ACS Chemical Biology最新文献

Side chain-to-side chain peptide macrocyclization or stapling is a chemical modification that is frequently used to increase the metabolic stability, the cell permeability, and/or the binding affinity of peptide drugs. Interestingly, it was recently reported that α-helical stapling can also protect the amyloidogenic peptide hormone islet amyloid polypeptide (IAPP) from aggregation and amyloid-associated toxicity. IAPP is the major component of insoluble amyloid deposits found in diabetic patients, and its derivatives constitute potential therapeutic candidates to treat metabolic disorders. Herein, we investigated the effects of macrocyclization chemistry on amyloid formation and cytotoxicity by comparing different stapling strategies: lactamization, azide-alkyne click chemistry, and formation of thioether link. The (i, i + 4) intramolecular macrocyclization of IAPP between positions 13 and 17 imposed, or not for some derivatives, a local stability of the helical secondary structure, modulating the propensity of the peptide to self-assemble into amyloid fibrils. The helically constrained derivatives inhibited the aggregation of unmodified IAPP and showed a reduced capacity to perturb the cell plasma membrane and to induce cell death. This study offers key molecular insights into the use of stapling strategies as a chemical approach to prevent the aggregation of peptide therapeutics and to inhibit the cytotoxicity of amyloidogenic peptides associated with protein misfolding disorders.

Biosynthetic pathways of specialized metabolites utilize protein–protein interactions (PPIs) to facilitate metabolic flux and sequester reactive intermediates. The monoterpene indole alkaloid pathway of Catharanthus roseus contains several metabolic branch points that may be mediated via transient PPIs. We investigated one branch point of this pathway that is responsible for the conversion of the intermediate dehydrosecodine into three possible cyclized alkaloid scaffolds, which act as intermediates en route to medicinally important alkaloids, such as vinblastine. We verified previously observed PPIs between reductase-cyclase pairs and additionally uncovered PPIs between evolutionarily related protein homologues. Through structural analysis of dehydrosecodine cyclases, we identified surface residues that appear to mediate interaction with the upstream reductase. We then demonstrated, via in vitro competition assays, that these residues impact the distribution of downstream products. These results highlight the significance of transient PPIs in the control and regulation of specialized metabolite pathways.

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules designed to induce the degradation of specific proteins within a cell. While most PROTACs are noncovalent interactors, covalent PROTACs may benefit from improved selectivity and pharmacodynamics, yet remain largely understudied. Here, a covalent gold-based PROTAC (AuPROTAC) was synthesized, featuring a Au(III)-warhead, known to induce cysteine-arylation in a gold-templated two-step mechanism, linked to a cereblon binding moiety. The degradome of the AuPROTAC was characterized by establishing a cycloheximide chase assay in a nonproliferative steady-state HL-60 cell culture, enabling the identification of PROTAC degradation targets uncoupled from confounding effects originating from cell-cycle-dependent translational patterns. The method was verified using the known SMARCA2 and PBRM1-degrader ACBI2. AuPROTAC could degrade the oncogenic tyrosine kinase MERTK and the thioredoxin-like 1 protein TXNL1. Their degradation was successfully rescued by proteasome inhibition. Proteome-wide degradation selectivity was further characterized by ranking the degraded targets according to the reduction extent of their protein half-lives. Interestingly, the AuPROTAC degraded a relatively limited number of proteins (95) when compared to ACBI2 (221).

Ferroptosis is a form of iron-mediated regulated cell death driven by lipid peroxidation (LPO). It has not only further improved our understanding of the cell death mechanism but also shown enormous potential in therapeutic applications. While the precise subcellular itinerary of ferroptotic cell death remains a subject of ongoing debate, radical-trapping antioxidants (RTAs) are widely recognized as efficient antiferroptotic agents due to their ability to interrupt LPO chain propagation. Here, we highlight recent pioneering works in the field, showing how probes derived from RTAs serve as powerful chemical tools for resolving the mechanism of ferroptosis across multiple cellular compartments.

Protein-based bispecific degraders, known as bioPROTACs, have emerged as powerful tools for targeted protein degradation through the ubiquitin-proteasome system (UPS). However, the relative efficacy of various recruitment domains within these degraders remains poorly understood. To address this knowledge gap, we conducted a comprehensive comparison of recruitment domains in bioPROTACs, utilizing eGFP as a proof-of-principle degradation target and an eGFP-binding DARPin with known structure as an adapter. Our innovative approach combined microinjection and live-cell microscopy, enabling a detailed assessment of directly measured degradation rates as a single-cell kinetic readout, unaffected by uptake or biosynthesis rates of the degrader, and across the different chemical classes. We examined nine degron peptides, three E3 ligase domains or adapters, and two series of small-molecule binders, linked in various geometries. Our results revealed that bioPROTACs based on E3 or adapter protein domains and small molecules generally exhibited the highest degradation rates, while most degron peptides showed comparatively low efficacy. Notably, for VHL-ligand-1 and thalidomide, the placement of the coupling site and linker position significantly influenced performance. This study provides crucial insights into the design and optimization of bioPROTACs, paving the way for the development of more effective degraders for specific applications. Our findings contribute to the growing field of targeted protein degradation and offer valuable guidance for researchers seeking to enhance the efficacy of bioPROTAC-based therapeutic approaches.

Aggregates of the protein α-synuclein may initially form in the gut before propagating to the brain in Parkinson’s disease (PD). Indeed, our prior work supports that enteroendocrine cells, specialized intestinal epithelial cells, could play a key role in the development of this disease. Enteroendocrine cells natively express α-synuclein and form synapses with enteric neurons as well as the vagus nerve. Severing the vagus nerve reduces the load of α-synuclein aggregates in the brain, suggesting that this nerve is a conduit for gut-to-brain spread. Enteroendocrine cells line the gut lumen; as such, they are in constant contact with metabolites of the gut microbiota. We previously found that when enteroendocrine cells are exposed to nitrite─a potent oxidant produced by gut bacterial Enterobacteriaceae─a biochemical pathway is initiated that results in α-synuclein aggregation. Here, we detail the cellular and molecular mechanisms involved. First, we holistically profiled nitrite-exposed enteroendocrine cells through untargeted proteomics. Next, we performed targeted analyses that specifically probed the mechanistic role of dopamine, as our prior findings suggested that dopamine is critical for nitrite-induced α-synuclein aggregation. In dopamine-free HeLa cells treated with nitrite, α-synuclein aggregation was indeed suppressed. Proteomic signatures in dopamine-free cells treated with nitrite were distinct from those in nitrite-treated enteroendocrine cells, highlighting pathways relevant to intestinal development of PD. Intriguingly, we observed that enteroendocrine cells maintain viability upon exposure to nitrite and in the presence of α-synuclein aggregates. This cellular robustness suggests that these cells may be a reservoir of toxic α-synuclein aggregates. As a possible antidote, our findings show that benserazide and α-methyl tyrosine─chemical inhibitors of dopamine biosynthesis─limited aggregation. Curious about mechanisms of disease etiology outside of α-synuclein aggregation, we also profiled the enteroendocrine cell lipidome─an emerging area of interest in PD research─to motivate future targeted studies delineating the roles of dysregulated lipid metabolism in disease onset. Overall, these studies lay a foundation for mechanistically informed therapeutic targets to prevent the intestinal formation of α-synuclein aggregates before they spread to the brain.

