Journal of the American Chemical Society

Cells utilize liquid-liquid phase separation to organize biochemical reactions within biomolecular condensates, which function as membraneless organelles. Although these assemblies are known to enhance reaction rates by concentrating reactants, the mechanisms beyond simple mass-action effects remain poorly understood. Here, we examined how the physicochemical microenvironment within condensates modulates reaction kinetics using spontaneous protein ligation as a model reaction, conducting a systematic analysis across various condensates, ranging from structured scaffolds (PRM-SH3 systems) to intrinsically disordered protein (IDP)-based scaffolds such as LAF, TAF, and FUS. We designed a FRET-based proximity-sensitive client probe to quantify increases in effective local concentration arising from excluded-volume effects. In parallel, we measured internal hydrophilicity and water activity, revealing them as additional key determinants of reaction acceleration. Together, the findings presented here elucidate how phase-separated compartments regulate biochemical reactions through the interplay of physical (effective concentration) and chemical (hydrophilicity and water activity) microenvironments and provide mechanistic insights for engineering condensates with tunable reactivity.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

Nicotinamide adenine dinucleotide (NAD+) is crucial for cellular functions including DNA repair and metabolism. Nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymes catalyze the final step of NAD+ synthesis from NMN and ATP. There are three NMNAT isoforms: NMNAT1, NMNAT2, and NMNAT3, located in the nucleus, cytoplasm, and mitochondria, respectively. Nuclear NAD+ promotes disease progression in NAD+-dependent cancers, and it is hypothesized that targeting NMNAT1 with small-molecule inhibitors could be an effective therapeutic strategy. Here, we identify an NMNAT1 inhibitor from a bioactive compound screen and report its effects on NAD+ levels and the viability of NMNAT1-dependent cancer cell lines. The compound AMI-1 is a known inhibitor of Protein Arginine N-Methyltransferase 1, and we find that it also inhibits NMNAT1 with similar potency. Additionally, we determined a cryo-EM structure of NMNAT1 bound to AMI-1 and revealed its mechanism of inhibition. This provides proof of principle for inhibiting NMNAT1 to target NAD+ metabolism in dependent cancers, while also highlighting that caution is warranted when interpreting studies using AMI-1 as a PRMT1 inhibitor, given its effect on NAD+ through NMNAT1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/716846v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@59933borg.highwire.dtl.DTLVardef@d1298borg.highwire.dtl.DTLVardef@1fe902dorg.highwire.dtl.DTLVardef@1abb3cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

Direct solar-to-chemical conversion offers a compelling route to clean, dispatchable energy. Photosystem I (PSI), an evolutionarily optimized light-driven oxidoreductase central to oxygenic photosynthesis, can be repurposed for direct solar-fuel production by efficiently coupling its photochemistry to catalysts, thereby storing sunlight as chemical energy in the H-H bond of H2. One promising architecture integrates PSI with Pt nanoparticle (PtNP) catalysts to create photocatalytic PSI-PtNP biohybrids. Advancing these systems requires molecular-level insight into protein-nanoparticle interactions and the bio-nano electron transfer pathways that govern activity; however, progress has been constrained by limited structural data to guide rational design. Here, we present two molecular structures of active PSI-PtNP assemblies that (a) compare thermophilic and mesophilic PSI scaffolds and (b) probe how removal of the terminal [4Fe-4S] clusters and stromal subunits in PSI reshapes protein-nanoparticle interfaces and photocatalysis. Structural analyses and molecular dynamics simulations define the interface topology, electrostatics, and cofactor-to-nanoparticle distances, revealing key molecular features that control biohybrid formation and electron transfer efficiency. These data establish mechanistic links between scaffold composition, bio-nano interface geometry, and catalytic performance, yielding design principles for optimizing PSI-PtNP architectures. The resulting structure-function insights provide a blueprint for engineering PSI-based solar-fuels systems and, more broadly, inform the design of protein-nanomaterial interfaces for light-driven catalysis.

