Nature Chemical Biology

G-protein-coupled receptors (GPCRs) signal through multiple heterotrimeric G proteins, {beta}-arrestins, GPCR kinases (GRKs), and downstream effectors, whose combinatorial interactions shape cellular responses. These events are typically measured with separate assay formats that each capture only part of the network, making comparison across signaling layers difficult. Here, we consolidate a broad set of previously reported GPCR signaling interactions and assay concepts, together with newly designed sensors, into a single NanoBiT split-luciferase framework, allowing multiple layers of signal transduction to be examined side by side in living cells. We show that rational sensor engineering, in particular the positioning of NanoBiT fragments and targeted modification of the tagged proteins, is essential for detecting transient protein-protein interactions. The framework implements assays for G-protein dissociation, {beta}-arrestin recruitment, conformational activation and trafficking, and GRK recruitment, and extends to G-protein-effector and inter-effector interactions across diverse G, G{beta}, G{gamma}, {beta}-arrestin, GRK, adenylyl cyclase, PLC{beta}, and RhoGEF subtypes. It also enables real-time monitoring of the difficult-to-access G12/13-RhoGEF-RhoA pathway. Together, these assays provide a unified NanoBiT readout for systematic, side-by-side dissection of GPCR signaling.

Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced anti-leukemic properties via orthogonal chemical screening. Through multi-omics profiling and genome-scale CRISPR/Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate (PI4P) signaling at the endoplasmic reticulum (ER)-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.

The regulation of post-translational modifications (PTMs) is central to cellular biology and disease. Induced-proximity strategies enable manipulation of PTMs by recruiting modifying enzymes to proteins of interest, but identifying effective effector enzymes typically requires extensive heterobifunctional molecule synthesis before biological validation. Here we report a modular platform that enables rapid evaluation of PTM editing enzymes against defined protein substrates in living cells using compound-dependent or nanobody-mediated induced proximity. Using lysine acetylation as a model system, we demonstrate programmable acetylation of GFP, histone H3, and p53 through recruitment of diverse acetyltransferases. Effector identity dictates site-specific acetylation patterns, enabling selective PTM deposition across substrates and cellular compartments. This platform enables rapid identification of productive effector-substrate relationships prior to heterobifunctional molecule development, accelerating the design of induced-proximity chemical probes for targeted PTM editing.

Biomedical research overlooks most genes in favor of a well-studied minority, yet whether analogous blind spots exist in metabolomics remains unknown. We show that reductive amination, forming secondary amines from aldehydes or ketones and amines, generates a previously hidden class of metabolites we term alkamines. Multiplexed synthesis of 8,475 alkamines combined with MS/MS searches across 1.7 billion spectra identified 1,626 candidates across multiple species and organs. Of these, 56 were confirmed in biological samples, including 27 steroid- and 12 drug-derived alkamines matching prescription patterns. Notably, 77% of synthesized alkamines are absent from PubChem. This combinatorial logic likely explains why alkamines have evaded detection and suggests drug metabolism frameworks substantially underestimate drug-derived metabolite diversity. Reductive amination is an overlooked route modifying steroids, bile acids, and xenobiotics.

We describe an engineered "writer/reader" framework for programming post-translational control into synthetic mammalian signaling proteins. In this approach, a bacterially-derived biotin protein ligase (BirA) was used as a "writer" element for the modification of artificial receptors and transcription factors containing a biotin acceptor peptide (AP) fusion tag. To enable modification events to transmit biochemical information, we designed encodable "reader" modules using sequences from a biotinamide-binding antibody. Proteins fused to reader domains were able to interact with AP-tagged polypeptides in a biotinylation-dependent manner, and control over the timing and extent of these interactions could be modulated through both genetic and chemically-based strategies. Genetic and cell-specific control over AP-reader module interactions was achieved via regulated BirA expression, and the interaction states of both intra-and inter-cellular complexes could be modulated with biotinamide-based and bioorthogonally-functionalized compounds. The utility of this approach was demonstrated by installing post-translational and chemogenetic control into synthetic Notch ("SynNotch")-based systems.

