Cell Chemical Biology

Reversible inactivation of protein tyrosine phosphatases by reactive oxygen species (ROS) is essential to the phosphorylation of growth factor receptors. An important outcome of the inactivation of protein tyrosine phosphatase 1B (PTP1B) by ROS involves the conformational change of its phosphotyrosine binding loop which adopts a solvent exposed position in its oxidized form. We previously demonstrated that 14-3-3{zeta} binds to the phosphotyrosine binding loop of the oxidized form of PTP1B. Using a rational approach, we developed a unique protein-protein interaction (PPI) inhibitor peptide derived from the phosphotyrosine binding loop of PTP1B designed to disrupt the interaction between PTP1B and the 14-3-3{zeta}-complex. Exploiting this cell-permeable peptide, we showed decreased association between PTP1B and the 14-3-3{zeta}-complex in cells treated with epidermal growth factor (EGF). We also demonstrated that preventing the association of this 14-3-3{zeta}-complex to PTP1B deterred oxidation and inactivation of PTP1B following EGF receptor (EGFR) activation and generation of ROS. Treating cells with our PPI inhibitor decreased EGFR phosphorylation on PTP1B-specific sites. Furthermore, treating EGFR-driven epidermal cancer cells with our PPI inhibitor also significantly inhibited colony formation and cell viability, consitent with increased activation of PTP1B. These data highlight the ability of PTP1B to downregulate critical signaling pathways in cancer when activated using peptide drugs such as our protein-protein interaction inhibitor. We anticipate that preventing or destabilizing the reversible oxidation of other members of the protein tyrosine phosphatase superfamily using PPI inhibitors may offer a foundation for a broad therapeutic approach to rectify dysregulated signaling pathways in vivo. Significance StatementLimited understanding of redox mechanisms regulating PTP catalytic activity is a major knowledge gap that has hampered our efforts to develop activation strategies. In its reversibly oxidized and inactivated form, conformational changes of PTP1B influence its association with regulatory proteins. We demonstrate that designing a cell-permeable peptide based on a loop of PTP1B that becomes exposed during oxidation can block its interaction with the 14-3-3{zeta}-multiprotein complex and activate the phosphatase. Moreover, activating PTP1B using our protein-protein interaction inhibitor peptide decreases the phosphorylation of its substrate EGFR and decreases the effectiveness of cancer cells to form colonies. This study provides important insights into the therapeutic potential of protein-protein interaction inhibitors that regulate the redox cycle of PTPs to reestablish physiological signaling.

In the effort to develop efficacious antibacterials that engage targets for which there is no pre-existing resistance, inhibition of the enoyl-acyl carrier protein reductase FabI has shown promise, with triclosan and fabimycin as representative members of the two major drug classes that show activity against important bacterial pathogens. Here, we use a morbidostat and whole genome sequencing to comprehensively evaluate the resistance profiles that arise in pathogenic bacteria in response to these FabI inhibitors. When assessed against E. coli, fabimycin and triclosan were found to induce primarily non-overlapping resistance profiles leading to minimal cross-resistance between the two compounds. Furthermore, in vivo evaluation of the prominent resistant mutants indicates poor fitness, with the most fit mutant still susceptible to fabimycin. Collectively, these results suggest the combination use of two antibiotics that engage different positions on the same target as a means to kill pathogenic bacteria and limit resistance.

Neurosteroids are endogenous neuromodulators and emerging therapeutics, but understanding but understanding how these compounds modulate receptor signaling within defined neuronal populations and networks has been limited by an inability to deliver these molecules with receptor-level and cell-type specificity. Here, we developed a neurosteroid DART (Drug Acutely Restricted by Tethering) that combines the GABAA receptor subunit-selectivity of a neuroactive steroid (NAS) with the cell-type specificity of the DART platform. Screening of seventeen NAS analogs identified seven scaffolds suitable for further engineering, and structure-activity analysis revealed that DART linker attachment at the C11 position preserved NAS activity on GABAA receptors, whereas C2 and C17 attachment failed to exhibit activity. Functional profiling of C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The most selective compound, YX85.1DART.2, potentiated GABA-evoked currents in neurons expressing pharmacogenetically isolated 4/{delta}-containing GABAA receptors but not in {gamma}2-expressing neurons. A previously validated BZP.1DART.2 produced complementary selectivity on the two receptor populations. Together, these findings establish new tools for interrogating subunit-specific NAS actions on inhibitory signaling in defined neuronal populations.