Mutant KRAS is a potent oncogene, serving as a tumor driver in many solid human cancers. Current small-molecule inhibitors target the highly conserved G-domain, but to gain further mechanistic insight into the roles of different isoforms, we investigated the strategy of sterically shielding the unstructured hypervariable regions (HVRs). KRAS HVRs undergo a series of post-translational modifications that enable intracellular trafficking and membrane attachment. Previous attempts to drug KRAS by preventing its post-translational modification, based on inhibition of the involved prenylation enzymes have been largely unsuccessful. In this study, we explored the property of Designed Armadillo Repeat Proteins (dArmRPs) to specifically bind unstructured regions. We assembled a dArmRP to recognize the unstructured KRAS4B-HVR and developed it into a high-affinity binder by directed evolution. The resulting dArmRP recognizes the 14 C-terminal residues of unprocessed KRAS4B, thereby blocking the farnesyltransferase-binding epitope. This steric shielding disrupts KRAS4B post-translational modification and thereby significantly reduces its plasma membrane localization, while demonstrating complete selectivity over KRAS4A, NRAS, and HRAS. This work establishes the shielding of intrinsically disordered regions as a precise biochemical strategy to control protein function and provides an isoform-specific tool to dissect KRAS biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/712636v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@791ac4org.highwire.dtl.DTLVardef@cc4c91org.highwire.dtl.DTLVardef@b6c920org.highwire.dtl.DTLVardef@4e8a9c_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical representation of how the unstructured KRAS4B-HVR is occupied by a dArmRP, making it inaccessible for the FTase.

Therapeutic peptides combine high target specificity with potent biological activity.1 However, treatment success is often limited by rapid clearance and the need for frequent injections.2, 3 This challenge is particularly acute for therapeutic peptides used in obesity, where clinical benefit must be balanced against dose-dependent adverse effects. In nature, these constraints are overcome by storing hormones as reversible fibrils,4 but pharmacokinetic control is essential for widespread adoption of bio-inspired self-assembled depots for therapeutic peptides. Here, we show that tuneable pharmacokinetics can be achieved and modelled by mapping the fundamental chemical parameters of reversibly self-assembly in vitro. We demonstrate this approach for the amylin analogue pramlintide. Amylin analogues are under development for the next generation of diabetes and obesity treatments, with improved mechanism of action e.g. preserving lean body mass.5-8 Pramlintide is an approved drug with a well-established safety profile, however, it has a comparable half-life to native amylin.8-12 In a pilot study, we achieve in vitro-in vivo correlation, increasing the half-life of pramlintide 20-82-fold in rats, while controlling burst release. These findings demonstrate that the optimisation of pharmacokinetics can be decoupled from peptide engineering, establishing a generalisable framework for generating long-acting peptide formulations by emulating native storage mechanisms.

Lanthipeptides represent the largest group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides offer promising avenues for discovering new antibacterial and antifungal agents. Here, we identify and structurally analyze the product of the tla BGC, which encodes a class II lanthipeptide in the thermophilic bacterium Thermoactinomyces sp. DSM 45891. Heterologous co-expression of the lanthipeptide synthetase TlaM resulted in modification of the two precursor peptides TlaA1 and TlaA2, which share 58% identity. TlaA1 was dehydrated seven times and TlaA2 six times. In both peptides, four thioether rings were formed with two overlapping DL-(methyl)lanthionine rings at the C-terminus. Both peptides also contain two central and N-terminal non-overlapping DL-methyllanthionines. These findings demonstrate that these peptides deviate from the general rule of stereoselective LL-(methyl)lanthionine formation from a DhxDhxXxxXxxCys motif (Dhx = dehydroalanine or dehydrobutyrine). AspN-cleaved TlaM-modified TlaA1 displayed anti-microbial activity against a subset of bacteria including Gram-negative ESKAPE pathogens. We named the lantibiotic thermolanthin.

