Journal of Medicinal Chemistry

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [≥]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [≥]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@abe1c9org.highwire.dtl.DTLVardef@746a08org.highwire.dtl.DTLVardef@dd5861org.highwire.dtl.DTLVardef@1d572c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alzheimers disease (AD) is a multifactorial disease with mixed pathologies. Consequentially, drugs targeting multiple pathological processes may offer synergistic benefits. While histone deacetylase (HDAC) inhibitors have demonstrated efficacy in alleviating AD-related pathologies in animal models, the neuroprotective Wnt/{beta}-catenin signaling pathway remains compromised in AD brain. CI-994 is a class I HDAC inhibitor containing N-(2-aminophenyl)-benzamide. Our recent studies indicate that CI-994 is also an activator of Wnt/{beta}-catenin signaling by stabilizing Wnt co-receptor LRP6. We herein use CI-994 as a scaffold to develop novel potent dual modulators of class I HDACs and Wnt/{beta}-catenin signaling for AD therapy. Our lead compound, W2A-28, selectively inhibits class I HDAC1, 2 and 3 with IC50 values of 0.51 M, 0.68 M, and 0.22 M, respectively, and shows no inhibitory activities on other HDACs. Furthermore, W2A-28 potently activates Wnt reporter activity with an EC50 value of 1.61 M in Wnt-3A-expressing HEK293 cells. As expected, activation of Wnt/{beta}-catenin signaling by W2A-28 is associated with elevated LRP6 protein level. Importantly, W2A-28 displays excellent microsomal stability in both mouse and human liver microsomal stability assays, alongside high permeability and a lack of active efflux in MDR1-MDCKII models. Critically, W2A-28 treatment significantly enhances histone acetylation, activates Wnt/{beta}-catenin signaling, and suppresses tau phosphorylation in AD patient-specific cerebral organoids carrying APOE {varepsilon}4/{varepsilon}4 or APOE {varepsilon}3/{varepsilon}4 with PSEN1 M146V mutation. Our findings position W2A-28 as a promising multi-target drug candidate for AD therapy.

CAPON (NOS1AP) is an adaptor protein involved in neuronal nitric oxide synthase (nNOS) signaling and has been implicated in Alzheimers disease (AD), excitotoxicity, and tau-associated neurodegeneration. Here, we report the identification of cyclic peptide ligands targeting CAPON using phage display screening of a disulfide-constrained peptide library. Phage enrichment, ELISA validation, microscale thermophoresis (MST), and biolayer interferometry (BLI) identified CAP1 as the lead peptide, exhibiting low micromolar binding affinity toward CAPON. Computational studies further supported stable CAPON-CAP1 interactions through complementary hydrophobic and electrostatic contacts. Functionally, CAP1 attenuated A{beta}42-induced neuronal toxicity, suppressed NMDA-driven nitric oxide production, and reduced pathological tau phosphorylation in neuronal models under AD-relevant stress conditions. In addition, CAP1 demonstrated favorable preliminary pharmacokinetic properties, including good aqueous solubility, plasma stability, and measurable membrane permeability. Collectively, these findings establish the first cyclic peptide ligands targeting CAPON and identify CAP1 as a promising scaffold for modulation of CAPON-dependent neurodegenerative signaling.

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly-conserved ATP binding pocket but diverge in their substrate binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed, however it remains challenging to achieve on-target selectivity across different kinases and target specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a non-conserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@287a47org.highwire.dtl.DTLVardef@17407eborg.highwire.dtl.DTLVardef@b40435org.highwire.dtl.DTLVardef@804352_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study presents the structural verification of baicalin isolated from a hydroethanolic extract of an in vitro Scutellaria baicalensis root culture using X-ray diffraction analysis and a set of NMR spectroscopy techniques. The crystalline molecular structure of the sample was found to correspond to baicalin. The 1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC spectra confirmed that the chemical shifts, signal multiplicities, integral intensities, and spin-spin coupling constants were fully consistent with the structure of the target compound. Minor impurity signals were detected in the aliphatic region of the spectra, with a total content not exceeding 5 mol%. These results confirm the high purity and structural individuality of baicalin, a biologically active flavonoid glycoside of considerable interest.