Many genetically validated targets in cancer, including the transcription factor {beta}-catenin ({beta}-cat), have historically been viewed as undruggable. Cell-based phenotypic screening of chemical compounds can reveal new biological and pharmacological principles. Natural products are powerful probes because of their superior structural diversity, drug-like properties, and biological activities as compared to unoptimized synthetic compounds. We screened 326,304 natural product mixtures (40,744 extracts and 285,560 fractions derived from them) using mammalian cells expressing an oncogenic version of {beta}-cat fused to a suicide protein. Multiple fractions degraded the {beta}-cat fusion protein or drove it into a compartment where both fusion partners were apparently inactive. The active natural product from one of the latter specifically activates novel, but not classical, protein kinase Cs (PKCs) and thereby relocates {beta}-cat to juxtamembrane vacuolar structures. These findings suggest a path for inactivating oncogenic {beta}-cat and underscore the power of screening natural product collections with robust phenotypic assays.

MicroRNAs (miRNAs) typically regulate gene expression by promoting mRNA degradation, but select miRNAs, such as miR-466l-3p (miR-466), can instead stabilize transcripts in coordination with RNA-binding proteins (RBPs) like HuR. We identify conserved AU-rich elements (cAREs) within the 3'UTRs of IL-17A, GM-CSF, and IL-23A as critical cis-regulatory binding sites where miR-466 facilitates HuR recruitment to promote mRNA stability. Using site-directed mutagenesis, RNA pulldown, and MS2-TRAP assays to capture miRNA-mRNA complexes, we demonstrate that HuR binding depends on prior engagement by miR-466. Disrupting this interaction with rationally designed Target Site Blockers (TSBs) oligonucleotides destabilizes target mRNAs and suppresses cytokine expression in vitro and in vivo. TSBs directed against IL-17A, GM-CSF, and IL-23A selectively blocked miR-466 binding, reduced transcript stability, and lowered cytokine production without affecting unrelated mRNAs. In murine models of LPS-induced inflammation, psoriasis, and autoimmunity, TSBs exhibited therapeutic efficacy and cytokine specificity, outperforming monoclonal antibodies in some settings. Phosphorothioate-modified TSBs enabled systemic delivery and retained activity in human T cells, underscoring translational potential. Similar to antisense oligonucleotides, TSBs trigger RNase H1-mediated degradation while also blocking miRNA-mRNA interactions. These findings establish miR-466-HuR cooperation as a therapeutically targetable axis through TSBs without affecting global miRNA function. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/709388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@82c326org.highwire.dtl.DTLVardef@da15e7org.highwire.dtl.DTLVardef@1d3fc69org.highwire.dtl.DTLVardef@607656_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TEXTBOXMechanism of TSB-mediated disruption of cooperative miRNA-HuR-dependent mRNA stabilizationA: In the canonical model, destabilizing miRNAs (e.g., miR-16) bind to their target sites within the 3'UTR, recruiting the RNA-induced silencing complex (miRISC) to promote mRNA decay or translational repression. B: In contrast, a newly identified class of miRNAs--stabilizing miRNAs (E-miRNAs), such as miR-466l-3p--bind to specific target sequences within AU-rich elements (AREs) in the 3'UTR. This binding facilitates cooperative recruitment of the RNA-binding protein HuR (ELAVL1), resulting in enhanced mRNA stability and/or translation. C: Target site blockers (TSBs) designed to occlude miRNA-binding sites competitively inhibit miRISC loading, thereby disrupting HuR engagement and reversing stabilization. This selective disruption leads to transcript-specific mRNA destabilization without affecting global miRNA function. C_TEXTBOX