Modern drug discovery demands efficient strategies for generating structurally diverse compound libraries. Skeletal editing--a transformative paradigm enabling precise atom-level modifications within molecular frameworks, offers a sustainable alternative to traditional synthetic routes. While carbene insertion-mediated approaches have dominated single-carbon insertion strategies, current methodologies are limited by their reliance on hazardous, unstable carbene precursors and harsh reaction conditions. Herein, we report a multicopper oxidase (MCO)-catalyzed skeletal editing that enables the direct, one-step transformation of phenolic and indole derivatives into functionalized tropones and quinoline analogues through exogenous single-carbon insertion. This platform employs stable and safe nitroalkanes as carbon sources and O2 as the sole terminal oxidant. It accommodates a broad substrate scope and yields products with superior antibacterial activity against to multidrug-resistant strains relative to their parent compounds. This work introduces the first biocatalytic platform for exogenous single-carbon insertion skeletal editing. This sustainable and scalable strategy overcomes key limitations of synthetic approaches, offering efficient skeletal remolding and rapid expansion of bioactive compound libraries. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/714988v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@ed9336org.highwire.dtl.DTLVardef@15beeeaorg.highwire.dtl.DTLVardef@a26525org.highwire.dtl.DTLVardef@19e7707_HPS_FORMAT_FIGEXP M_FIG C_FIG

Life is predicated on chirality, a molecular asymmetry akin to the left and right versions of human hands. Here we show that privileged protein residues are predisposed for chiral regulation. We developed enantiomeric oxaziridine reagents that systematically identify pro-(S) and pro-(R) methionine oxidation sites across proteomes that can be erased by stereospecific methionine sulfoxide reductase enzymes A and B, respectively. These probes reveal that chiral regulation of methionine oxidation-reduction processes can allosterically regulate protein function, as shown in cell and murine models of oxidative stress where selective (R)-methionine sulfoxide formation on M69 of biphenyl hydrolase-like protein leads to hydrolase inhibition and amplification of proteome N-homocysteinylation modifications. This work introduces a platform for characterizing sites of asymmetric methionine oxidation and the functional consequences concomitant with an individual chiral single-atom modification.

We discovered that palmatine (PAL), a well-known natural product, was inducing the fluorogenic fixation of live cells upon visible light irradiation. This ultrafast phenomenon proceeded under high spatiotemporal control down to single cells (SC), with persistence of well-preserved fixed-labeled cells. Cell "optofixing" was mediated by PAL interaction with nuclear and mitochondrial DNA, yielding reactive oxygen species (ROS) mainly singlet oxygen (1O2), lipid peroxidation (LPO) and LPO-derived fixing aldehydes. We found that other DNA dyes including conventional trackers were also capable of optofixing cells, furnishing a consistent methodology (fluorophore-mediated optofixation, FLUMO) across the visible spectrum. Our results pave the way for the functional ablation and labeling of target cell populations using small fluorophores, with applications in SC, organoid and whole organism biology.

