ACS Central Science

Despite their therapeutic potential across a wide range of central nervous system (CNS) disorders, nucleic acid-based therapeutics are limited by inefficient delivery to deep brain regions at clinically viable doses. Transferrin receptor 1 (TfR1) has emerged as an attractive target for receptor-mediated transcytosis across the blood-brain barrier (BBB), enabling systemic delivery of biologics such as lysosomal enzymes and monoclonal antibodies. In this study, we demonstrated the translational potential of recently described TfR1-targeting camelid-derived single-domain antibodies (VHHs) for CNS delivery of siRNAs. When conjugated 1:1 to different tool siRNAs, these VHHs promote rapid and robust intracellular uptake, resulting in potent RNAi activity at low nanomolar concentrations in neural cells. Systemic administration of VHH-siRNA conjugates in wild-type mice, hTfR1 transgenic-mice and non-human primates revealed a favourable pharmacokinetic profile characterized by rapid TfR-dependent distributional clearance and efficient functional uptake in deep brain structures. This translated into durable target knockdown of 50-80% at both mRNA and protein levels and with ED50 below 1 mg/kg siRNA. Collectively, these findings establish our TfR1 targeting VHHs as a fit-for-purpose platform for the systemic delivery of therapeutic oligonucleotides to deep brain structures at clinically relevant doses, opening new avenues for the treatment of diverse CNS disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/726486v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13668eorg.highwire.dtl.DTLVardef@1b1feeeorg.highwire.dtl.DTLVardef@d7be2dorg.highwire.dtl.DTLVardef@6b221_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bioluminescence resonance energy transfer (BRET) systems are widely used for live-cell spectroscopy and biosensor engineering, yet the intrinsic pH sensitivity of commonly used BRET components has not been systematically examined. Here, we show that major BRET luciferase donors, fluorescent acceptors, and donor-acceptor assay pairs exhibit pronounced pH-dependent spectroscopic behavior across physiologically relevant conditions, identifying environmental pH responsiveness as a fundamental property of widely used BRET systems and a potential source of previously underappreciated assay artifacts. Leveraging these principles, we engineered ORION (ratiOmetRIc prOton seNsor), a genetically encoded ratiometric BRET pH sensor based on the NanoLuc-mVenus fusion. ORION exhibited strong brightness, an approximately 9-fold dynamic range, and robust responsiveness across a substantially broader pH range than that of existing genetically encoded sensors. Compared to pHluorin2, ORION maintained substantially improved quantitative performance at acidic pH values below 6.0. To demonstrate its utility in a biological application, we applied ORION across diverse cancer cell models and identified heterogeneous acid imprinting states, suggesting that tumor cells can retain persistent physiological memory of adaptation to acidic microenvironments even after prolonged ex vivo culture. Together, these findings establish pH responsiveness as a fundamental property of BRET systems and position ORION as a best-in-class platform for interrogating and quantifying pH regulation of biology in living systems.

Spatiotemporal regulation of mRNA translation is central to gene expression. Over the past decade, translation has become directly observable in live cells at single-mRNA resolution by tagging nascent chains with tandem arrays of short epitope tags recognized by genetically encodable fluorescent intracellular antibodies (intrabodies). While this technology has revolutionized our understanding of translation regulation, the current toolbox of tagging systems remains limited. Here, we developed a novel and tight-binding intrabody against a short (11-amino acid) HIV protease epitope (named UTag). To ensure robust intracellular folding of the anti-UTag intrabody, we further engineered a cysteine-free variant that folds and functions independently of disulfide-bond formation, as validated by X-ray crystallography. The cysteine-free anti-UTag intrabody retains high binding affinity comparable to the parental intrabody while exhibiting significantly improved thermostability ([~]80 {degrees}C). Importantly, the cysteine-free UTag system enables real-time tracking of single-mRNA translation in live cells with performance on par with the parental UTag system as well as the established SunTag and ALFA-tag. Collectively, these results demonstrate that the newly developed UTag system expands the toolbox for live-cell translation tracking and provides complementary tools for multiplexed applications.

