ACS Central Science

Antibiotic-resistant (AMR) bacterial infections are a major global health threat. Despite the critical need for new antimicrobials, progress is constrained by protracted development timelines, as well as the requirement for chemical novelty to avoid cross-resistance. Although advances in high-throughput screening, genome mining, and machine learning have greatly accelerated antimicrobial discovery, insufficient separation between antibacterial efficacy and host toxicity remains a bottleneck, precluding the clinical development of many promising compounds. Here, we establish a generalizable, two-component strategy to engineer antimicrobial safety and mobilize otherwise inaccessible chemical space for antimicrobial therapy, using calicheamicin, a potent cytotoxin with unacceptable host toxicity, as a proof of concept. In the first arm, we engineer a conditionally-active drug conjugate that limits calicheamicin activity to infected tissue, thereby reducing systemic toxicity. In the second arm, we co-administer a re-engineered self-resistance enzyme from Micromonospora echinospora, the natural producer of calicheamicin, as an "antidote" to neutralize calicheamicin present outside of infected tissue, further mitigating off-target toxicity. The conditionally-active conjugate exhibits activity against Gram-negative and Gram-positive pathogens in response to a protease present within the infected microenvironment. When delivered in combination with the antidote, antibacterial efficacy is maintained while off-target toxicity is reduced in mouse models of Gram positive and negative bacterial pneumonia. We anticipate that our dual strategy, which engineers, rather than selects for enhanced drug safety, by combining conditional drug activity with antidote-driven neutralization of off-target effects, provides a generalizable framework for mobilizing other promising but toxic compounds as antimicrobials.

Pathological seeding of protein misfolding is a hallmark of proteinopathies. However therapeutic strategies to clear these aggregates are lacking, impairing both study of their biological importance in disease etiology and progression as well as development of therapeutics. This is due in part to the need to selectively clear oligomerized proteins whilst leaving functional monomers intact, as well as the challenge of developing molecules that act on the full complement of misfolds the protein can adopt throughout the course of disease. In this work, we describe a dopant system consisting of an engineered alpha-synuclein protein construct that rapidly co-aggregates into existing WT alpha-synuclein oligomers, enabling rapid degradation of the entire assembly in the presence of a small molecule trigger. This work provides proof-of-principle for an approach that transforms pathological seeding from a disease-driver into a therapeutic vulnerability, and is potentially applicable to any proteinopathy without requiring a small molecule binder of the pathologic species.

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacteriums complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.

Single-agent clinical response data for widely used chemotherapies have remained difficult to analyze because they are scattered across decades of print literature. We consolidated these sources into NCI1970-Meta, a 49,002-patient dataset covering 30 drugs across 18 cancers, enabling the first quantitative comparison of clinical outcomes, laboratory potency metrics, clinical exposure, and literature-derived biomarkers. Clinical patterns were strongly lineage-driven: cancer type explained far more ORR variability than drug identity (37.3% vs 15.6%; F = 13.43 vs 3.29). FDA approvals reflected these same patterns where ORR strongly predicted indication status (F = 98.3-104.1). In contrast, laboratory efficacy metrics did not track clinical activity. Raw in vitro AUC showed no association with ORR (R2 = 0.00; 95% CI: 0.00-0.01) and was dominated by drug identity rather than cancer lineage (85.2% vs 8.6%; F = 212.2 vs 21.6). Instead, AUC correlated with clinical exposure: unbound Cmax (R2 = 0.21 [0.21-0.34]) and therapeutic minimum concentrations (R2 = 0.69 [0.64-0.73]). This indicates that standard assay ranges capture exposure requirements rather than true efficacy. Normalizing potency by exposure restored the expected clinical relationships and resolved drug-specific anomalies such as gemcitabine. Biomarkers showed consistent behavior across clinical and laboratory settings. Among 314 biomarker-drug pairs, correlation directions were significantly conserved (R2 = 0.17 [0.10-0.25]; p = 2.8x10-14). Literature-defined sensitivity and resistance annotations were enriched in vitro (OR = 3.7; p = 2.36x10-8) and in the clinic (OR = 3.9; p = 6.52x10-9), with stronger performance for correlations >0.1 (OR = 13.3 in vitro; OR = 8.4 clinically). Simple biomarker-sum models performed well across drugs, consistent with multi-pathway pseudo-first-order behavior. Overall, NCI1970-Meta provides a quantitative framework linking laboratory pharmacology to real-world clinical efficacy. Biological signal is reliably preserved within drugs, while cross-drug comparisons require explicit exposure normalization. This resource offers a statistical foundation for improving drug prioritization, biomarker development, and translational pharmacodynamic modeling.

Antibody-drug conjugates (ADCs) have experienced a surge in clinical approvals in the past five years. Despite this success, a major limitation to ADC efficacy in solid tumors is poor tumor penetration, which leaves many cancer cells untargeted. Increasing antibody doses or co-administering ADC with an unconjugated antibody can improve tumor penetration and increase efficacy when target receptor expression is high. However, it can also reduce efficacy in low-expression tumors where ADC delivery is limited by cellular uptake. This creates an intrinsic problem because many patients express different levels of target between tumors and even within the same tumor. Here, we generated High-Avidity, Low-Affinity (HALA) antibodies that can automatically tune the cellular ADC delivery to match the local expression level. Using HER2 ADCs as a model, HALA antibodies were identified with the desired HER2 expression-dependent competitive binding with ADCs in vitro. Multi-scale distribution of trastuzumab emtansine and trastuzumab deruxtecan co-administered with the HALA antibody were analyzed in vivo, revealing that the HALA antibody increased ADC tumor penetration in high-expression systems with minimal reduction in ADC uptake in low-expression tumors. This translated to greater ADC efficacy in immunodeficient mouse models across a range of HER2 expression levels. Furthermore, Fc-enhanced HALA antibodies showed improved Fc-effector function at both high and low expression levels and elicited a strong response in an immunocompetent mouse model. These results demonstrate that HALA antibodies can expand treatment ranges beyond high expression targets and leverage strong immune responses.

Antibodies and other protein therapeutics have revolutionized medicine, but their application is largely limited to extracellular targets. The lack of efficient intracellular delivery methods remains a major bottleneck. Here, we engineered a family of small ([~]90 amino acids), metabolically stable membrane translocation domains (MTDs) by modifying the loop sequences of a human fibronectin type III (FN3) domain. The most potent variant, MTD4, is highly cell-permeable and can be recombinantly fused to the N- or C-terminus of any peptide or protein, serving as a versatile "plug-and-play" vehicle. We demonstrate that MTD4 fusions efficiently deliver a wide variety of functional peptides and proteins into the cytosol and nucleus of eukaryotic cells, both in vitro and in vivo. Following systemic administration, MTD4 fusion proteins exhibit broad biodistribution and homogenous tissue penetration in mice. Importantly, MTD4 is effective at low nanomolar (nM) concentrations, making it a promising platform for addressing a vast range of intracellular and previously "undruggable" targets.

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named Template-assisted covalent modification, where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

Proteolysis-targeting chimeras (PROTACs) represent a transformative therapeutic modality, yet the viability of covalent PROTACs remains debated, as irreversible binding seemingly contradicts the catalytic mechanism central to their function. Here, we develop and characterize PSIRC3, a highly potent covalent PROTAC for Brutons tyrosine kinase (BTK) that addresses this ambiguity. PSIRC3 induces potent and selective BTK degradation with a sub-nanomolar DC50 of 0.75 nM and a Dmax greater than 85%, while its non-covalent counterpart is completely inactive. This degradation activity is strictly dependent on covalent bond formation with the Cys481 residue, as evidenced by a total loss of efficacy against the C481S BTK mutant. PSIRC3 acts with remarkable speed, achieving maximum BTK degradation within 30 minutes, a kinetic profile linked to rapid cell permeation and efficient ternary complex formation. In vivo, a single administration of PSIRC3 leads to substantial BTK degradation in both PBMCs (>80%) and splenocytes (>50%). Computational modeling, parameterized with experimental data, reveals that degradation efficacy is governed by a delicate balance between E3 ligase and target protein affinities. Specifically, excessively high E3 affinity is detrimental by inducing a hook effect, while higher target affinity is generally beneficial. Our findings provide strong evidence that covalent engagement can drive potent and selective protein degradation, challenging the prevailing notion that catalytic turnover is indispensable for PROTAC efficacy. This work establishes a new benchmark for covalent degraders and opens new avenues for targeting previously intractable proteins.

The burgeoning field of targeted protein degradation (TPD) has opened new avenues for modulating the activity of previously undruggable proteins of interest. To date, TPD has been dominated by small molecules containing separate linked domains for protein engagement and recruitment of cellular degradation machinery. The process of identifying active compounds has required tedious optimization and has been successful largely against a limited set of targets with well-defined, suitable docking pockets. Here we present a polymer chemistry approach termed the HYbrid DegRAding Copolymer (HYDRAC) to overcome standing challenges associated with the development of TPD. These copolymers densely display either peptide-based or small molecule-derived degradation inducers and target-binding peptide sequences for the selective degradation of disease-associated proteins. HYDRACs are synthesized in a facile manner, are modular in design, and are highly selective. Using the intrinsically disordered transcription factor MYC as an initial proof-of-concept, difficult to drug protein target, HYDRACs containing a MYC-inhibitory peptide copolymerized with a validated degron, showed robust and selective degradation of the target protein. Treatment of tumor-bearing mice with MYC-targeted HYDRACs showed decreased cell proliferation and increased tumor apoptosis, leading to significantly suppressed tumor growth in vivo. The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained MYC degradation. To demonstrate generalizability, HYDRACs were further designed against a second elusive target of clinical interest, KRAS, by employing a consensus RAS binding motif. RAS-targeted HYDRACs showed degradation in two cell lines harboring separate KRAS alleles, suggesting potential pan-KRAS activity. We envision the HYDRAC platform as a generalizable approach to developing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for TPD.

