Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide-MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

The microbiota generates diverse metabolites to modulate host physiology and disease, but their protein targets and mechanisms of action have not been fully elucidated. To address this challenge, we explored microbiota-derived indole metabolites and developed photoaffinity chemical reporters for proteomic studies. We identified many potential indole metabolite-interacting proteins, including metabolic enzymes, transporters, immune sensors and G protein-coupled receptors. Notably, we discovered that aromatic monoamines can bind the orphan receptor GPRC5A and stimulate β-arrestin recruitment. Metabolomic and functional profiling also revealed specific amino acid decarboxylase-expressing microbiota species that produce aromatic monoamine agonists for GPRC5A-β-arrestin recruitment. Our analysis of synthetic aromatic monoamine derivatives identified 7-fluorotryptamine as a more potent agonist of GPRC5A. These results highlight the utility of chemoproteomics to identify microbiota metabolite-interacting proteins and the development of small-molecule agonists for orphan receptors.

With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the 'polarity reversal' of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein-protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.

Acinetobacter baumannii is a nosocomial Gram-negative pathogen that often displays multidrug resistance. Discovering new antibiotics against A. baumannii has proven challenging through conventional screening approaches. Fortunately, machine learning methods allow for the rapid exploration of chemical space, increasing the probability of discovering new antibacterial molecules. Here we screened ~7,500 molecules for those that inhibited the growth of A. baumannii in vitro. We trained a neural network with this growth inhibition dataset and performed in silico predictions for structurally new molecules with activity against A. baumannii. Through this approach, we discovered abaucin, an antibacterial compound with narrow-spectrum activity against A. baumannii. Further investigations revealed that abaucin perturbs lipoprotein trafficking through a mechanism involving LolE. Moreover, abaucin could control an A. baumannii infection in a mouse wound model. This work highlights the utility of machine learning in antibiotic discovery and describes a promising lead with targeted activity against a challenging Gram-negative pathogen.

The complement receptors C3aR and C5aR1, whose signaling is selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR1 signaling lead to multiple inflammatory disorders, including sepsis, asthma and acute respiratory distress syndrome. The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR1 remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo-C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understanding of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.

Histone acetyltransferases (HATs) are implicated as both oncogene and nononcogene dependencies in diverse human cancers. Acetyl-CoA-competitive HAT inhibitors have emerged as potential cancer therapeutics and the first clinical trial for this class of drugs is ongoing (NCT04606446). Despite these developments, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Having discovered that multiple regulators of de novo coenzyme A (CoA) biosynthesis can modulate sensitivity to CBP/p300 HAT inhibition (PANK3, PANK4 and SLC5A6), we determined that elevated acetyl-CoA concentrations can outcompete drug-target engagement to elicit acquired drug resistance. This not only affects structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B HAT inhibitor that is currently in Phase 1 clinical trials. Altogether, this work uncovers CoA metabolism as an unexpected liability of anticancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.

Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, stoichiometric measurement of individual Ψ sites in human transcriptome remains unaddressed. Here we develop 'PRAISE', via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional bisulfite treatment, our approach is based on quaternary base mapping and revealed an ~10% median modification level for 2,209 confident Ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1, with TRUB1 targets showing the highest modification stoichiometry. In addition, we quantified known and new Ψ sites in mitochondrial mRNA catalyzed by PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide Ψ; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

RNA-binding proteins (RBPs) play an important role in biology, and characterizing dynamic RNA-protein interactions is essential for understanding RBP function. In this study, we developed targets of RBPs identified by editing induced through dimerization (TRIBE-ID), a facile strategy for quantifying state-specific RNA-protein interactions upon rapamycin-mediated chemically induced dimerization and RNA editing. We performed TRIBE-ID with G3BP1 and YBX1 to study RNA-protein interactions during normal conditions and upon oxidative stress-induced biomolecular condensate formation. We quantified editing kinetics to infer interaction persistence and show that stress granule formation strengthens pre-existing RNA-protein interactions and induces new RNA-protein binding events. Furthermore, we demonstrate that G3BP1 stabilizes its targets under normal and oxidative stress conditions independent of stress granule formation. Finally, we apply our method to characterize small-molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile dynamic RNA-protein interactions in cellular contexts with temporal control.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy-lysosomal pathway.