Despite intensive efforts, the ferroptosis gatekeeper glutathione peroxidase 4 (GPX4) remains difficult to selectively target due to stringent structural constraints surrounding its catalytic selenocysteine, which impose tight requirements on warhead reactivity and geometry. Here, leveraging a chemoproteomic approach, we characterize a potent and selective covalent GPX4 inhibitor featuring a pyrimidinylmethyl isourea warhead and define the chemical features underlying its proteome-wide selectivity. This chemotype enables tunable electrophile reactivity through steric and electronic modulation of leaving group ability, suggesting potential broader utility for targeting other recalcitrant proteins. Building on this scaffold, we further develop two selective GPX4 degraders - one CRBN-dependent and the other CRBN-independent - enabling complementary modulation of GPX4 through both inhibition and degradation. Together, these molecules expand the GPX4 chemical toolbox for more nuanced interrogation of GPX4 biology.

Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.

Antibody and protein-drug conjugates (XDCs) have emerged as promising cancer therapeutics, yet their clinical utility remains constrained by dose-limiting toxicities and narrow therapeutic windows. These safety challenges stem primarily from two factors: premature payload release during systemic circulation, and poor physicochemical properties inherent to the hydrophobic cytotoxic drugs they carry. Prior strategies attempted to address these limitations by appending water-soluble tags to reduce overall conjugate hydrophobicity, but achieved only modest improvements. As a result, the hydrophobic nature of cytotoxic payloads has remained a persistent obstacle in XDC development. Here, we report a fundamentally different chemical strategy that reframes this liability as a design opportunity. Rather than masking drug hydrophobicity, we exploit it as the driving force for self-assembly of facially amphiphilic protein-drug conjugates with programmable drug moieties (PDCs). In this architecture, the hydrophobic cytotoxic drug and the hydrophilic protein serve as the core and shell, respectively, spontaneously assembling into monodisperse, well-defined spherical protein nanotherapeutics of controlled size. This design principle transforms a longstanding physicochemical challenge into a functional engineering tool, enabling precise nanostructure formation without sacrificing potency. In vitro studies confirm that the resulting nanotherapeutics effectively kill cancer cells, establishing a strong foundation for further therapeutic development.

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

Photoswitchable ligands enable photocontrol of biomolecular activity by binding to targets in an isomer-dependent, light-responsive manner. Recent developments in ionizable photoswitchable ligands greatly expand their applications but introduce a major design challenge: light-responsive binding can depend on isomeric form, chemical substitution, and binding-induced shifts in protonation equilibria. These effects are tightly coupled, subtle in magnitude, and difficult to predict. Consequently, few computational methods have been developed and systematically benchmarked for quantitatively predicting them. Here, we establish a multiscale free-energy method and benchmark it against experimental data for a series of recently developed photoswitchable inhibitors of Escherichia coli dihydrofolate reductase (eDHFR), a crucial target in photopharmacology. Constant pH replica-exchange molecular dynamics and quantum mechanics/molecular mechanics umbrella sampling quantitatively characterize the ligands protonation-state change upon binding to the eDHFR active site. Thermodynamic integration simulations using alternative alchemical pathways, thermodynamic cycles, and protonation-state assignments were evaluated for predicting light-responsive affinity differentials and substituent effects. Direct cis-to-trans transformations with explicit treatment of environment-dependent protonation states best reproduce experimental trends. Compound-to-compound pathways are less reliable because force-field inaccuracies introduce large pK errors that are difficult to correct when protonation/deprotonation processes implicitly enter the thermodynamic cycle. TI simulations that ignore binding-induced protonation-state changes fail to consistently reproduce experimental trends. Protein-ligand and ligand-water interaction analyses further reveal the energetic and structural origins of isomer-dependent binding. This study establishes a systematic free-energy method for designing ionizable photoswitches in photopharmacology.