Antimicrobial peptides (AMPs) that also form functional amyloids exhibit remarkable environmental sensitivity, yet the physicochemical rules governing their structural switching remain unresolved. Here, we investigate how surfactant charge and assembly dynamics regulate the antimicrobial-amyloidogenic transition of Uperin 3.5, a 17-residue amphibian AMP with pronounced conformational plasticity. Using an integrated approach combining all-atom molecular dynamics simulations with circular dichroism and thioflavin T fluorescence assays, we systematically probe the effects of surfactant identity, concentration relative to the critical micelle concentration (CMC), peptide stoichiometry and ionic strength. We show that -helical stabilisation and antimicrobial-like behaviour scale directly with surfactant charge: anionic Sodium dodecyl sulphate (SDS) induces the highest helicity in monomeric Uperin 3.5 ({approx}80-90%), followed by zwitterionic dodecyl-phosphocholine (DPC) ({approx}35-45%), while cationic Cetyltrimethylammonium bromide (CTAB) fails to stabilise secondary structure. This charge-ordered trend is mirrored in oligomer remodelling, with SDS driving the most rapid dissociation of {beta}-sheet tetramers, DPC inducing slower partial disassembly and CTAB exhibiting minimal effect. Above the CMC, micellar environments stabilise amphipathic -helical states and efficiently dissolve amyloid assemblies. In striking contrast, under below-CMC conditions, limited SDS availability combined with peptide crowding promotes cooperative aggregation, where surfactant monomers act as dynamic scaffolds that nucleate N-terminal {beta}-sheet interactions--an effect strongly accelerated by physiological salt. Large-scale simulations reveal mixed /{beta} aggregates whose formation is governed by electrostatic screening and surfactant-mediated co-assembly. Together, these findings establish surfactant charge and assembly state as quantitative, environment-dependent regulators of functional amyloidogenesis in antimicrobial peptides. More broadly, they suggest that controlled modulation of membrane-mimetic environments can be exploited to bias peptides toward antimicrobial or amyloidogenic states, offering conceptual avenues for therapeutic strategies targeting peptide misfolding and neurodegenerative disorders.

The targeted modulation of gene expression with bifunctional small molecules enables the precise control of cellular and biological processes. To screen for ligands that could be used to induce gene expression, we conjugated the high affinity FKBP(F36V) binder, AP1867, to known high-affinity binders of activating epigenetic machinery. We tested these bifunctionals in a FKBP(F36V)-tagged transcription factor reporter system and found bifunctional induced transactivation is relatively common, being observed for bifunctionals with BET ligand JQ1, p300/CBP ligand GNE-781, CDK9 ligand SNS-032, and BRD9 ligand iBRD9. aTAG-2 (mAP1867-C8-GNE781) was identified as the strongest and most potent transactivator, possessing single-digit nanomolar activity. When tested in models where oncogenic RNA binding protein-transcription factor fusion proteins have been FKBP(F36V)-tagged, we unexpectedly observed rapid collapse of the fusion transcriptional program. In a tagged Ewing sarcoma model, aTAG-2 exhibits at least three distinct mechanisms of action: i) RIPTAC mediated p300/CBP inhibition, ii) ubiquitination- and ternary complex-dependent EWS/FLI degradation, and iii) replacement of p300 with CBP at EWS/FLI bound chromatin loci. Together, these data establish bifunctionals targeting p300/CBP that toggle between a program of ultra-potent transactivation and repression depending on cellular context. Overall demonstrating that induced proximity with a given ligand does not encode a fixed functional outcome.

KRAS mutations drive multiple cancers and represent an important therapeutic target, together with other oncogenic regulators such as MYC, KIT, and BCL2 that are critically involved in pancreatic cancer. Here we describe a novel therapeutic strategy based on stable nucleolipid-modified G-quadruplexes (NLG4). Cell viability assays demonstrate that NLG4 strongly inhibit pancreatic cancer cell proliferation, whereas non-lipidic G-quadruplex sequences display minimal activity under comparable conditions. Owing to their distinctive physicochemical properties, including stabilization of parallel G-quadruplex structures and self-assembly into micellar aggregates, NLG4 efficiently internalize into cells and interact with key G-quadruplex unfolding factors such as UP1. This interaction leads to a marked downregulation of KRAS, c-MYC, c-KIT, and BCL2 expression. Suppression of these oncogenes profoundly affects pancreatic cancer cell fate, as evidenced by reduced expression of proliferation (Ki67) and anti-apoptotic (BCL2) markers. In addition, NLG4 treatment decreases inflammatory signaling mediated by NF-{kappa}B and inhibits major pro-proliferative kinase pathways, including ERK, AKT, and phosphorylated AKT. The therapeutic relevance of this decoy strategy is further supported by the observed potentiation of gemcitabine antitumor activity. Overall, these findings highlight NLG4 as a promising anticancer approach that simultaneously targets multiple oncogenic pathways through G-quadruplex-based decoy mechanisms, with translational potential for future pancreatic cancer treatment.