Adaptors serve as hubs to regulate diverse protein complexes in cells. This multitude of functions can complicate the study of adaptors, as their genetic disruption may simultaneously impair the activities of several compositionally distinct complexes (or adaptor complexoforms). Here we describe the chemical proteomic discovery of bicyclopyrrolidine acrylamide stereoprobes that react with cysteine-100 (C100) of the methyltransferase (MT) adaptor TRMT112 in human cells. Curiously, the stereoprobes showed negligible reactivity with uncomplexed recombinant TRMT112, and we found that this interaction was restored excluively in the presence of METTL5, but not other MTs. A co-crystal structure revealed stereoprobe binding to a composite pocket proximal to C100 of TRMT112 that is templated by METTL5 and absent in other TRMT112:MT complexes. Structural rearrangements promoted by stereoprobe binding in turn lead to allosteric agonism of METTL5, thus revealing how covalent ligands targeting a pleiotropic adaptor can confer partner-specific functional effects through reactivity with a single complexoform.

Nitrate functions as a signaling molecule beyond its metabolic intermediate role. Despite progress in plants, the mechanisms underlying mammalian nitrate sensing and signaling remain unclear. The accompanying study identifies Sialin2--a proteolytic fragment of nitrate transporter Sialin--as a mammalian nitrate sensor mediating cellular responses. Here, we demonstrate that nitrate triggers endocytosis, inducing Sialin proteolysis and Sialin2 generation. Nitrate-induced Sialin2 scaffolds Lyn kinase with epidermal growth factor receptor (EGFR) at endosomes, activating phosphatidylinositol 3-kinase (PI3K)-AKT-nitric oxide synthase (NOS) pathway to stimulate localized nitric oxide (NO) production, enhancing angiogenesis and cell survival. In hypertensive rats, nitrate supplementation restores endothelial function and reduces blood pressure through AKT/eNOS-dependent signaling. Unlike the classical nitrate-nitrite-NO pathway, the Sialin2-PI3K-AKT-NOS axis confines NO synthesis to endosomal microdomains, enabling spatiotemporally precise vasodilation. By establishing Sialin2 as a mammalian nitrate sensor, this study unveils a novel paradigm in nitrogen homeostasis and provides targeted therapeutic strategies for vascular disorders.

Lipids exhibit extraordinary molecular diversity, yet tools to selectively manipulate defined lipid classes in living cells are lacking. Here we show that lipid tail structure biases metabolic fate, enabling the design of synthetic lipid analogs with programmable metabolic selectivity. This approach enables selective cellular production of distinct lipid species or subclasses, including types of neutral lipids, phospholipids, sphingolipids, and ether lipids, without genetic or enzymatic perturbation. We further couple metabolic selectivity to chemical functionalization using bifunctional lipids, in which one modification directs metabolic flux and a second enables bioorthogonal tagging. Using this strategy, we achieve selective in situ labeling of different lipid pools in living cells. Together, our work establishes a chemical biology strategy that enables unprecedented precision in modulating, functionalizing, and rewiring the mammalian lipidome.

Efforts to transform polyethylene terephthalate (PET) deconstruction products using live cells have been limited by terephthalic acid (TPA) uptake. Here, we used an intracellular carboxylate reduction assay to show that apparent TPA uptake in E. coli cells that lack a dedicated TPA transporter sharply increases between pH 5-6. Furthermore, we discovered that glycol ester deconstruction products, mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), surprisingly each result in rapid pH-independent uptake. We exploited glycol ester uptake along with deletion of 22 cellular oxidoreductases to design intracellular hydrolysis routes for synthesis of upcycled reduction products from BHET at >90% yields, and from real PET wastes after tandem catalytic glycolysis and cell-based valorization at >80% combined yields. Our work has important ramifications for PET utilization by cells and adds new perspectives on the evolution of the PETase/MHETase system.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane embedded signaling proteins. These receptors are involved in a wide array of physiological processes, marking them as attractive targets for drug development. Bitopic ligands, which are comprised of a pharmacophore that targets the receptor orthosteric site and a linked moiety that binds to a separate site, have considerable potential for addressing GPCR function. Here, we report the synthesis and evaluation of novel bitopic conjugates consisting of a small molecule pharmacophore that activates the adenosine A2A receptor (A2AR) linked to antibody fragments (nanobodies, Nbs). This approach leverages the high affinity and specificity binding of Nbs to non-orthosteric sites on engineered A2AR variants to provide bitopic Nb-ligand conjugates that stimulate strong and enduring signaling responses. We further demonstrate that such bitopic conjugates can induce activation by spanning two distinct receptor protomers. This property enables the selective targeting of receptor pairs over either individual receptor, as a form of "logic-gated" activity. We showcase the broad applicability of bitopic conjugates in this context by demonstrating their activity in targeting several pairs of co-expressed receptors, including GPCR monomers from different classes. Furthermore, we demonstrate that this dual-targeting strategy initiates signaling responses that diverge from those induced by monovalent ligands. The ability to target receptor pairs using nanobody-ligand conjugates offers a powerful strategy with potential for cell type-selective signaling and implications for GPCR drug discovery efforts more broadly.