G-quadruplex (GQ) structures within the HIV-1 long terminal repeat (LTR) regulate viral transcription and represent promising antiviral targets; however, detailed mechanistic understanding of their ligand recognition at the molecular level remains limited and has largely been investigated under dilute conditions despite the crowded and compartmentalized nature of intracellular environment. Here, we investigate the interaction of the cationic porphyrin TMPyP4 with the HIV-1 LTR-III GQ under dilute conditions and inside protein-rich phase-separated condensates that mimic intracellular biocondensates. Steady-state and time-resolved fluorescence measurements reveal a dual binding behavior that is not discernible from absorption spectroscopy. A high-affinity guanine-rich binding mode leads to efficient fluorescence quenching through electron transfer from ground-state guanine to excited TMPyP4, whereas a weaker non-guanine binding mode gives rise to enhanced and long-lived emission. Nucleotide-specific control experiments validate the origin of these distinct binding environments. Molecular docking and molecular dynamics simulations further support preferential binding of TMPyP4 at the terminal G-quartet together with a secondary binding mode near the quadruplex-duplex junction. Importantly, both TMPyP4 and LTR-III GQ preferentially partition into the condensates, where the hybrid GQ structure, dual binding behavior, and associated excited-state signatures remain preserved despite the crowded and viscous environment. Although a slight reduction in binding affinity is observed inside the condensates, the overall binding mechanism remains largely preserved due to compensatory effects arising from the condensate microenvironment. Overall, this work demonstrates that ligand recognition of viral GQ remains preserved within protein condensates and establishes fluorescence spectroscopy as a sensitive approach for resolving hidden binding heterogeneity in GQ-ligand interactions.

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@287a47org.highwire.dtl.DTLVardef@17407eborg.highwire.dtl.DTLVardef@b40435org.highwire.dtl.DTLVardef@804352_HPS_FORMAT_FIGEXP M_FIG C_FIG

The eukaryotic translation initiation factor 4E (eIF4E) is a central regulator of cap-dependent translation and a compelling pharmacological target in disorders marked by protein synthesis dysregulation, including cancer and Fragile X Syndrome (FXS). Among endogenous eIF4E regulators, the CYFIP1-eIF4E interaction is uniquely selective, offering a framework for designing targeted translation modulators. Here, we report Cy-9B, a rationally engineered, stapled peptidomimetic derived from CYFIP1 that binds eIF4E, disrupts eIF4E-eIF4G complex, and suppresses cap-dependent translation. Enhanced-sampling free-energy simulations reveal that Cy-9B engages eIF4E through a non-canonical binding mode. Cy-9B exhibits drug-like properties, including high proteolytic stability and nanomolar affinity. Functionally, Cy-9B inhibits lung cancer cell proliferation, migration, and invasion. In neurodevelopmental disease models, Cy-9B partially normalizes excessive translation in FXS hippocampal neurons and rescues social behavior deficits in a Cyfip1 haploinsufficient Drosophila melanogaster model, restoring wild-type-like performance. Cy-9B emerges as a first-in-class therapeutic candidate for disorders sharing translational dysregulation, highlighting targeted modulation of eIF4E as a broadly applicable and physiologically compatible therapeutic strategy.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@abe1c9org.highwire.dtl.DTLVardef@746a08org.highwire.dtl.DTLVardef@dd5861org.highwire.dtl.DTLVardef@1d572c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 [A] resolution, revealing the molecular basis for improved M4 mAChR PAM activity. MK-97 adopts a distinctive boomerang-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y922.64 and a {pi}-{pi} stacking interaction with W4357.35. Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M4 mAChR PAMs with improved pharmacological properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723386v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1ab9c78org.highwire.dtl.DTLVardef@1adb532org.highwire.dtl.DTLVardef@152f9f7org.highwire.dtl.DTLVardef@990768_HPS_FORMAT_FIGEXP M_FIG C_FIG