Targeted protein degradation (TPD) is a therapeutic strategy to remove disease-causing proteins by routing them to the ubiquitin-proteasome, autophagy, or lysosme machineries. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic hetero-bifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging and the number of currently addressable ubiquitin E3 ligases is limited. Here we demonstrate hetero-bifunctional de novo designed proteins as alternatives for TPD to access more targets and ligases. First, we develop a stable and highly adaptable helix-turn-helix scaffold for presenting different binding sites. Next, we use computational protein design to incorporate and embellish hot-spot- binding sites to target BCL-xL, plus short linear motifs (SLiMs) for KLHL20 ligase recruitment. The resulting mono- and bi-functionalised proteins bind the targets in vitro, and the latter degrade BCL-xL in cells leading to apoptosis.

Methyl-coenzyme M reductase (MCR) is a crucial enzyme for methanogenesis and harbors several unusual post-translational modifications. Recent studies have identified glutamine C-methyltransferase (QCMT), as a B12-dependent radical SAM enzyme responsible for methylating a glutamine residue within the MCR active site. B12-dependent radical SAM enzymes have the remarkable ability to alkylate unactivated Csp2- and Csp3-atoms in a stereoselective manner. However, the factors influencing the stereo-selectivity and catalytic properties of this emerging superfamily of enzymes remain poorly understood. In this study, we report the mechanistic, structural, and biochemical investigation of several QCMTs. Our findings reveal significant differences among them, notably in their ability to bind cobalamin. In addition, our data support that C H-atom abstraction and methyl transfer are not concerted but rather independent processes that require motion within the enzymes active site. We also demonstrate that QCMT can catalyze novel reactions, including the formation of unnatural C-methylated residues, peptide epimerization, reversible H-atom abstraction, and the direct conversion of glycine into O_SCPLOWDC_SCPLOW-alanine. Overall, our data are consistent with QCMT being a unique and versatile biocatalyst allowing for the installation of unnatural post-translational modifications and provide a structural and biochemical rationale for the control of the stereochemistry by B12-dependent radical SAM enzymes.

Protein self-assembly enables precise nanoscale organization but rarely translates into macroscopic materials that retain functionality beyond aqueous environments. Here, we report that a bacterial microcompartment (BMC) trimer fused with SpyTag (T1-SpyTag), when expressed as a standalone component, undergoes rapid and spontaneous self-assembly into macroscopically visible fibers and layered sheets. These structures span from the nanoscale to the millimeter scale, forming robust three-dimensional protein materials that remain structurally intact and functionally accessible in both solution and dried states. Unlike previously reported SpyTag-enabled BMC systems that function primarily as passive cargo-loading modules, T1-SpyTag macromolecular structures exhibit emergent material behavior, including chemical robustness under denaturing conditions, while preserving covalent reactivity toward SpyCatcher-fused cargos. The multilayered architecture enables tunable surface display, access to ultrathin, processable protein films, and surface renewability through layer-by-layer removal and regeneration. This work demonstrates how a minimal genetic modification of a native protein building block can drive the formation of functional, macroscopic protein materials, thus expanding the design space of BMC-derived assemblies for biointerfaces, catalysis, and sustainable protein materials engineering.

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD*YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD*YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD*YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD*YAP inhibitors.

