ACS Chemical Biology最新文献

ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD⁺ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC₅₀ values below 5 μM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC₅₀ value of 1.3 μM. We observed structural differences in the NAD⁺ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds.

AlpJ-family oxygenases catalyze distinctive oxidative B-ring cleavage and rearrangement reactions during the biosynthesis of atypical angucycline natural products, which are characterized by unique chemical structures and diverse biological activities. While the individual functions of a few AlpJ-family enzymes have been reported, there is a lack of systematic exploration and functional comparison within this enzyme family, hindering a comprehensive understanding of the AlpJ-family oxygenases. In this study, we have systematically explored and analyzed AlpJ-family oxygenases, identifying 49 representative homologues, which can be classified into two distinct evolutionary groups. We revealed that enzymes from different groups exhibit clear functional differentiation, catalyzing the same angucycline substrate dehydrorabelomycin into distinct products, whereas enzymes within the same group display more similar catalytic functions with varying degrees of functional overlap. This underscores the intriguing functional conservation and divergence of the AlpJ-family oxygenases. In addition, we report the first crystal structure of a Group I enzyme, PenE. Structural analysis and site-directed mutagenesis identified key structural features and residues within AlpJ-family oxygenases, which harbor hydrophobic substrate-binding pockets at both the N- and C-termini, both of which are essential for function. Our findings provide valuable insights into the evolution, catalytic mechanisms, and functional divergence of this unique family of oxygenases. Further investigation of these newly identified AlpJ homologues and their associated biosynthetic gene clusters will facilitate the discovery of enzymes with unique catalytic mechanisms and bioactive atypical angucyclines with novel structures.

Telomerase reverse transcriptase is a ribonucleoprotein complex that maintains telomere length in rapidly dividing cells, thus enabling cellular immortality. Despite being recognized as an important cancer target for decades, no small molecule telomerase inhibitors have been approved as anticancer therapeutics to date. Several limitations, including the absence of high-throughput screening tools, have posed challenges to the telomerase drug discovery field. Here, we describe a high-throughput, fluorescently coupled screening methodemploying a chemically modified reporter nucleotide. We utilize the Tribolium castaneum telomerase as a surrogate model as it shares a high degree of active site homology with the human enzyme . We piloted this tool by screening a chemical library of ∼3600 nucleoside mimetics todemonstrate excellent assay quality, and identified 2 compounds with inhibitory activity that were further validated in a direct enzymatic assay. Our work introduces a method that has the potential to uncover novel telomerase inhibitors for further drug discovery efforts.

Oxidoreductase enzymes are widely used biocatalysts due to their high enantioselectivity and broad substrate compatibility in useful transformations. Many oxidoreductases require nicotinamide cofactors (i.e., NAD(P)H). To replace this costly natural cofactor, synthetic nicotinamide cofactor biomimetics (NCBs) offer different shapes, binding affinities, and reducing potentials that exceed the capabilities of wild-type NAD(P)H. However, the ill-defined structure-activity relationships (SARs) of various NCBs slow rationally guided innovation, such as customized reducing potentials. Here, we dissect two essential elements of NCB design, holding the nicotinamide invariant. First, the linker length between the nicotinamide and an unconjugated aromatic ring uncovered unexpected benefits to redox activity for two or three carbon linkers. Second, substitution on this unconjugated aryl group (Ring 2) might not be expected to affect activity. However, SAR trends demonstrate substantial benefits to reductive potential conferred by electron-donating functionalities on Ring 2. Furthermore, catalysis by two enzymes demonstrates enzyme-dependent tolerance or sensitivity to the NCB structures. Density functional theory (DFT) and computational modeling provide a theoretical framework to understand and build upon these observations. Ring 2 reaches up to the nicotinamide to stabilize its positive charge after oxidation through π-π stacking and charge transfer. Thus, the systematic examination of NCB's stability, electrochemical redox potentials, and kinetics uncovers trends for the improved design of NCBs.

Nitrogen mustards are a family of clinically used anticancer drugs that contain a DNA-alkylating bis(2-chloroethyl)amino group. Appending the bis(2-chloroethyl)amino alkylating agent to noncovalent DNA-binding groups such as intercalators, polyamides, or polyamines has the potential to yield DNA-targeted anticancer agents with improved potency. In the work reported here, substituted quinoxaline groups were explored as minimal intercalators expected to confer noncovalent DNA-binding properties on a bis(2-chloroethyl)anilino mustard alkylating unit. A quinoxaline unit with a cationic dimethylamino-containing side chain was found to be a more potent DNA-alkylating and cross-linking agent than the clinically used mustard chlorambucil (Chb). The results of dye displacement and multiple DNA alkylation assays showed that the quinoxaline ring binds noncovalently to duplex DNA, likely via intercalation. The quinoxaline-mustard conjugate was more active than Chb against a variety of cancer cell lines. Evidence is presented, showing that both the quinoxaline-mustard and the clinically used drug Chb formed aggregates in aqueous buffer; however, the results clearly show that the propensity to form aggregates clearly does not abrogate the DNA-alkylating properties or bioactivity of these compounds.