Targeting membrane receptors underlies the success of antibody-drug conjugates (ADCs), yet single-receptor formats can be limited by heterogeneous expression, compensatory signaling, and variable internalization. Here we developed Multivalent Interchangeable Nanobody Degradation System (MINDS), a modular nanobody-Fc chassis that co-engages multiple membrane receptors, promotes their lysosomal co-depletion, and enables delivery of diverse intracellular payloads. As a proof of concept, we generated Tritazumab, a trispecific nanobody-Fc targeting three oncogenic receptors EGFR, cMET, and TfR1. Tritazumab incorporates a high-affinity, non-transferrin-competing anti-TfR1 nanobody that drives efficient uptake and lysosomal trafficking, enabling coordinated depletion of all three receptors. Across non-small cell lung cancer models, Tritazumab achieved rapid and sustained multi-receptor surface loss with picomolar degradation potency, reaching near-maximal depletion within approximately 1.5 hours. Conjugation of Tritazumab to MMAE preserved receptor binding and produced substantially greater antiproliferative activity and improved tumor selectivity relative to clinical ADCs in matched cell models, along with potent in vivo tumor growth inhibition and acceptable tolerability in a xenograft model. Extending the platform beyond cytotoxic payloads, a BRD4 molecular glue conjugate improved the selectivity window by > 100-fold and showed marked in vivo efficacy, while an EZH2-targeting PROTAC conjugate achieved an approximately 1,000-fold increase in intracellular degradation potency relative to the free PROTAC. These findings establish MINDS as a modular multispecific degrader-payload platform that integrates receptor co-depletion to enhance anticancer selectivity and efficacy.

Different small-molecule drugs targeting the same protein can produce divergent clinical outcomes through poorly characterized interactome changes. Existing proximity labeling approaches for target identification suffer from background biotinylation independent of small-molecule recruitment, obscuring true drug targets and their binding partners. Here, we incorporate a destabilizing domain (DD) into the biotin targeting chimera (BioTAC) framework to create ddBioTAC, wherein the proximity labeling enzyme TurboID is selectively stabilized only upon binding of a bifunctional targeting molecule. Using the bromodomain-targeting molecule NICE-01 in HeLa cells, we demonstrate that, in the absence of the bifunctional targeting molecule the destabilized TurboID enzyme (TurboID-DD) exhibits reduced protein levels and biotinylation activity compared to the control TurboID-FKBP (FK506-binding protein), while recovering comparable activity upon NICE-01 treatment. This results in an eightfold improvement in specific enrichment of the known target bromodomain containing protein 4 (BRD4) and its interactors, including MED1 and EF1D. Proteome-wide mass spectrometry confirms that ddBioTAC more accurately discriminates drug targets and proximal interactors from non-specific background, advancing unbiased drug-induced interactome profiling.

Crystalline inclusion proteins CipA and CipB from Photorhabdus luminescens serve as versatile scaffolds for clustering genetically fused heterologous enzymes into crystalline inclusion bodies. Although engineered Cip crystals are known to function as solid biocatalysts for improving metabolite production in bacterial cells, the phase separation behavior of Cip proteins in non-bacterial cellular environments, as well as their biochemical attributes in a soluble, non-crystalline state, remain poorly understood. This study demonstrates that CipA and CipB efficiently undergo crystallization in the cytosol of human embryonic kidney cells both at normal and hypothermic cell culture conditions. Within 72 hours post-transfection, CipA and CipB become the most abundant proteins in transfected cells and produce distinctive cytosolic crystals often exceeding 10 m at least in one of the dimensions. Co-expression of CipA and CipB drives spontaneous demixing into two distinct crystal populations, and the orthogonality is maintained even when an unrelated third protein crystallizes in the same cytosol, permitting three crystal types to coexist simultaneously. Intracellular crystals are readily isolable from cells, and once purified, these crystals are stable under physiological pH conditions. However, CipA and CipB show notable differences in their crystal dissolution kinetics and protein oligomerization states when solubilized under acidic or alkaline conditions. These findings suggest that CipA/CipB forms a robust orthogonal self-assembly pair and establish CipA/CipB crystals as an efficient platform for producing biochemically programmable intracellular crystals. These properties should extend the Cip-based scaffolding approach to mammalian cell systems for synthetic biology applications.