Age-accompanied chronic, low-grade systemic inflammation (inflammaging) drives the onset and progression of neurodegenerative disorders like Parkinsons disease (PD). Currently, no disease-modifying therapies are available for PD. Exposure to environmental toxicants, including paraquat (PQ), rotenone, and neurotoxic metals, increases disease risk. Conversely, sustained consumption of dietary soft electrophiles, such as flavonoids, carotenoids, vitamin E vitamers, and essential fatty acids, has been associated with increased lifespan and delayed age-related neurological decline. Omega-3 and select omega-6 fatty acids also serve as precursors of lipid-derived specialized pro-resolving mediators (SPMs), which exert potent anti-inflammatory and inflammation-resolving activities. Here, we report the development of a robust analytical method to quantify pro-resolving oxylipins in a PQ-induced Drosophila melanogaster model of PD, enabling investigation of how dietary phytochemicals modulate anti-inflammatory and pro-resolving lipid metabolism in vivo. We hypothesized that plant-derived soft electrophiles promote active resolution of neuroinflammation by enhancing the production of pro-resolving oxylipins derived from essential fatty acids, and that their neuroprotective effects are linked to their soft electrophilic properties. Our results demonstrate that specific lipophilic plant-derived soft electrophiles significantly upregulate pro-resolving oxylipins in Drosophila heads following PQ exposure. We identify a subset of flavones and structurally related phytochemicals that selectively enhance SPM biosynthesis and show that this response involves the NF-{kappa}B orthologue relish. Additionally, feeding modality and sex-specific dimorphisms were found to influence oxylipin production. Collectively, these findings indicate that structurally related dietary soft electrophiles enhance endogenous pro-resolving lipid pathways, promote resolution of toxin-induced neuroinflammation, and have potential preventive and therapeutic relevance for neuroinflammation-associated neurodegenerative diseases. HighlightsO_LIQuantification of pro-resolving lipids in a Drosophila Parkinsons model. C_LIO_LISpecific structural features of phytochemicals contribute to in vivo bioactivity. C_LIO_LILipophilic soft electrophiles show therapeutic potential against neuroinflammation. C_LIO_LIFeeding modality and sexual dimorphism also regulate oxylipin production. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/714080v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@2088cforg.highwire.dtl.DTLVardef@1f5d026org.highwire.dtl.DTLVardef@134aa44org.highwire.dtl.DTLVardef@965e28_HPS_FORMAT_FIGEXP M_FIG C_FIG

Steroid-based fluorescent-quencher probes now enable real-time, residue-level mapping of previously inaccessible cholesterol-binding sites on G-protein-coupled receptors. We designed Tide Quencher 1 (TQ1) conjugated steroids that target two distinct peripheral sites on the M1 muscarinic receptor. One near the extracellular N-terminus and another adjacent to the intracellular C-terminus. Using pregnanolone glutamate as a versatile scaffold, we synthesised a library of probes varying in C-3 linker length ({gamma}-aminobutyric acid vs. L-glutamic acid) and C-3/C-5 stereochemistry (3/3{beta}/5/5{beta}). Fluorescence-quenching assays with CFP-tagged receptors revealed that TQ1 probes consistently outperformed Dabcyl, delivering up to 40 % quenching within minutes and sub-micromolar EC50 values. The most potent N-terminal probe (35-PRG-Glu-TQ1 (5)) achieved 300 nM potency, while the best C-terminal probe (35{beta}-PRG-Glu-TQ1 (3)) reached 1 {micro}M potency with rapid association. Molecular docking and MD simulations identified key residues (K20, Q24, W405 at the N-site; K57, Y62, W150 at the C-site) mediating binding, a prediction confirmed by alanine-scan mutagenesis that markedly reduced quenching at the N-terminus and only modestly affected the C-terminus. Competition experiments with non-quenching analogues further validated probe specificity. Crucially, the pregnane core proved essential; alternative steroid backbones failed to generate robust quenching. This fluorescence-quenching platform overcomes the limitations of traditional radioligand assays, providing kinetic insight, high-throughput compatibility, and the ability to dissect lipid-GPCR interactions in native membranes. The approach is readily extensible to other GPCR families, opening new avenues for structure-guided drug discovery targeting allosteric cholesterol sites.