Cell Chemical Biology

Glycosylation depends on tightly regulated pools of nucleotide-sugars (NS), yet the mechanisms controlling mammalian NS homeostasis and their downstream effects on glycoprotein biosynthesis remain poorly understood. UDP-galactose 4'-epimerase (GALE) catalyzes the reversible interconversion of UDP-galactose/UDP-glucose and UDP-N-acetylgalactosamine/UDP-N-acetylglucosamine, making it a central regulator of glycan precursor pools and an excellent model enzyme for studying NS metabolism. Here, we report the discovery of a cell-active small molecule inhibitor of human GALE through a high-throughput chemical screening strategy. Using a coupled luminescence-based assay, we identified the FDA-approved drug disulfiram as a GALE inhibitor. Biochemical analyses demonstrated that disulfiram directly inhibits GALE through covalent modification of cysteine residues, including C153, likely via its reactive metabolite diethyldithiocarbamate. In cultured human cells, disulfiram treatment phenocopied genetic GALE deletion, reducing terminally sialylated glycans, mucin-type O-glycans, and properly glycosylated mucin-domain glycoproteins. These effects were rescued by galactose supplementation, consistent with a mechanism of on-target GALE inhibition. Similar phenotypes were observed in human lung adenocarcinoma cells, supporting a broader role for GALE in regulating glycosylation and mucin biosynthesis across tissue types. Together, these studies establish a platform for the discovery of pharmacological GALE inhibitors as new research tools, identify disulfiram as a cell-active chemical probe for studying NS regulation, and suggest that targeting GALE might modulate mucin hypersecretion in muco-obstructive diseases and mucinous cancers.

Tumor reliance on antioxidant defenses creates a vulnerability to ferroptosis, yet strategies to therapeutically disable these systems remain limited. Here, we identify targeted degradation of the selenium uptake receptor LRP8 as an effective approach to decrease the abundance of the ferroptosis-protective enzyme glutathione peroxidase 4 (GPX4). Using bispecific cytokine receptor-targeting chimeras (KineTACs) that couple LRP8 to cytokine receptor internalization pathways, we selectively direct LRP8 to the lysosome for degradation. LRP8 degradation reduces the abundance of several selenoproteins, including GPX4, lowering the cellular threshold for lipid peroxidation and sensitizing cancer cells to ferroptosis. These findings establish receptor-mediated selenium uptake as a critical, targetable node in ferroptosis resistance and demonstrate that extracellular protein degradation can be leveraged to reprogram intracellular translational dependencies in cancer cells. More broadly, this work provides a framework for exploiting nutrient acquisition pathways to overcome therapy resistance.

Plant monoterpene indole alkaloids (MIAs) exhibit important pharmacological activities, yet understanding of their biosyntheses remains incomplete. Since protein-protein interactions (PPIs) represent a conserved regulatory mechanism in MIA-producing plants, we developed a large-scale, yeast-based screening pipeline to profile PPIs of a key enzyme, strictosidine {beta}-D-glucosidase (SGD) from Mitragyna speciosa (kratom). This screen identified six novel medium-chain dehydrogenases/reductases (MDRs) as high-confidence interaction partners of SGD. Biochemical characterization revealed that all six MsMDRs produce an MIA we named charlamine by acting directly on the reactive strictosidine aglycone intermediate, preventing its spontaneous rearrangement and establishing a functional rationale for SGD-MDR interaction. One MsMDR additionally catalyzed the reduction of vallesiachotamine, derived from the spontaneous rearrangement of strictosidine aglycone, to another previously unreported MIA, vallesiachotaminol. Parallel transcriptomics and genomics analyses uncovered a biosynthetic gene cluster containing a dihydrocorynantheine aldehyde esterase, functioning downstream of MsMDRs. Collectively, these findings demonstrate the utility of interactomics-driven plant pathway discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/722234v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ac5843org.highwire.dtl.DTLVardef@1d6d51dorg.highwire.dtl.DTLVardef@1417446org.highwire.dtl.DTLVardef@38afb2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antibiotic discovery has long relied on occupancy-driven inhibition, leaving a vast number of potential bacterial targets undrugged.1 Targeted protein degradation offers a mechanistically distinct alternative to inhibition, yet its application to antibacterial drug discovery remains largely unexplored.2-4 Here we describe the development of first-in-class heterobifunctional bacterial proteolysis targeting chimeras (BacPROTACs) directed against an essential Mycobacterium tuberculosis protein, 4-phosphopantetheinyl transferase (PptT).5 Leveraging the modular architecture of BacPROTACs, we repurposed PptT inhibitors by incorporating them into degraders, yielding compounds with markedly improved antimycobacterial activity. Integrating in vitro and cellular approaches, we developed a characterisation pipeline to assess protein degradation in bacteria, applicable to future BacPROTAC programmes. Our study establishes targeted protein degradation as a strategy for antibacterial drug discovery.

Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole-core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the {Omega}perX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 g mL1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.

Oncogenic MYC transcription factors profoundly alter cellular programs, imposing dependencies that can be therapeutically exploited in MYC-driven cancers such as high-risk neuroblastoma. However, dissecting such synthetic lethal vulnerabilities using controlled, tunable gene expression within a uniform genetic background remains challenging. Widely used tetracycline-regulated systems rely on inducers known to perturb mitochondrial function, introducing significant off-target effects that may confound interpretation. To overcome this limitation, we established novel cumate (p-isopropylbenzoate)-inducible neuroblastoma models that enable physiologically unbiased regulation of MYC(N) expression. Functional validation demonstrated that cumate itself does not induce off-target effects on neuroblastoma cell viability, mitochondrial membrane potential, morphology, proteostasis, or stress signaling, even at the highest recommended dose. The developed SHEP-CuO-MYC and -MYCN models show efficient, titratable, and reversible upregulation of c-MYC and N-MYC, respectively, recapitulating the expression levels observed in MYC(N)-amplified neuroblastoma. As a proof-of-concept, we applied these models to mechanistically validate the recently proposed mitoribosomal synthetic lethality, providing fully unbiased evidence that elevated c-MYC/N-MYC levels sensitize neuroblastoma cells to inhibitors of mitochondrial gene expression. Although impairing mitochondrial translation activated mitochondrial integrated stress response in both MYC-on and MYC-off states, it led to dramatic MYC downregulation coupled with enhanced caspase-dependent cell death in MYC-on cells. These findings reveal that MYC(N) overexpression confers a selective, proliferation-independent mitochondrial vulnerability that can be therapeutically targeted by repurposing well-tolerated mitochondrial ribosome-targeting antibiotics. Collectively, our models provide a robust platform for studying the MYC-mitochondria interplay and can be directly adapted for drug repurposing screens targeting mitochondrial dependencies in neuroblastoma and, potentially, other MYC-driven tumors. HIGHLIGHTSO_LICumate shows no inducer-associated mitochondrial or cytotoxic off-target effects C_LIO_LICumate-inducible MYC models enable mechanistic studies of mitochondrial synthetic lethality C_LIO_LIMYC overexpression drives neuroblastoma sensitivity to mitochondrial translation inhibition C_LIO_LICommon ribosomal antibiotics trigger caspase-dependent cell death in MYC-driven tumor cells C_LIO_LIContext-specific MYC downregulation links mitochondrial stress to MYC synthetic lethality C_LI