N6-methyladenosine (m6A) is a major epitranscriptomic modification that regulates RNA metabolism and affects the replication and latency reversal of human immunodeficiency virus type 1 (HIV-1) in cells. Methyltransferase-like 3 (METTL3) is the principal catalytic enzyme responsible for m6A deposition, and its pharmacological inhibition has emerged as a potential therapeutic strategy for cancer and viral infections. However, the relative potency of METTL3 inhibitors in reducing m6A levels and their effects on HIV-1 latency reversal remain undefined. Here, we compared three commercially available METTL3 inhibitors (STM2457, STM3006, and STC-15) to evaluate their ability to reduce RNA m6A levels, suppress HIV-1 latency reversal, and affect cell viability in latently infected J-Lat cells and primary CD4+ T cells. In J-Lat cells, STM3006 and STC-15 were more potent than STM2457 in reducing RNA m6A levels at 24 and 48 hours post-treatment, as reflected by lower half-maximal inhibitory concentrations (IC50). However, STM3006 and STC-15 exhibited significant cytotoxicity at concentrations above 2 {micro}M at 48 hours post-treatment, whereas STM2457 displayed minimal toxicity across all tested doses. In primary CD4+ T cells from three healthy donors, all three inhibitors reduced RNA m6A levels but induced greater cytotoxicity compared with J-Lat cells, with comparable effects at optimized concentrations. Notably, reduced RNA m6A levels correlated with diminished HIV-1 latency reversal in both J-Lat cells and a primary central memory CD4+ T cell model. Together, these findings demonstrate differential potency and cytotoxicity among METTL3 inhibitors and support a critical role for m6A RNA modification in regulating HIV-1 latency reversal.

AO_SCPLOWBSTRACTC_SCPLOWCytosolic NAD{square} synthesis supports ovarian cancer growth by enabling PARP16-dependent mono(ADP-ribosyl)ation (MARylation) of ribosomal proteins, thereby fine-tuning translation and maintaining protein homeostasis. While genetic depletion of PARP16 disrupts ribosome MARylation and impairs tumor cell growth, the therapeutic potential of pharmacologic PARP16 inhibition in this pathway remains unexplored. Here, we characterized the effects of DB008, a tool compound that functions as a selective inhibitor of PARP16, in ovarian cancer cells. Biochemical analyses demonstrated that PARP16 undergoes NAD{square}-dependent auto-MARylation and that NMNAT-2 supplies NAD{square} to support this activity. DB008 potently inhibited PARP16 auto-MARylation in vitro. In ovarian cancer cells, DB008 engaged PARP16, reduced its MARylation, and decreased ribosome-associated MARylation. Consistent with PARP16 depletion, DB008 enhanced global protein synthesis, increased protein aggregation, and suppressed cell growth and anchorage-independent colony formation. CRISPR-mediated deletion of the PARP16 gene in ovarian cancer cells abolished the effects of DB008 on translation, protein aggregation, and proliferation, demonstrating on-target activity. Moreover, cells expressing a PARP16 mutant resistant to DB008 were unaffected by inhibitor treatment, further confirming that the cellular effects of DB008 require on-target inhibition. Finally, DB008 significantly inhibited tumor growth in OVCAR3 xenografts, with on-target engagement of PARP16 in the xenograft tumors. Collectively, these findings establish PARP16 as a druggable regulator of ribosome MARylation and protein homeostasis in ovarian cancer and provide pharmacologic proof-of-concept that disrupting ribosomal MARylation impairs tumor growth.

The ability of Plasmodium falciparum gametocytes to remain quiescent within the vertebrate host but poised for rapid onward development in the mosquito is an adaptation essential to maximise the onward spread of malaria. In this dormant state, mature infectious stage V gametocytes are largely unaffected by most antimalarial drugs and our limited understanding of how gametocytes prepare for mosquito transmission has hindered the identification of new molecular targets for transmission-blocking therapeutics. In this study, we move beyond the total proteome of gametocytes and define the translatome of mature stage V gametocytes using L-azidohomoalanine incorporation into nascent proteins, click chemistry purification and proteomic analysis. We identify the proteins and pathways that gametocytes sustain in preparation for transmission during this dormant period and through genetic disruption, we validate this approach by demonstrating the importance of parasite pyridoxal 5-phosphate biosynthesis for mosquito transmission.