Bacteria cope with the limitation of iron by producing siderophores, small molecules they export that have high affinity for iron. Once complexed, the ferric siderophore is transported into the cell through specialized receptors allowing the iron to be released and used in a variety of biological processes. Many peptide siderophores that use catechol, phenolate, or oxazoline/thiazoline groups to coordinate iron are produced by a family of enzymes called nonribosomal peptide synthetases (NRPSs). Alternately, a smaller family of NRPS-independent siderophores (NISs) is produced by a different biosynthetic strategy. The NIS pathways employ one or more NIS synthetases that combine an amine commonly harboring a hydroxamate with a carboxylate substrate. Discovered in 2007 in an uncharacterized Nocardia species, a siderophore called nocardichelin was identified and chemically characterized that contained features of both NIS and NRPS siderophores. Nocardichelin contains an N-salicyloxazoline moiety, predicted to be built by a modular NRPS, and a dihydroxamate containing N-hydroxy-N-succinylcadaverine and N-hydroxy-N-tetradecenoylcadaverine groups. To explore this potential hybrid NRPS/NIS, we identified a biosynthetic gene cluster in Nocardia carnea containing 13 enzymes and four proteins involved in transport. We have functionally characterized four of the enzymes for their activity and substrate specificity and further solved the structures of two enzymes. We present our discovery and initial characterization of this cluster, describe remaining questions for elucidation of the unusual siderophore, and discuss the potential for use in downstream biocatalytic applications.

The nematode Caenorhabditis elegans produces a large family of ascaroside pheromones, which it uses in chemical communication to coordinate the development and behavior of the population. The acyl-CoA oxidase (ACOX) enzymes, which catalyze the first rate-limiting step in peroxisomal β-oxidation, act as gatekeepers for the biosynthesis of ascarosides with specific side-chain lengths. By performing unbiased comparative metabolomics on acox-1.1, -1.2, -1.3, -1.4, and -3 mutant worms and acox-1.1;acox-3 double mutant worms, we provide a comprehensive view of the different roles of these enzymes in ascaroside biosynthesis and implicate them in a number of additional biosynthetic pathways. Our data show that acox-1.1 and acox-3 are required for the biosynthesis of a broad range of medium- and long-chain ascarosides, while acox-1.2, acox-1.3, and acox-1.4 specialize in ascarosides with specific side-chain lengths. Specific acox mutants accumulate a variety of modified ascarosides that are likely shunt products. Furthermore, we show that acox-1.1 and acox-3, but not other acox genes, are required for the biosynthesis of a specific subset of N-acylethanolamines (NAEs), many of which have hydroxyl groups at specific positions in their fatty acyl side chains. Through stable-isotope labeling, feeding experiments, and chemical synthesis, we characterize the structures of these NAEs and show that their fatty acyl groups are derived from both bacteria and nematode sources. One of the most strongly acox-dependent NAEs that has a β-hydroxy fatty acyl group is attractive to C. elegans at attomolar concentrations, whereas a closely related NAE with a γ-hydroxy fatty acyl group is not, indicating that a subset of secreted NAEs may influence worm behavior.

Self-assembled peptides are promising templates for the design of inhibitors of protein-protein interactions (PPIs) because they can be endowed with affinity- and selectivity-defining amino acids alongside favorable physicochemical properties such as solubility and stability. Here, we describe a tunable coiled-coil scaffold and its interaction with MCL-1, an α-helix-binding antiapoptotic protein and important target in oncology. We explore the role of oligomerization, multivalency, and cooperativity in PPI inhibition. Hot-spot residues from an MCL-1 binding peptide (NOXA-B) are grafted onto the outer surfaces of homo- and heterodimeric coiled-coil peptides to obtain inhibitors with mid-nM potency and selectivity over BCL-x_L. Binding of homodimeric coiled coils to MCL-1 is positively cooperative, resulting in stabilization of both partners. Homodimeric coiled coils support the binding of two copies of the target protein. Modification of the coiled-coil sequence to favor assembly of higher-order scaffolds (trimer and tetramer) negatively impacts inhibitory potency, with AlphaFold2 modeling and biophysical data indicating a complex interplay between coiled-coil oligomerization and target binding. Together, these data establish dimeric coiled coils as the most promising of such scaffolds to develop inhibitors of α-helix-mediated PPIs.

Brucellae are pathogenic bacteria responsible for a worldwide zoonosis called brucellosis. In this study, we exploit the d-mannose central metabolism for the selective labeling of lipopolysaccharide (LPS), a key virulence factor in Gram-negative bacteria. Our approach provides chemical tools to allow selective derivatization of bacterial membranes in vivo and a handle for imaging studies. Using Brucella abortus mutants, we demonstrate that the clickable monosaccharides are exclusively incorporated into the lateral branch of the core LPS glycan but not in the O-chain or any other cell wall component. The metabolic route followed by the mannose analogues was also evidenced and showed that phosphomutase ManB, whose XRD 3D-structure was solved, was the metabolic entry of azidosugars, which do not follow a salvage pathway. Site-specific incorporation of mannose in the LPS core opens new perspectives such as the identification of macromolecules binding this important structure for the host-pathogen interaction.

Dysregulation of lipid homeostasis is associated with a wide range of pathologies encompassing neurological, metabolic, cardiovascular, oncological, and renal disorders. We previously showed that lipid droplet (LD) accumulation in podocytes contributes to the progression of diabetic kidney disease (DKD) and reducing LDs preserves podocyte function and prevents albuminuria. Here, we sought to identify compounds that treat pathological LD accumulation. We developed a phenotypic assay using human podocytes and deployed it to screen a combinatorial library comprising over 45 million unique small molecules. This led to the identification of a compound series that effectively reduces LD accumulation in stressed podocytes. Mechanistic studies revealed that these compounds activate lipophagy, reduce LD accumulation, and rescue podocytes from cell death. In contrast, compounds known to induce general autophagy failed to mimic these effects, indicating a novel lipophagy-specific mechanism of action (MoA), which was confirmed by unbiased phenotypic profiling. An advantage of this therapeutic strategy is its potential to not only halt the progression of pathological lipid accumulation but also reverse it. These compounds will serve as tools for uncovering novel drug targets and therapeutic MoAs for treating DKD and other diseases with similar etiologies.