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

Prostate cancer (PCa) is one of the principal contributors to health burden in the aging male population. PCa develops through dysregulation of androgen receptor (AR) signaling pathways. Despite improvements in diagnostic techniques and interventions, no pharmacological measures with long term efficacy have been established once PCa advances to castration resistant prostate cancer (CRPC). To circumvent this issue, tetra-aryl cyclobutanes (CBs) have been proposed as structurally distinct compounds with a mechanism of action differing from traditional androgen receptor signaling inhibitor (ARSIs). Here, we apply principles of crystal engineering and solid state synthesis to expand the class of CBs through strategic derivatization. The synthesis of the CB occurs quantitatively, producing no side products and eliminating the need for product purification. We demonstrate how head-to-tail stacking interactions of halo-pyrimidine rings can be exploited to stack and align unsymmetrical alkenes to undergo [2+2] photodimerization to generate the CB in the solid state. We examine the structure-function relationships of CBs in vitro by profiling AR mediated transcriptional activity, receptor translocation, and cell viability. Moreover, we explore and identify putative binding interactions within CB/AR complexes and establish an adaptive ligand-binding potential using molecular docking platforms. In total, our data suggests that CBs have unexploited therapeutic potential in CRPC and that green chemistry and crystal engineering principles offer a unique route to generating these drug candidates.

Homeodomain-interacting protein kinase 4 (HIPK4) is a dual-specificity kinase that is predominantly expressed in differentiating spermatids, required for sperm development, and a promising target for nonhormonal male contraception. Genetic and functional studies have established an essential role for HIPK4 in spermiogenesis, where it acts at least in part through regulation of the F-actin-scaffolded acroplaxome during spermatid head shaping. The direct molecular targets of HIPK4 and their downstream effectors remain poorly defined, and small-molecule probes would be versatile tools for further investigating HIPK4 functions. Synthetic HIPK4 ligands could also be valuable leads for the development of nonhormonal male contraceptives. Here, we report the discovery of a cyanoquinoline-based series of HIPK4 inhibitors with nanomolar potency. Our lead compounds are selective for HIPK4, both within the HIPK family and across the broader kinome, establishing this scaffold as a useful starting point for probe and lead development. Unexpectedly, we found that a subset of these cyanoquinolines also perturbs HIPK4 proteostasis in a cell type-specific manner. In spermatids, these compounds induce the formation of detergent-insoluble HIPK4 aggregates and promote interactions between this kinase and the autophagy receptor Tax1-binding protein 1 (TAX1BP1). Together, our findings establish cyanoquinoline ligands as a new chemotype for probing HIPK4 biology and advancing male contraceptive discovery.

The rise of new, rapidly mutating viruses presents increasing challenges for drug developers. Traditional methods, such as high-throughput screening and drug repurposing against mutagenic viral targets, have recently shown their limitations. Our current rational molecular engineering approach offers a sustainable solution by targeting viral ion channels, which generally have low mutation rates. First, extending the amantadine molecule led to the development of new compounds that better match the alternating hydrophobic and hydrophilic patterns of the inner walls of ion channels--a common feature across many viruses. Then, simplifying the structure yielded a cyclohexylamine-based minimalist scaffold that effectively blocks the ion channel and demonstrates improved antiviral activity compared to well-known agents such as amantadine and arterolane. SARS-CoV-2 variants served as test systems in laboratory experiments. The new molecular scaffolds presented here provide a strong foundation for designing potent, broad-spectrum viral ion channel blockers.