Hyperactivated glycolysis is a metabolic hallmark of most cancer cells. Although sporadic information has revealed that glycolytic metabolites possess nonmetabolic functions as signaling molecules, how these metabolites interact with and functionally regulate their binding targets remains largely elusive. Here, we introduce a target-responsive accessibility profiling (TRAP) approach that measures changes in ligand binding-induced accessibility for target identification by globally labeling reactive proteinaceous lysines. With TRAP, we mapped 913 responsive target candidates and 2,487 interactions for 10 major glycolytic metabolites in a model cancer cell line. The wide targetome depicted by TRAP unveils diverse regulatory modalities of glycolytic metabolites, and these modalities involve direct perturbation of enzymes in carbohydrate metabolism, intervention of an orphan transcriptional protein's activity and modulation of targetome-level acetylation. These results further our knowledge of how glycolysis orchestrates signaling pathways in cancer cells to support their survival, and inspire exploitation of the glycolytic targetome for cancer therapy.

O-linked β-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including glucose and glutamine; its dysregulation leads to molecular and pathological alterations that cause diseases. Here we report that O-GlcNAc directly regulates de novo nucleotide synthesis and nicotinamide adenine dinucleotide (NAD) production upon abnormal metabolic states. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1), the key enzyme of the de novo nucleotide synthesis pathway, is O-GlcNAcylated by O-GlcNAc transferase (OGT), which triggers PRPS1 hexamer formation and relieves nucleotide product-mediated feedback inhibition, thereby boosting PRPS1 activity. PRPS1 O-GlcNAcylation blocked AMPK binding and inhibited AMPK-mediated PRPS1 phosphorylation. OGT still regulates PRPS1 activity in AMPK-deficient cells. Elevated PRPS1 O-GlcNAcylation promotes tumorigenesis and confers resistance to chemoradiotherapy in lung cancer. Furthermore, Arts-syndrome-associated PRPS1 R196W mutant exhibits decreased PRPS1 O-GlcNAcylation and activity. Together, our findings establish a direct connection among O-GlcNAc signals, de novo nucleotide synthesis and human diseases, including cancer and Arts syndrome.

Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.

Cancer-associated chromosomal rearrangements can result in the expression of numerous pathogenic fusion proteins. The mechanisms by which fusion proteins contribute to oncogenesis are largely unknown, and effective therapies for fusion-associated cancers are lacking. Here we comprehensively scrutinized fusion proteins found in various cancers. We found that many fusion proteins are composed of phase separation-prone domains (PSs) and DNA-binding domains (DBDs), and these fusions have strong correlations with aberrant gene expression patterns. Furthermore, we established a high-throughput screening method, named DropScan, to screen drugs capable of modulating aberrant condensates. One of the drugs identified via DropScan, LY2835219, effectively dissolved condensates in reporter cell lines expressing Ewing sarcoma fusions and partially rescued the abnormal expression of target genes. Our results indicate that aberrant phase separation is likely a common mechanism for these PS-DBD fusion-related cancers and suggest that modulating aberrant phase separation is a potential route to treat these diseases.

Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

Amyloid fibril is an important pharmaceutical target for diagnostic and therapeutic treatment of neurodegenerative diseases. However, rational design of chemical compounds that interact with amyloid fibrils is unachievable due to the lack of mechanistic understanding of the ligand-fibril interaction. Here we used cryoelectron microscopy to survey the amyloid fibril-binding mechanism of a series of compounds including classic dyes, (pre)clinical imaging tracers and newly identified binders from high-throughput screening. We obtained clear densities of several compounds in complex with an α-synuclein fibril. These structures unveil the basic mechanism of the ligand-fibril interaction, which exhibits remarkable difference from the canonical ligand-protein interaction. In addition, we discovered a druggable pocket that is also conserved in the ex vivo α-synuclein fibrils from multiple system atrophy. Collectively, these findings expand our knowledge of protein-ligand interaction in the amyloid fibril state, which will enable rational design of amyloid binders in a medicinally beneficial way.