Liquid–liquid phase separation (LLPS) is pivotal in generating membraneless organelles and assembling cellular inclusions. Interactions mediated by RNA and intrinsically disordered regions of proteins are ubiquitous mechanisms that drive their LLPS. Here, we identify that a site-specific interaction stimulates the LLPS of WDR5, a chromatin-associated protein hub. Our study proves that WDR5 undergoes self-association between its N-terminal intrinsically disordered region and a multitasking binding site. This mechanism facilitates the formation of liquid droplets in a cell-free environment. Notably, WDR5 undergoes phase separation in mammalian cells, forming nuclear puncta (NP) in response to osmotic stress. Further, nuclear WDR5 condensates encompass a critical oncoprotein transcription factor, MYC, and WDR5-binding RNA under hyperosmotic conditions. Our findings suggest that RNA modulates WDR5 phase separation and influences nuclear puncta formation, potentially serving as a general stress response mechanism. These outcomes illuminate a distinctive mechanochemical signaling process, highlighting the functional interplay among WDR5, RNA, and MYC at the chromatin level, particularly during osmotically induced LLPS.

Mimicry of protein secondary structure elements, such as α-helices and β-sheets, using conformationally constrained peptide macrocycles, can be utilized to disrupt native protein–protein and protein-nucleic acid interactions. Although α-helical stapled peptides have been extensively studied as pharmacological probes, the application of β-sheet and β-hairpin mimetics remains comparatively limited. Less is known about the structural and biophysical consequences of β-hairpin macrocyclization in the context of target binding. In this work, we use a poxvirus immune antagonist protein 018 as a template for the structure-based design of β-hairpin mimetic macrocyclic peptides targeting the STAT1 transcription factor. We demonstrate that successive orthogonal cyclizations have additive effects on the thermodynamic and kinetic properties of peptide binding, most notably slowing the dissociation from the target. We elucidate the structural and dynamic consequences of interstrand and head-to-tail cross-linking and propose a kinetic model explaining the gains in target residence. Finally, we highlight the pharmacological potential of these peptides by competitive inhibition of STAT1 binding to its cognate interferon receptor docking site. These data suggest that β-hairpin macrocyclization may represent a general strategy to extend target engagement, with implications for peptidic probe design.

The 8-OH of rifamycin is essential for its bioactivity, while its naphthalene ring formation with 8-OH has remained unclear. Biochemical and structural analysis has demonstrated that a pair of rare NAD⁺-dependent dehydrogenases, RifS and RifT, forms a S₂T₂ structure to dehydrogenate at C8 in a structure-dependent manner. RifS catalyzes 8-dehydrogenation through a canonical NAD⁺-dependent mechanism, while RifT acts as a noncatalytic partner. Finally, we proposed a plausible pathway for the transformation of benzene-type prorifamycin A (1) to naphthalene-type 34a-deoxyrifamycin W (1a) bearing the 8-OH group. These results provided direct evidence for the branch point of rifamycin and 8-deoxyrifamycin biosynthesis and paved an approach to engineering novel ansamycins.

Only a few sesquarterpenes (C₃₅ terpenes) have been found in nature, highlighting a chemical space requiring focused exploration of compounds. Noncanonical class IB terpene synthases (IB-TPSs) are the only enzymes that can cyclize C₃₅ prenyldiphosphate. Currently, ca. 6000 IB-TPS homologues from bacteria have been registered in the NCBI database. However, only two sesquarterpenes have been identified as IB-TPS products. In this study, we performed genome mining of 11 IB-TPSs from phylogenetically diverse bacterial species using an expression system in Bacillus subtilis. We revealed that 8 of the 11 homologues with ≥30% identity to Bacillus subtilis TPS (BsuTPS) synthesize the same product, tetraprenyl-β-curcumene, as BsuTPS. The absolute stereochemistry of the tetraprenyl-β-curcumene formed by BsuTPS and one of its homologues was determined to be (−)-7R using vibrational circular dichroism and specific optical rotation analyses. In contrast, 3 of the 11 IB-TPS homologues with <30% identity to BsuTPS produced 3 novel cyclic sesquarterpenes, 1 of which had an unprecedented 5/3-fused bicyclic sesquarterpene skeleton. These new sesquarterpenes could be synthesized through various carbocation quench mechanisms that differ from those of previously identified sesquarterpenes. In this study, we demonstrated that novel sesquarterpenes can be discovered with a high probability (3 out of 4) among IB-TPS homologues with <30% identity to BsuTPS, thereby expanding the structural diversity of sesquarterpenes.