Host-targeting antivirals offer a promising strategy for combating emerging viral threats by targeting cellular pathways required for infection. The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated as a host dependency factor exploited by multiple viruses, including coronaviruses, making it an attractive antiviral target. Here, we show for the first time that targeted degradation of p38 using the proteolysis-targeting chimera (PROTAC) NR-7h potently inhibits coronavirus infection. NR-7h induced substantial degradation of p38 in multiple cell lines and inhibited infection of two seasonal coronaviruses OC43 and 229E, providing broad pan-coronavirus activity. Infectious viral titres and viral RNA levels were significantly reduced without any detectable cytotoxicity. NR-7h exhibited greater antiviral potency than conventional p38 small-molecule inhibitors, with an IC50 of 1.0 nM compared with 648.4 nM for LY2228820, while the parent kinase inhibitor PH-797804 did not achieve 50% inhibition at the highest concentration tested. Pseudovirus and time-of-addition studies indicated that antiviral activity occurred at a post-entry stage of infection. Importantly, antiviral activity was eliminated by inhibition of proteasome function or E3 ligase activity, demonstrating dependence on PROTAC-mediated degradation. Our findings provide a proof-of-concept that targeted degradation of host kinase p38 can function as an antiviral modality and suggest PROTAC-based host-directed therapeutics may offer advantages over conventional kinase inhibition for broad-spectrum antiviral development.

Covalent inhibitors are rapidly becoming the standard of care for treatment of a range of disease states. Covalent inhibitors bind irreversibly to their target using a reactive electrophile (or warhead). Acrylamide and 2-butynamide are the most commonly used cysteine targeting electrophiles. These warheads are chosen for their efficient and selective modification of the protein and are presumed to be otherwise functionally inert. Using a panel of BTK covalent inhibitors (Tirabrutinib, Acalabrutinib, Ibrutinib and Zanubrutinib), we show that the 2-butynamide warhead on Tirabrutinib and Acalabrutinib, unlike the acrylamide warhead on Ibrutinib and Zanubrutinib, induces conformational heterogeneity in key regions required for BTK signaling. Tirabrutinib or Acalabrutinib bound BTK adopt multiple conformational states that are in dynamic exchange, show increased binding to the substrate PLC{gamma} and are less effective at inhibiting PLC{gamma} signaling when compared to Ibrutinib. Swapping only the warheads between Tirabrutinib and Ibrutinib leads to a corresponding switch in BTK dynamics and inhibitor efficacy. The unanticipated warhead-specific allosteric effects raise interesting possibilities regarding inhibitor-specific mechanisms of resistance. SIGNIFICANCE STATEMENTTreatment of B-cell cancers such as Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma has been revolutionized by the development of covalent inhibitors that target Brutons Tyrosine Kinase (BTK). These orally bioavailable cancer drugs are highly effective in interfering with B-cell growth and provide patients with long lasting remission. These treatments do come with vulnerabilities as inhibitor-specific resistance mutations emerge in a subset of patients. Here we investigate how chemical differences among available BTK inhibitors drive differential protein dynamics and signaling interactions that could foreshadow specific resistance mechanisms. As continuous use of BTK inhibitors progresses in time, the field will continue to learn which drugs, and which structural features of these drugs, either limit resistance or provide alternatives to established resistance.

Disturbances in protein homeostasis are a defining feature of aging and many neurodegenerative conditions. However, the balance of protein synthesis and clearance (i.e. protein turnover), remains challenging to quantify and to pharmacologically interrogate in post-mitotic human neurons at screening scale. Here, we establish a fluorescent timer-based, live-cell high-content imaging platform for protein turnover perturbation in human embryonic stem cell-derived neurons. We screened for 5,897 small molecules and identified 199 compounds that enhanced protein turnover. Dose-response assay of 47 prioritized candidates revealed several robust turnover modulators spanning diverse target classes. Transcriptome profiling and label-free quantitative proteomics on neurons treated with three selected compounds revealed upregulated expression of translation-associated genes. These compounds suppressed pathogenic [a]-synuclein (pS129) in a mouse primary neuron model of Lewy body-like pathology, and for one of them in human dopaminergic neurons. Together, this work provides a scalable discovery framework for protein turnover modulation in healthy and pathological contexts. TeaserA protein turnover screen identifies compounds increasing turnover in human neurons and reducing -synuclein pathology.