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinsons disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, -fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing a metabolite likely to worsen Parkinsons phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

Summary/AbstractThe central AAA+ ClpC/ClpP protease in Gram-positive bacteria is crucial for virulence and stress resistance and has been recognized as drug target. Natural cyclic peptides deregulate the essential Mycobacterium tuberculosis ClpC1 and cause cell death. Similarly, overactivated mutants of the non-essential Staphylococcus aureus ClpC homologue cause uncontrolled proteolysis and severe toxicity in vivo. However, no chemical modulators of S. aureus ClpC have been described. Here, using a biochemical high-throughput screen we identify eight chemically distinct bona fide small molecules that robustly stimulate ClpC ATPase and proteolytic activity in vitro. Structural, computational, and mutational analyses define two ligandable regulatory sites within the ClpC N-terminal domain (NTD) as compound targets: a conserved hydrophobic groove and an allosteric pArg1 pocket, both engaged in substrate recognition. These findings establish S. aureus ClpC as chemically targetable and provide mechanistic insight into its regulatory architecture, enabling future development and optimization of chemical probes to deregulate AAA+ protease control.

Taxol is a blockbuster chemotherapeutic derived from the Pacific Yew tree. Recent work in our group has identified a complete pathway to baccatin III, a key intermediate, that hinges on a novel accessory protein, Facilitator of Taxane Oxidation (FoTO1). This protein dramatically improves yield and alters enzyme product profiles when reconstituting the Taxol pathway in N. benthamiana. FoTO1 has been shown to act early in the biosynthetic pathway improving the yields of product generated by the combination of a plastidial diterpene synthase (taxadiene synthase) and an endoplasmic reticulum (ER) localized cytochrome P450 (T5-alpha-hydroxylase). Here, we show that FoTO1 is an enzyme capable of converting taxadiene-(4),5-epoxide, the likely product of T5H oxidation, into taxadien-5-ol. FoTO1 is also functional in yeast, resolving a key bottleneck for development of a bioproduction route to Taxol in this host. Targeted mutagenesis of key catalytic residues in FoTO1 abrogates function in vitro but not in planta, suggesting non-catalytic contributions of FoTO1 to the taxane pathway. A combination of proximity labelling, bimolecular fluorescence complementation assays, and co-immunoprecipitation studies revealed that FoTO1 interacts with and organizes various P450s in the Taxol pathway. These approaches highlight the importance of both FoTO1s catalytic and non-catalytic functions in improving yields in the early Taxol pathway. Beyond Taxol biosynthesis, FoTO1 boosts yields for diverse diterpene pathways from across phylogeny, suggesting a general role of this protein class in mediating metabolism across the plastid and ER in plants.

New antimalarial compounds are urgently required to overcome artemisinin partial resistance that has emerged in Asia and now Africa. Ozonides are promising next-generation artemisinins that offer the improved pharmacokinetic property of a prolonged in vivo half-life. To assess the potential for parasite resistance to ozonides in an artemisinin-resistant background, we subjected Cambodian Kelch13 (K13) mutant parasites to increasing artefenomel (OZ439) pressure up to in vivo physiological concentrations. Whole-genome sequencing identified a novel non-propeller K13 A212T mutation in OZ439-resistant parasites. Gene editing and drug susceptibility assays revealed that the K13 double mutation R539T+A212T is a determinant of OZ439 resistance. In extended parasite recovery assays, this resistance mechanism was associated with accelerated parasite recrudescence following OZ439 or OZ277 exposure. This phenotype was also observed in K13 C580Y+A212T double mutant parasites. Global metabolomic profiling revealed no changes in the levels of hemoglobin-derived peptides in OZ439-resistant parasites, suggesting that resistance is not associated with drug activation. Instead, double mutant parasites exhibited increased levels of metabolites linked to glutathione, nucleotide, and aspartate-glutamate metabolism, suggesting a higher capacity for redox regulation to tolerate drug-induced oxidative damage. Our findings demonstrate that ozonide resistance can emerge through a novel K13 mutation on the background of existing artemisinin-resistance k13 alleles.

The ubiquitin specific protease 28 (USP28) is implicated in tumorigenesis by controlling the turnover of the oncogene c-MYC and the ubiquitin ligase FBW7. Here, we describe small molecule inhibitors of USP25 and USP28, leading to cancer cell cycle arrest and death. However, genetic deletion of USP25/28 does not replicate this effect. An integrated -omics approach revealed off-target effects for thienopyridine carboxamide compounds upon the translation apparatus. Chemoproteomics and CRISPR-GOF analyses suggested binding of the compound to a region near the ribosome complex polypeptide exit tunnel. Structural analysis of a USP28-inhibitor complex enabled the design of modified USP25/28 inhibitor molecules which minimized translation-related off-target effects. In distinction to earlier compounds, the optimized inhibitors were non-toxic to breast cancer cells yet retained potent anti-proliferative activity in squamous lung carcinoma cells, where USP28 is associated with disease progression. Together, our results demonstrate that refined USP25/28 inhibitors can selectively suppress tumor growth by targeting the TP63-FBW7-c-MYC signaling axis, offering a more precise therapeutic strategy for treating squamous lung cancers whilst minimizing undesired cytotoxicity.