Immune inhibitory signaling in microglia contributes to impaired amyloid clearance and neuroinflammation in Alzheimers disease (AD), yet small molecule modulators targeting these pathways remain largely unexplored. Here, we report the development of a high-throughput cellular thermal shift assay (HT-CETSA) platform for identification of small molecule binders targeting the inhibitory immune receptor ILT3 (LILRB4). Screening of [~]40,000 compounds yielded multiple validated hits, including IB15C, a submicromolar ILT3 binder identified through preliminary structure-activity relationship optimization. Orthogonal validation by microscale thermophoresis, surface plasmon resonance, docking, and site-directed mutagenesis confirmed direct and target-specific ILT3 engagement. Functionally, IB15C disrupted the ILT3-ApoE interaction and restored microglial activity in human iPSC-derived microglia, reducing SHP1/2, suppressing cytokine secretion, and enhancing amyloid uptake. IB15C also demonstrated favorable in vitro pharmacokinetic and safety properties, supporting further development of ILT3-targeted neuroimmune therapeutics.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Class B1 G-protein-coupled receptors (GPCRs), such as the calcitonin gene-related peptide (CGRP) receptor and parathyroid hormone 1 (PTH1) receptor, require native lipid interactions to maintain signalling-competent conformations. However, conventional detergents disrupt these environments. Amphipathic copolymers offer a detergent-free alternative, yet the field still lacks a clear understanding of which polymer architectures best preserve active-state GPCR pharmacology, limiting their broader translational utility. Here, we examine how distinct copolymer chemistries influence the functional integrity of class B1 GPCRs by comparing SMA 2000, DIBMA-12, and the electroneutral sulfo-DIBMA. Using NanoLuciferase bioluminescence resonance energy transfer (NanoBRET) ligand-binding, competition, and mini-G-protein recruitment assays on nanodisc-encapsulated receptors, we show that all three copolymers maintain high-affinity extracellular ligand binding but differ markedly in their ability to preserve intracellular signalling. Despite lower receptor extraction efficiency, only sulfo-DIBMA support mini-Gs engagement at the CGRP receptor and enable G-protein-dependent allosteric modulation at the PTH1 receptor, including conserved ligand affinity and prolonged residence time. These data reveal that polymer charge and backbone chemistry, rather than extraction yield, determine whether native-like nanodiscs retain the conformational landscape required for active-state signalling. Controlling non-specific ligand binding to the copolymer is a key requirement for a successful assay. Our findings identify sulfo-DIBMALP as a particularly superior environment for preserving native signalling behaviour in class B1 GPCRs, highlighting copolymer chemistry as an important determinant in detergent-free membrane protein studies. HIGHLIGHTSO_LISulfo-DIBMA encapsulated nanodiscs preserve active-state conformation of human calcitonin gene-related peptide receptor and parathyroid hormone 1 receptor. C_LIO_LIAll three copolymers (SMA 2000, DIBMA-12 and sulfo-DIBMA) preserve extracellular ligand binding but only sulfo-DIBMA preserves intracellular functional competence, including mini-Gs recruitment and G-protein-dependent allosteric modulation. C_LIO_LICopolymer chemistry, particularly the electroneutral, aliphatic nature of sulfo-DIBMA, may influence the preservation of signalling-competent states in two class B1 GPCRs by minimising charge-driven perturbations during solubilisation. C_LIO_LISulfo-DIBMALP provides a novel platform for studying dynamic membrane proteins with potential to provide mechanistic insights and facilitate drug discovery programmes in the future. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/724797v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12db163org.highwire.dtl.DTLVardef@d8efb3org.highwire.dtl.DTLVardef@610dbaorg.highwire.dtl.DTLVardef@1cc3ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Leukocyte immunoglobulin-like receptor B4 (LILRB4, ILT3) is an inhibitory immune checkpoint expressed on myeloid cells, where it contributes to immunosuppression within the tumor microenvironment. Secretogranin 2 (SCG2) has recently been identified as a functional ligand of LILRB4, yet small molecule modulators of this interaction remain unexplored. Here, we report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to interrogate the LILRB4 (ILT3)-SCG2 interaction. The assay demonstrated robust performance and was validated using a blocking anti-LILRB4 antibody, consistent with orthogonal ELISA measurements. Pilot screening of chemical libraries identified 23 primary hits, of which two compounds, BMS-813160 and PSB-603, showed reproducible, dose-dependent inhibition with TR-FRET IC50 values of 26.7 {+/-} 1.03 {micro}M and 37.2 {+/-} 2.14 {micro}M, respectively. Activity was confirmed by ELISA, supporting the robustness of the assay. This platform enables high-throughput discovery of first-in-class small molecule modulators of the LILRB4-SCG2 immune checkpoint and provides a foundation for targeting myeloid-driven immunosuppression.

Selective inhibition of phosphodiesterase 4B (PDE4B) remains a promising strategy for preserving the anti-inflammatory benefit of PDE4 inhibition in chronic obstructive pulmonary disease while reducing PDE4D-associated tolerability liabilities. This study integrated SHAP-interpretable machine learning, natural product virtual screening, hierarchical docking, post-docking MM-GBSA, isoform cross-docking, binding-pocket comparison, ADMET prediction, and 100 ns molecular dynamics simulations to identify PDE4B-selective inhibitors from the LOTUS natural product database. A Random Forest classifier trained on curated ChEMBL PDE4B bioactivity data achieved an external performance with AUC-ROC = 0.955, accuracy = 0.893, F1-score = 0.896, MCC = 0.785, and prioritized 119,698 predicted actives from 276,518 LOTUS compounds. SHAP analysis identified BertzCT and TPSA as major contributors to predicted activity. Sequential Lipinski, PAINS, and QED filtering retained 14,210 candidates for structure-based evaluation. Extra precision docking identified four leads with PDE4B docking scores of -9.123 to -12.080 kcal/mol, all outperforming roflumilast (-7.658 kcal/mol). Cross-docking and post-docking MM-GBSA supported preferential PDE4B binding for three candidates. The top lead, LTS0048837, maintained a stable PDE4B-bound pose during simulation, with comparatively stronger interaction persistence than its PDE4D complex and the roflumilast reference. These findings nominate LTS0048837 as a computationally prioritized PDE4B-selective natural product lead requiring experimental enzyme, cellular, and pharmacokinetic validation.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG