonical conformation of the covalent "acyl-enzyme complex." To clarify how protein-ligand interactions mediate inhibitor binding, we applied molecular modeling and molecular mechanics-based dynamics analyses. Our analyses illustrate how seemingly minimal changes to inhibitor structure modulate β-lactam binding mode and inhibitor potency, as described by the metric k_inact/K_I. Furthermore, we demonstrate that persistent interaction in the covalent acyl-enzyme complex between the inhibitor carboxylate and a highly conserved three-residue motif is not fully predictive of k_inact/K_I for PBP1b. In silico modeling suggests that the noncovalent preacyl complex may leverage this interaction, but a postacylation change in ligand conformation may accompany acylation in some inhibitors. The elucidation of key PBP1b ligand-receptor interactions pre- and postacylation will inform the rational design of novel PBP inhibitors and probes.