While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70s ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

Novel antibodies have been created for targeted degradation of extracellular and membrane proteins in the lysosome. The mechanism of degradation of target proteins for these antibodies has involved either chemical conjugation of synthetic mannose 6-phosphate (M6P) or engineered bispecific antibodies. Currently, recombinant antibodies cannot be produced with naturally phosphorylated N-glycans. Here, we report the development of a novel platform technology for producing bifunctional therapeutic antibodies with high levels of M6P-bearing glycans directly from producing cells. The antibodies designated as phosphorylated N-glycosylated peptide chimeric antibodies (PNCA) maintain their affinity for antigens with concurrent high affinity binding to cell surface cation-independent mannose-6-phosphate receptors that facilitate internalization and delivery of antibody/antigen complexes to lysosomes for efficient degradation of both target extracellular soluble and membrane proteins. This PNCA approach provides a simple, scalable, and viable approach for producing naturally phosphorylated bifunctional antibodies from production cell lines for targeted protein degradation in lysosomes.

Fluorescence lifetime imaging microscopy (FLIM) can visualize multiple targets in a single spectral window, making it a powerful tool to overcome multiplexing limitations during live cell fluorescence microscopy. Here we show that small molecule probes-which are well-suited for imaging applications due to high specificity, low toxicity, and the elimination of transfection requirements-can be fine-tuned via bioorthogonal chemistry to exhibit predictably different fluorescent lifetimes suitable for FLIM multiplexing.

Bisbenzylisoquinoline alkaloids (bisBIAs) are pharmacologically valuable plant metabolites with complex stereochemical architectures, yet the catalytic principles governing their assembly have remained largely unclear. Here, we elucidate the enzymatic pathway to cyclic bisBIAs and uncover a non-canonical redox-mediated mechanism for post-assembly stereochemical control. We identify cytochrome P450 enzymes that catalyze regioselective oxidative dimerization and macrocyclization of benzylisoquinoline monomers, establishing the macrocyclic scaffold. Subsequent stereochemical specification is achieved by a paired oxidase-reductase module that selectively epimerizes a single stereocenter through a transient imine formation, converting (R,S)-configured intermediates to (S,S)-products. Reconstitution of the pathway in yeast enabled production of both native bisBIAs and non-natural analogs, demonstrating pathway modularity and engineering potential. These results establish the biochemical principle underlying bisBIA biosynthesis and provide a framework for programmable biosynthesis of these complex natural products.

C-Terminal cyclic imides are posttranslational modifications on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by Western blot and enrichment from whole cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

Substance use and related mental health epidemics are causing increasing suffering and death in diverse communities.1,2 Despite extensive efforts focused on developing pharmacotherapies for treating substance use disorders, there is an urgent need for radically different therapeutic approaches.3,4 Ibogaine provides an important drug prototype in this direction, as a psychoactive iboga alkaloid suggested to have the ability to interrupt opioid use in drug-dependent humans.5 However, ibogaine and its major metabolite noribogaine present considerable safety risk associated with cardiac arrhythmias.6 We introduce a new class of iboga alkaloids - "oxa-iboga" - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds act as potent kappa opioid receptor agonists in vitro and in vivo, but exhibit atypical behavioral features compared to standard kappa psychedelics. We show that oxa-noribogaine has greater therapeutic efficacy in rat models of opioid use, and no cardiac pro-arrhythmic potential, in contrast to noribogaine. Oxa-noribogaine induces long-lasting suppression of morphine and fentanyl intake after a single dose, persistent reduction of morphine intake and reinforcing efficacy after a short treatment regimen, and suppression of morphine and fentanyl drug seeking in relapse models. Oxa-noribogaine also induces a lasting elevation of neurotrophin proteins in the ventral tegmental area and medial prefrontal cortex, consistent with targeted neuroplasticity induction and alteration of addiction-like states. As such, oxa-iboga compounds represent candidates for a novel type of pharmacotherapy for treatment of opioid use disorder.