Aberrant HGF-c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression and a clinically validated therapeutic axis, but current inhibitors predominantly target the intracellular kinase domain and remain vulnerable due to limited selectivity and resistance development. We therefore pursued an upstream strategy based on small molecules that target the extracellular HGF-c-MET interaction interface. We combined large-scale virtual screening of more than one million compounds from the ChemDiv and Enamine libraries with molecular dynamics (MD) simulations, steered MD, MM/GBSA profiling, and iterative lead optimization to identify candidate c-MET inhibitors targeting its extracellular (EC) domain. In HGF-stimulated HuH7 cells, selected compounds suppressed c-MET autophosphorylation, reduced cell viability, and inhibited long-term colony formation. Surface plasmon resonance (SPR) further confirmed direct binding of L083-1287 and 8008-3424 to the recombinant c-MET ectodomain. Mechanistic analyses identified previously unrecognized hotspot residues on the c-MET EC domain and a novel inhibitory network spanning multiple c-MET ectodomain interfaces. L083-0077 displayed the most consistent interaction pattern within this framework, including stabilization of key hotspot residues and preserved binding under acidic conditions relevant to the tumor microenvironment. Zebrafish xenograft assays with selected early hit compounds revealed compound-dependent developmental liabilities supporting the use of this model as an early in vivo prioritization step during lead optimization. These findings establish EC interface-directed c-MET inhibition as a promising therapeutic strategy in HCC and provide a mechanism-guided platform for the development of selective, upstream c-MET inhibitors with the potential to complement or overcome limitations of kinase-directed therapies.

Human N-glycoproteins constitute a market worth hundreds of billions of dollars. However, their production in yeast is often limited by misfolding and subsequent degradation, largely due to differences in N-glycan-dependent protein quality control (QC) systems between humans and yeast. Notably, yeast lacks the UGGT-mediated reglucosylation-refolding cycle that rescues misfolded glycoproteins, and its degradation pathway involves fewer rate-limiting steps. To address this, we engineer the glycoprotein QC system in Kluyveromyces marxianus, a promising host for protein production, by introducing key human components and modifying native pathways. Expression of human UGGT1 or UGGT2 enhances the soluble and secretory production of glycoproteins in an activity-dependent manner. This effect is further improved by co-expression of the UGGT cochaperone SEP15 and by reducing native glucosidase II trimming activity. In addition, introduction of human EDEM2, a rate-limiting enzyme in glycoprotein degradation, delays ER-associated degradation and increases secretion. Integration of these engineering strategies substantially enhances the production of several high-value human-derived glycoprotein therapeutics, including etanercept, dulaglutide, and abatacept, with up to a [~]12-fold increase. These findings demonstrate that engineering a human-like glycoprotein QC network in yeast is an effective strategy to improve glycoprotein folding and secretion.

Many diseases arise from dysfunction of defined cell populations, yet most therapeutics distribute broadly, limiting efficacy and causing toxicity. We developed ExACT, a platform for cell-type-selective intracellular delivery that exploits membrane transporters. In vivo screening of combinatorial fluorescent small-molecule libraries in mouse brain identified chemistries whose uptake is dictated by endogenous transporter expression, yielding compounds with preferential entry into neurons, astrocytes, pericytes and endothelial cells. One series showed strong selectivity for brain and retinal endothelium, where Slco1a4 mediated uptake. This selectivity principle extended to the human orthologue SLCO1A2, highly expressed in brain endothelium and oligodendrocytes, where it mediated selective uptake in a humanized mouse model and human iPSC-derived oligodendrocytes. Ectopic expression of SLCO1A2 in neurons via gene therapy created a synthetic entry port, conferring ExACT conjugate uptake on otherwise inaccessible cells. Bifunctional compounds linking transporter-targeting motifs to antisense oligonucleotides or small-molecule drugs retained pharmacological activity while conferring transporter-dependent cell-type selectivity, illustrating how transporter diversity can be harnessed for precision pharmacotherapy.