Lanthipeptides represent the largest group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides offer promising avenues for discovering new antibacterial and antifungal agents. Here, we identify and structurally analyze the product of the tla BGC, which encodes a class II lanthipeptide in the thermophilic bacterium Thermoactinomyces sp. DSM 45891. Heterologous co-expression of the lanthipeptide synthetase TlaM resulted in modification of the two precursor peptides TlaA1 and TlaA2, which share 58% identity. TlaA1 was dehydrated seven times and TlaA2 six times. In both peptides, four thioether rings were formed with two overlapping DL-(methyl)lanthionine rings at the C-terminus. Both peptides also contain two central and N-terminal non-overlapping DL-methyllanthionines. These findings demonstrate that these peptides deviate from the general rule of stereoselective LL-(methyl)lanthionine formation from a DhxDhxXxxXxxCys motif (Dhx = dehydroalanine or dehydrobutyrine). AspN-cleaved TlaM-modified TlaA1 displayed anti-microbial activity against a subset of bacteria including Gram-negative ESKAPE pathogens. We named the lantibiotic thermolanthin.

Proteolysis-targeting chimeras (PROTACs) are emerging as potent tools for targeted protein degradation that overcome many of the limitations of traditional small molecule inhibitors. Yet how these hetero-bifunctional therapeutics enter cells remains a mystery. While passive diffusion is conventionally assumed, the bulky structure of PROTACs suggests that active transport may be required. Recently, the fatty acid transporter CD36 was identified as a key receptor for PROTACs. However, because the uptake mechanism of CD36 is itself unknown, how PROTACs enter cells remains a mystery. Here we show that PROTAC uptake and function require clathrin-mediated endocytosis. We uncover previously unrecognized clathrin adaptor-binding motifs in the CD36 C-terminus and use live-cell imaging to visualize the recruitment of both CD36 and PROTACs to sites of clathrin-mediated endocytosis on the cellular plasma membrane. Strikingly, disruption of clathrin assembly through either genetic or pharmacological means abolishes all detectable PROTAC-induced protein degradation, demonstrating that the clathrin pathway is required for the function of PROTACs that utilize diverse E3 enzymes against multiple targets. These results elucidate the molecular mechanism of PROTAC entry into cells, providing critical information for optimizing cellular uptake and response to targeted degraders.

The Niemann-Pick Type C1-Related protein of the malaria parasite Plasmodium falciparum, PfNCR1, is a promising anti-malarial drug target facilitating cholesterol homeostasis at the interface of the malaria parasite with its host-red blood cell. PfNCR1 is localized to otherwise functionally uncharacterized regions covering [~]half of the host-parasite interface. These regions are defined by exceptionally narrow membrane contact sites, leaving only [~]3-4 nm vertical aqueous space in between the membranes. Determining the origin and functional consequence of localization to the closely apposed membrane is central for our understanding of PfNCR1 as drug target but also offers a window into the mechanism of the group of homologous proteins, associated with congenital conditions and cancer. Here we define the mechanism of PfNCR1s membrane contact site localization and its implication for cholesterol transport. We identified a 141 amino acid long (amphipathic) helix - linker - (amphipathic) helix domain ("HLH domain") unique to Plasmodium spp., that is necessary for efficient localization of PfNCR1 to the narrow membrane contact sites. Mechanistically, we show that this localization relies on the HLH domains physicochemical properties. GPI-anchoring the isolated HLH domain or a version of the HLH domain in which the helices are replaced by the amphipathic helix of human ATG3 are sufficient to target a fluorescent protein to regions of endogenous PfNCR1. Functionally, we demonstrate that the degree of PfNCR1 localization to narrow contact sites qualitatively correlates with its ability to maintain cholesterol homeostasis, linking PfNCR1s membrane contact to its recognized transport function. Collectively, the results establish the HLH domain as key element for PfNCR1s localization and effectiveness in cholesterol transport while also opening avenues to probe the narrow membrane contact site regions with engineered proteins.

