Anabaenopeptins are a family of cyanobacterial cyclic peptides that display potent enzyme inhibition, particularly against carboxypeptidases A and B, as well as the serine/threonine phosphatases PP1 and PP2A. Defined by a 19-membered macrocyclic ring and a ureido-linked exocyclic amino acid, these compounds vary considerably in their amino acid composition, influencing both potency and selectivity. This review compiles published IC50 values for natural and synthetic anabaenopeptins, organizing them by enzyme target, and highlighting recurring structural motifs that drive inhibitory activity. Through this comparative analysis, we identify emerging trends in structure-activity relationships and underscore gaps in assay standardization and structural validation. These insights provide a critical foundation for advancing the biological evaluation of anabaenopeptins as environmental contaminants, mechanistic probes, and candidate scaffolds for therapeutic development.

4-Methylazetidinecarboxylic acids (MeAZEs) are unusual nonproteinogenic amino acids found in nonribosomal peptides, such as bonnevillamide D and vioprolide A. The biosynthesis of MeAZEs is thought to proceed from S-adenosyl-l-methionine (SAM) via C-methylation and azetidine ring formation, but the order of these steps has remained unclear. Guided by our previous findings, we proposed that C4″-methylation of SAM by the cobalamin-dependent radical SAM enzyme BnvC, which is encoded in the biosynthetic gene cluster (BGC) of bonnevillamides, precedes azetidine ring formation. In this study, we identified the DUF364-containing enzyme Orf5, which is associated with the bnvC gene in the BGC of bonnevillamides, converts (4″R)-4″-methyl-SAM into cis-MeAZE. Further, we found that VioH, previously shown to catalyze azetidine ring formation from SAM in vioprolide A biosynthesis, transforms (4″S)-4″-methyl-SAM into trans-MeAZE. Structural modeling indicated that the C-methyl group was essential for efficient cyclization. These results elucidated the biosynthetic logic of MeAZEs and established C-methylation as a prerequisite for azetidine ring formation.

Colibactin is a pseudo-C2-symmetric gut microbiome metabolite that induces DNA interstrand cross-links and plays a causal role in colorectal cancer. Since efforts to isolate colibactin have not been successful, we developed colibactin 742 (3a/b) as a stable colibactin mimetic. However, colibactin 742 (3a/b) exists as a mixture of ring and chain isomers, which complicates analysis of its activity. We report here the discovery of colibactin 686 (9) as a superior colibactin mimetic. Colibactin 686 (9) is more potent than colibactin 742 (3a/b) and recapitulates the bacterial genotoxic phenotype. Colibactin 686 (9) possesses a C2-symmetric structure, which will expedite its synthesis, and is incapable of ring-chain isomerization, which will simplify analysis of its biological activity. We additionally establish that colibactins do not passively diffuse into cells, and are substrates for monocarboxylate transporter pumps. These latter findings have implications for trafficking of natural colibactin, which remains poorly understood.

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.

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.

Activity-Based Probes (ABPs) are invaluable tools for investigating enzymatic activity but can suffer from onerous syntheses and low stability in complex proteomes. Herein, we present the first synthesis of a robust, vinyl methyl ester (VME), bearing amino acid, which is compatible with solid phase peptide synthesis (SPPS). Novel peptidic probes incorporating the VME motif were prepared, and their labeling activity was investigated against the deubiquitinating enzyme (DUB) Otubain 1 (OTUB1), a critical cysteine protease DUB with remarkable specificity for Lys48 linked polyubiquitin chains. OTUB1 is implicated in DNA repair and immune response mechanisms and is currently considered a biomarker for tumorigenesis. A probe featuring the VME warhead demonstrated high reactivity and selectivity toward OTUB1, highlighting the significant potential of this approach to create robust and selective covalent tools for interrogating cysteine isopeptidases.

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.