The prokaryotic ubiquitin-like protein (Pup) conjugation system (PPS), which is essential for Mycobacterium tuberculosis (Mtb) virulence but absent in humans, presents an attractive drug target. Here, we report the discovery of ARQ-501, a quinone-based, covalent, substrate-competitive inhibitor of the Pup ligase PafA. ARQ-501 exhibited potent anti-mycobacterial activity against Mtb under host-mimicking stress conditions and within macrophages. We further identified the catalase-peroxidase KatG, essential for activation of the frontline prodrug isoniazid (INH), as a pupylation substrate. ARQ-501 inhibits KatG pupylation, causing its accumulation and creating a selective synergy with INH. This quantity over quality mechanism successfully rescued INH activation by the clinically prevalent KatG S315T mutant in enzymatic assays and enhanced INH efficacy against clinical S315T isolates to variable degrees. This work identifies a novel class of PafA inhibitors and a previously unrecognized role of pupylation in regulating KatG, offering a potential therapeutic avenue to combat drug-resistant tuberculosis.

We have developed and characterized a potent and cell active tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) inhibitor, 13. Compound 13 demonstrates in-cell, kinome-wide selectivity, and potently inhibits both TTBK1 and TTBK2. As part of our medicinal chemistry campaign, we also identified a structurally similar negative control, compound 5, which lacks in-cell affinity for TTBK1 and TTBK2. Based on their substrates, which include TDP-43, tau, and tubulin, TTBK1 and TTBK2 inhibition has been pursued as a therapeutic approach for Alzheimers disease, frontotemporal lobe dementia, and amyotrophic lateral sclerosis. TTBK2 is also an effector of ciliogenesis, acting in concert with CEP164, CP110, and CEP83 to initiate the biogenesis of primary cilia. The development of selective chemical tools for these kinases facilitates investigation into TTBK1/2-mediated pathways and potential disease-altering ramifications linked to their pharmacological perturbation.

The SARS-CoV-2 pandemic has underscored the urgent need for broad-spectrum antivirals in pandemic preparedness efforts. Nucleoside analogs targeting viral polymerases are often considered in this context. Here, we employ ensemble biochemical assays and single-molecule magnetic tweezers to characterize the detailed mechanism of action of the adenosine analog CMX521 (developed through Phase 1 clinical studies), a broad-spectrum antiviral against caliciviruses and coronaviruses, against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The triphosphate form of CMX521 is efficiently incorporated by RdRp, even against saturating ATP concentrations. Analog incorporation induces only a brief pause in nascent RNA synthesis. When embedded in the template strand, CMX521 causes the polymerase to stall ~9 s on average due to impaired uridine opposite incorporation. Multiple CMX521 residues in the template strand completely inhibit polymerase elongation. When the coronavirus polymerase is associated with the viral helicase, CMX521 strongly promotes copy-back RNA synthesis suggesting a second inhibitory mechanism for CMX521. Collectively, our findings establish a two-pronged mode of coronavirus polymerase inhibition by CMX521.

Kinases have proven to be one of the most fertile target classes for new drug approvals. However, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we systematically assessed the impact of minimal electrophilic modifications to ATP-site binding scaffolds, leading us to identify molecules that can control the activity and abundance of the master autophagy regulator, Unc-51-like autophagy activating kinase 1 (ULK1).

In vitro-transcribed messenger RNA (IVT mRNA) has emerged as a versatile protein expression platform with broad clinical potential. Current optimization strategies for IVT mRNA focus on untranslated regions (UTRs), mRNA stability, and codon usage, often guided by massively parallel screening and machine learning approaches. In contrast, the Kozak sequence, a key determinant of translation initiation, is often inconsistently incorporated into synthetic 5' UTR design, and its contribution to translation efficiency remains poorly defined. Here, we systematically varied the Kozak sequence across diverse UTR contexts and performed combinatorial optimization using synthetic, established, and viral UTRs to identify design principles for enhanced translation. We show that a single-nucleotide deviation from the consensus Kozak sequence consistently enhances protein expression across UTR contexts and coding sequences. This effect is conserved across in vitro and in vivo models, highlighting the generalizability of the optimized Kozak sequence. These findings redefine the role of the Kozak sequence in synthetic mRNA design and demonstrate its substantial contribution to translation efficiency when optimized, enabling improved mRNA-based therapeutics.