Drug-metabolizing enzymes determine therapeutic exposure, efficacy and toxicity, but defining their isoform-specific functions in vivo remains challenging. Cytochrome P450 enzymes (P450s) are central to drug metabolism and pharmacokinetics (DMPK) and mediate the phase I metabolism of [~]75% of all marketed drugs. However, conventional knockout models can induce develop-mental and compensatory adaptations, and selective inhibitors are unavailable for many P450 isoforms. Here, we report the use of an inducible chemical-genetic platform for acute and specific degradation of the endogenous P450 enzyme Cyp1a2 in mice. Using CRISPR-Cas9-mediated knock-in editing, we introduced an FKBP12F36V degron into the endogenous Cyp1a2 locus to generate Cyp1a2dTAG mice. Treatment with the dTAG degrader dTAG-13 recruited an E3 ubiquitin ligase to CYP1A2dTAG, resulting in rapid and reversible proteasomal depletion of CYP1A2dTAG in vivo. Temporally controlled CYP1A2dTAG loss altered caffeine pharmacokinetics as expected, validating this model as a functional tool for DMPK studies. By enabling reversible suppression of drug-metabolizing enzymes without permanent deletion or chronic inhibitor exposure, this work establishes targeted protein degradation as a broadly adaptable strategy for studying drug metabolism in vivo and provides a foundation for extending inducible DMPK control to other P450s, conjugating enzymes and transporters.

Prostate cancer (PCa) is one of the principal contributors to health burden in the aging male population. PCa develops through dysregulation of androgen receptor (AR) signaling pathways. Despite improvements in diagnostic techniques and interventions, no pharmacological measures with long term efficacy have been established once PCa advances to castration resistant prostate cancer (CRPC). To circumvent this issue, tetra-aryl cyclobutanes (CBs) have been proposed as structurally distinct compounds with a mechanism of action differing from traditional androgen receptor signaling inhibitor (ARSIs). Here, we apply principles of crystal engineering and solid state synthesis to expand the class of CBs through strategic derivatization. The synthesis of the CB occurs quantitatively, producing no side products and eliminating the need for product purification. We demonstrate how head-to-tail stacking interactions of halo-pyrimidine rings can be exploited to stack and align unsymmetrical alkenes to undergo [2+2] photodimerization to generate the CB in the solid state. We examine the structure-function relationships of CBs in vitro by profiling AR mediated transcriptional activity, receptor translocation, and cell viability. Moreover, we explore and identify putative binding interactions within CB/AR complexes and establish an adaptive ligand-binding potential using molecular docking platforms. In total, our data suggests that CBs have unexploited therapeutic potential in CRPC and that green chemistry and crystal engineering principles offer a unique route to generating these drug candidates.

Extracellular pH is a key microenvironmental factor shaping cell physiology and disease, creating a need for quantitative biosensors that can capture dynamic changes in pHe at the surface of individual living cells. Here, we develop a genetically encoded, ratiometric extracellular pH biosensor through systematic screening of a modular library of membrane-display designs that combine SEpHluorin with a pH-stable reference fluorophore. Screening identified a cell-surface-localised mKate2-SEpHluorin construct, named SurpHer, that exhibits dynamic ratiometric responses across the pHe range of 6 - 7.8. SurpHer shows robust membrane localisation and extracellular pH responsiveness across diverse human cell types including HEK293T, PANC-1 and MDA-MB231 cells. Following stable integration in MDA-MB-231 cells, SurpHer enabled time-course imaging of pHe gradients in a microfluidic platform for modelling tumour microenvironments. SurpHer enables real-time interrogation of the pericellular pH environment of tumor cells and, more broadly, provides a strategy to probe microenvironmental pH dynamics across diverse biological contexts.

Peptide mapping is a critical tool for characterizing biotherapeutic proteins and is essential for the development of monoclonal antibody drugs. Here we describe a new direct infusion technology that streamlines peptide mapping data collection and analysis, accelerating the method by up to 100-fold. This method, which we term RaPiD-mAb-MS, combines high-throughput plate-based sample preparation with direct infusion mass spectrometry analysis. RaPiD-mAb-MS allows analysis of 96 samples within [~] 1.5 to 2 hours, routinely achieves >95% sequence coverage, and has been successfully applied to 28 unique antibodies and over 2,000 samples. Here we demonstrate that RaPiD-mAb-MS detects and quantifies oxidation, deamidation, isomerization, glycosylation, and sequence variants with results comparable to conventional LC-MS based methods in a fraction of the time. Further, by eliminating chromatography, data analysis is greatly streamlined and simplified. By allowing for the collection of [~] 1,000 peptide maps per day, RaPiD-mAb-MS is positioned to accelerate all phases of antibody-based drug discovery & development and sets the stage for collection of massive datasets that would allow artificial intelligent prediction of optimal antibody variants and formulations.