Targeted protein degradation (TPD) is a therapeutic strategy to remove disease-causing proteins by routing them to the ubiquitin-proteasome, autophagy, or lysosme machineries. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic hetero-bifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging and the number of currently addressable ubiquitin E3 ligases is limited. Here we demonstrate hetero-bifunctional de novo designed proteins as alternatives for TPD to access more targets and ligases. First, we develop a stable and highly adaptable helix-turn-helix scaffold for presenting different binding sites. Next, we use computational protein design to incorporate and embellish hot-spot- binding sites to target BCL-xL, plus short linear motifs (SLiMs) for KLHL20 ligase recruitment. The resulting mono- and bi-functionalised proteins bind the targets in vitro, and the latter degrade BCL-xL in cells leading to apoptosis.

Chromatin accessibility is essential for maintaining the fidelity of gene regulation and is dynamically regulated by epigenetic enzymes that are often dysregulated in cancer. The most commonly mutated regulator is the modular, multi-subunit Brahma-associated factor (BAF) chromatin remodeling complex. Previous attempts to exploit BAF complex mutations as potential tumor vulnerabilities using loss-of-function approaches have shown limited clinical success. Here, we instead propose a gain-of-function (GOF) strategy and establish Transcriptional/Remodeling chemical Inducers of Proximity (TRIPs), a class of neomorphic molecules that recruit active BAF complexes to rewire an oncogenic repressor, B-cell lymphoma 6 (BCL6). TRIPs potently induce transcriptional de-repression and apoptosis in Diffuse Large B-cell Lymphoma (DLBCL), enabled by ternary complex formation between BCL6 and BAF. CRISPR knockout screening identifies the PBAF complex as an essential contributor to cellular TRIP efficacy. Finally, we demonstrate that TRIP induces chromatin enrichment of BAF at BCL6-bound sites, resulting in ATPase-dependent eviction of BCL6, and de-repression of pro-apoptotic BCL6 target genes. We establish BAF recruitment for targeted chromatin remodeling as a viable GOF pharmacological strategy for tackling diseases driven by aberrant gene repression.

Prostate cancer progression is characterized by dysregulated lipid metabolism, with fatty acid synthase (FASN), the rate-limiting step in de novo lipogenesis (DNL), resulting in significant accumulation of saturated lipids. Here, we investigate whether pharmacologic FASN inhibition creates a metabolic state that increases reliance on exogenous polyunsaturated fatty acids (PUFAs). Inhibition of FASN profoundly alters membrane phospholipid composition, driving compensatory incorporation of PUFAs into membrane phospholipids, thus increasing susceptibility to lipid peroxidation and oxidative damage. Combined FASN inhibition and PUFA exposure increased reactive oxygen species, induced mitochondrial hyperpolarization, and enhanced lipid peroxidation in both hormone-sensitive and castration-resistant prostate cancer models. Marked inhibition of human and murine prostate cancer organoids is achieved ex vivo. In genetically engineered, DNL-reliant Hi-Myc mice, a diet enriched in PUFAs significantly inhibited invasive carcinoma compared to a saturated fat-enriched diet. Environmental PUFAs modulate and enhance the therapeutic efficacy of FASN-targeted strategies. These findings set the stage for pharmacologic and dietary intervention in prostate cancer patients.

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in key cellular processes, including transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among human G4 motifs. The G4 Ligand-conjugated Oligonucleotide strategy addresses this need by combining the G4-binding capabilities of small-molecule G4-ligands with the sequence specificity of an oligonucleotide complementary to the flanking region of the target G4. Here, we systematically explore how the oligonucleotide component governs G4 binding and stabilization by varying its length, backbone composition, and sequence complementarity. This revealed that efficient G4 recognition depends on a strong interdependence between hybridization and G4-ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Central mismatches disrupt this dual recognition and reduce selectivity. While longer oligonucleotides hybridize more slowly, they form more stable complexes and show stronger G4 stabilization in thermal melting and polymerase stop assays. Replacing the DNA oligonucleotide with peptide nucleic acid enhances binding strength, thermal stability, and metabolic stability, but selective G4 stabilization is achieved only upon ligand conjugation. Together, these results show how rational oligonucleotide design enables selective and potent recognition of G4 structures using GL-Os.