Chemical modifications such as 2'-O-methyl (2'-OMe) are central to the efficacy and tolerability of RNA therapeutics. We recently identified that 2'-OMe RNA fragments as short as three nucleotides can exert opposing effects on Toll-like receptor 8 (TLR8) sensing in a motif-dependent manner. This discovery raises important considerations for degradation products of chemically modified RNA therapeutics, which may generate such immunologically active fragments. Here, leveraging their short length, we systematically map how base and sugar modifications within 3-mer oligonucleotides regulate TLR7 and TLR8 responses, and we resolve the structural basis for both TLR8 potentiation and TLR7/8 antagonism by RNA fragments. Building on these insights, we report the development of a dual TLR7/8 inhibitory oligonucleotide with therapeutic potential in autoimmune disease. Together, these findings provide unprecedented resolution of the immunomodulatory properties of oligonucleotide modifications on TLR7/8 and establish 3-mer oligonucleotides as the shortest functional class of RNA therapeutics described to date.

SARS-CoV, MERS-CoV, and SARS-CoV-2 exemplify the persistent threat posed by coronaviruses, with their capacity for zoonotic spill over, rapid transmission, and high mortality, and thus underscores the urgent need for broad-spectrum antiviral strategies. The nucleocapsid (N) protein, essential for RNA binding, genome packaging, and viral replication, is highly conserved among coronaviruses but remains an underexplored antiviral target. In our earlier work, we identified two small molecules, ceftazidime and sennoside A, that bind the N-terminal domain of the SARS-CoV-2 N protein and inhibit nucleic acid binding, and identified their binding sites using NMR chemical shift perturbation assays. Here, we observed that several residues involved in inhibitor binding are conserved across betacoronaviruses, suggesting a shared druggable vulnerability. We have purified recombinant N proteins from SARS-CoV, MERS-CoV, and SARS-CoV-2, and demonstrated by electrophoretic mobility shift assays that both compounds significantly reduced RNA binding. Their inhibitory concentrations (IC50) were determined using fluorescence polarization. The docking analyses indicated that both inhibitors target the RNA-binding pocket of the N-NTD, consistent with a conserved mechanism of action. Collectively, our findings reveal a conserved RNA-binding vulnerability in coronavirus N proteins and highlights the pan-coronavirus therapeutic potential of these inhibitors.

The cellular uptake and propagation of tau are central features of tauopathies, including Alzheimers disease, and are mediated by the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1). While prior studies have implicated LRP1 in tau binding and internalization, the biochemical features of this interaction and its suitability for therapeutic targeting remain incompletely defined. Here, we establish a quantitative and scalable framework to interrogate the tau-LRP1 interaction and identify small-molecule modulators. We engineered and purified the LRP1 ligand-binding domain 4 (BD4), a key region mediating tau interaction, and developed multiple orthogonal assays, including fluorescence polarization, split luciferase complementation, and time-resolved FRET, to measure LRP1-BD4 interactions with tau and a known peptide ligand. Across assay formats, we observe consistent binding affinities in the nanomolar range and demonstrate competitive displacement by tau, receptor-associated protein (RAP), and a peptide ligand, supporting overlapping binding interfaces. Leveraging these platforms, we performed small molecule high-throughput screening and identified a set of candidate inhibitors of the LRP1-BD4-tau interaction. Selected compounds reduced tau uptake in a cellular assay, phenocopying competitive inhibition by tau and a peptide ligand. Together, these studies define the LRP1-BD4-tau interaction as a biochemically tractable and druggable interface and establish an integrated discovery pipeline linking mechanistic characterization to functional cellular outcomes. This work provides a foundation for the development of therapeutic strategies targeting LRP1-mediated tau uptake.