Small molecules play indispensable roles in living systems as hormones, membrane bilayer constituents, enzyme cofactors, metal chelators, and defensive chemicals. The exceptional structural diversity of such molecules underpins their wide-ranging biological functions. Precise functional group insertion into various molecular scaffold classes is a hallmark of small molecule biosynthesis and is frequently important for biological activity. Functional group deletion is also important, but mechanisms are less well understood. Here, we report deletion of a cysteine-derived nitrogen atom during assembly of the conserved pharmacophore in the anticancer drug romidepsin, and related depsipeptide HDAC inhibitors. A shunt metabolite hydroxylated at the cysteine--carbon-derived position is a thousand-fold less active, indicating nitrogen deletion is important for potent HDAC inhibition. In vitro reconstitution and dissection of the complete nonribosomal peptide synthetase-polyketide synthase-mediated pathway for pharmacophore assembly reveal that cryptic S-octanoylation is catalysed by an atypical heterocyclisation domain, while multifunctional dehydratase and ketoreductase domains and trans-acting phosphotransferase and flavin-dependent oxidoreductase enzymes catalyse successive transformations in nitrogen deletion. Our findings significantly advance the understanding of heteroatom deletion mechanisms in small molecule biosynthesis and highlight the key role this can play in enhancing bioactivity. One-pot biocatalytic synthesis of the pharmacophore provides foundations for chemoenzymatic approaches to next-generation HDAC inhibitors.

Recent evidence indicates that mono - and poly-ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1-deoxyadenosine (N1-dA). Here, we report that PARP1 catalyzes N3-specific ADP-ribosylation of deoxycytidine (N3-dC) in single-stranded DNA. Analogous to N1-dA PARylation, which is prone to spontaneous adenine-to-inosine deamination, N3-dC PARylation promotes cytosine deamination, yielding N3-PARylated-deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC-MS/MS, namely N1-ribosyl-deoxyinosine (N1-r-dI) and N3-ribosyl-deoxyuridine (N3-r-dU). We synthesized both N1-r-dI and N3-r-dU as diagnostic standards and established absolute quantification of base ADP-ribosylations by LC-MS/MS. Quantitative analysis of PARylated dA and dC in ssDNA reveals pronounced sequence preferences of PARP1. Removal of these base modifications differs markedly, since ADP-ribose glycohydrolase TARG1 removes PAR from both dA and dC, whereas PARG acts exclusively on dA. Our results establish cytidine ADP-ribosylation as a novel DNA modification, with potential roles in DNA metabolism, epigenetic regulation, or genome stability.

Chimeric molecules, which bring together an effector enzyme and a protein-of-interest (POI) to add/remove post-translational modifications (PTMs), are furnishing transformative modalities (e.g., PROTACs). However, these chimeras scalability is limited as they employ rare, non-inhibitory binders of effectors. We report GRoup-transfer chimeras for Inducing Proximity (GRIPs) that employ abundantly available effectors inhibitors to append POI binder on the effector using group-transfer handles. To demonstrate scalability, we develop 6 GRIPs classes for 3 PTMs utilizing diverse inhibitor, spanning 16 effector-POI pairs. Furthermore, we report a toolbox of 42 tunable group-transfer handles for Cys/Lys residues and [~]5000 inhibitor-residue pairs for diverse effectors. Using global proteomics, we confirm the specificity for group transfer and PTM editing. GRIPs endowed new functionalities to POI drugs, including preventing rebound signaling upon drug withdrawal, a more potent/persistent inhibition, and inhibitor-induced pathway activation in 4 fully-endogenous systems. In diverse hemi-endogenous systems (tagged POI), GRIPs induced condensate formation with reduced off-targets, cleared pathogenic PTMs, and initiated PTM crosstalk. Overall, GRIPs provide a scalable and versatile platform for PTM editing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/706349v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1e443e5org.highwire.dtl.DTLVardef@1259756org.highwire.dtl.DTLVardef@f2b291org.highwire.dtl.DTLVardef@1a96191_HPS_FORMAT_FIGEXP M_FIG C_FIG