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). L,D-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of {beta}-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

The poor preclinical and clinical success rates of low molecular weight (LMW) compounds can be partially attributed to the inherent trial-and-error nature of pharmaceutical research, which is limited largely to retrospective data-driven, rather than prospective prediction-driven human relevant workflows stemming from: 1) inadequate scientific understanding of structure-activity, structure-property, and structure-free energy relationships; 2) disconnects between empirical models derived from in vitro equilibrium data (e.g., Hill and Michaelis-Menten models) vis-a-vis the native non-equilibrium cellular setting (where the pertinent metrics consist of rates, rather than equilibrium state distributions); and 3) inadequate understanding of the non-linear dynamic (NLD) basis of cellular function and disease. We argue that the limit of understanding of cellular function/dysfunction and pharmacology based on empirical principles (observation/inference) has been reached, and that further progress depends on understanding these phenomena at the first principles theoretical level. Toward that end, we have been developing and applying a theory (called "Biodynamics") on the general mechanisms by which: 1) cellular functions are conveyed by dynamic multi-molecular/-ionic (multi-flux) systems operating in the NLD regime; 2) cellular dysfunction results from molecular dysfunction; 3) molecular structure and function are powered by covalent/non-covalent forms of free energy; and 4) cellular dysfunction is corrected pharmacologically. Biodynamics represents a radical departure from the status quo empirical science and reduction to practice thereof, replacing: 1) the interatomic contact model of structure-free energy and structure-property relationships with a solvation free energy model; 2) equilibrium drug-target occupancy models with dynamic models accounting for time-dependent drug and target/off-target binding site buildup and decay; and 3) linear models of molecular structure-function and multi-molecular/-ionic systems conveying cellular function and dysfunction with NLD models that more realistically capture the emergent non-linear behaviors of such systems. Here, we apply our theory to COVID Mpro inhibition and overview its implications for a holistic, in vivo relevant approach to drug design.

Enzyme-based self-labeling tags enable covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing multifunctional ligands that contain both fluorophores and affinity tags or pharmacological agents and can still efficiently enter cells. Self-labeling tag ligands with short linkers can enter cells readily but often show less activity due to steric issues; ligands with long linkers can be more potent but show lower cell permeability. Here, we overcome this tug-of-war between efficacy and cell-permeability by devising a rational strategy for making cell permeable multifunctional ligands for labeling HaloTag fusions. We found that the lactone-zwitterion equilibrium sconstant (KL-Z) of rhodamines inversely correlates with their distribution coefficients (logD7.4), suggesting that ligands based on dyes exhibiting low KL-Z and high logD7.4 values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 from euchromatin to the nucleolus or heterochromatin. We discovered that translocation of BRD4 to constitutive heterochromatin in cells expressing HaloTag-HP1a fusion proteins can lead to apparent increases in transcriptional activity. These new reagents enable affinity capture and translocation of intracellular proteins in living cells and the use of Si-rhodamines and other low KL- Z/high logD7.4 dye scaffolds will facilitate the design of new multifunctional chemical tools for biology. SIGNIFICANCE STATEMENTUnderstanding cellular processes requires tools to measure and manipulate proteins in living cells. Self- labeling tags, such as the HaloTag and SNAP-tag, enable modification of cellular proteins with synthetic molecules. Creating ligands for these systems that have more than one chemical motif remains challenging, however, due to competing demands between cell permeability and functionality. We discovered that multifunctional ligands based on Si-rhodamines efficiently entered cells and enabled affinity purification of mitochondria or translocation of nuclear proteins; the performance of these molecules could be verified by fluorescence microscopy. These compounds should be useful for a variety of biological experiments and our general framework will allow the design of other multifunctional ligands to study living systems.

Synthesis of sequence-defined biomaterials whose monomer backbones diverge from canonical -amino acids represents the next frontier in protein and biomaterial evolution with the potential to yield better biological therapeutics, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials are {beta}-hydroxy acids. Many natural products contain isolated {beta}-esters, and polymeric {beta}-esters are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that {beta}2-hydroxy acids possessing both (R) and (S) absolute configuration are excellent substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro, and that (S)-{beta}2-hydroxy acids are substrates in cellulo. Using the MaPylRS/MatRNAPyl pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two (S)-{beta}2-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed enantioselective preference of the ribosome for the (S)-{beta}2-hydroxy acid backbone and mechanistic insights that inform future ribosomal engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that accepts a {beta}-backbone substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.

Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N-acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

Classifying the mechanisms of antibiotic failure has led to the development of new treatment strategies for killing bacteria. Among the currently described mechanisms, which include resistance, persistence and tolerance, we propose bacterial defiance as a form of antibiotic failure specific to prodrugs. As a prototypic model of a bacteria-activated prodrug, we construct cationic antimicrobial peptides (AMP), which are charge neutralized until activated by a bacterial protease. This construct successfully eliminated the vast majority of bacteria populations, while localizing activity to bacterial membranes and maintaining low active drug concentration. However, we observed defiant bacteria populations, which survive in the presence of identical drug concentration and exposure time. Using a multi-rate kinetic feedback model, we show that bacteria switch between susceptibility and defiance under clinically relevant environmental (e.g., hyperthermia) and genetic (e.g., downregulated protease expression) conditions. From this model, we derive a dimensionless quantity (Bacterial Advantage Heuristic, BAH) - representing the balance between bacterial proliferation and prodrug activation - that perfectly classifies bacteria as defiant or susceptible across a broad range of conditions. To apply this concept to other classes of prodrugs, we expand this model to include both linear and nonlinear terms and use general pharmacokinetic parameters (e.g., half-life, EC50, etc.). Taken together, this model reveals an analogous dimensionless quantity (General Advantage Key, GAK), which can applied to prodrugs with different activation mechanisms. We envision that these studies will enable the development of more effective prodrugs to combat antibiotic resistance.

Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m7GpppX cap present at the 5' terminus of coding mRNAs, with an m7G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technologys potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase - a critical enzyme in glycolysis - and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

Mucin proteins are essential for life but are challenging to study due to their complex glycosylation patterns. Synthetic mimics have become vital tools for understanding and modulating the roles of mucins in human health and disease. We developed a chemoenzymatic approach to prepare polypeptide-based synthetic mucins displaying a variety of glycans with native linkages and orientations. By combining the polymerization of glycosylated amino acid N-carboxyanhydrides with enzymatic sialylation and fucosylation, we produced a tunable panel of synthetic mucins. These polymers were recognized by natural glycan-binding and glycan-degrading enzymes, providing insights into the structural preferences of these proteins. Glycan- and linkage-dependent effects on proteolysis were observed. Further, investigation of the influence of glycans on peptide backbone secondary structure revealed that both sialylation and linkage at Ser vs. Thr have profound effects on hierarchical conformation. Overall, our methodology offers versatile tools for exploring the diverse glycobiology of mucins.

Many cancers depend on extracellular growth factors within the tumor microenvironment to drive aberrant signaling, proliferation, and survival. For example, anticancer therapies targeting vascular endothelial growth factor activity have been effective in blocking pro-angiogenic and pro-growth signals, including receptor tyrosine kinase inhibitors (e.g., Sunitinib and Sorafenib) and monoclonal antibodies (e.g., Bevacizumab). However, the effectiveness of these therapies is frequently limited by compensatory growth factor signaling and incomplete blockade, contributing to drug resistance and suboptimal long-term responses. To address these challenges, we developed Glycomimetic Lysosome-targeting Chimeras (GLYTACs) that sequester and degrade extracellular growth factors in the cancer cell environment. GLYTACs exploit growth factor interactions with cell-surface heparan sulfate (HS) glycans, a feature shared by many pro-tumorigenic signals and their receptors, by combining an HS-glycomimetic arm for growth factor binding with a polyvalent glycopolymer ligand targeting the lysosomal recycling cation-independent mannose 6-phosphate receptor (CI-M6PR) for efficient internalization and degradation. Treating HeLa cells with a heparin-based GLYTAC prototype drove rapid uptake and degradation of extracellular fibroblast growth factor 2 (FGF2). The capacity of heparin to promote the association of FGF2 with its cognate receptors (FGFRs) led to the degradation of the entire receptor-ligand complex, thereby reducing the availability of FGFRs at the cancer cell surface, which are necessary for sustained pro-oncogenic signaling. These findings highlight the potential of GLYTACs as an alternative to existing growth factor-blocking anticancer therapies and as a strategy to reshape the extracellular signaling environment of tumors.

Peptide-based therapeutics are currently in great demand but often suffer from rapid clearance and accumulation in off-target tissues which continue to present barriers in their clinical translation. Here, we developed an electrophilic peptide for the attachment of therapeutics to native immunoglobulin (IgG) in vivo, enabling the bioorthogonal covalent linkage, or painting, of peptide drugs of choice to circulating IgGs directly in live animals. Native IgG painting with glucagon-like peptide-1 (GLP-1) results in sustained body weight loss and prolonged blood glucose management after one dose. Such technology might revolutionize the next generation of long-acting peptide-based medicines.