ACS Medicinal Chemistry Letters

Metastatic melanoma is an aggressive cutaneous malignancy frequently driven by the oncogenic V600E mutation within the BRAF kinase. While first-generation Type IS BRAF inhibitors, such as dabrafenib, are currently prescribed to target this specific molecular vulnerability, paradoxical MAPK pathway activation, and acquired drug resistance necessitate the continuous development of structurally optimized lead molecules. In this study, chemical intuition, bioisosteric replacement, and computational molecular docking were employed to propose two novel BRAFV600E drug candidates. The proposed therapeutics, engineered to incorporate constrained sp3-hybridized aliphatic rings and a sulfoximine bioisostere, demonstrated thermodynamically superior docking scores within the mutant catalytic cleft compared to dabrafenib. Lastly, a homology analysis determined that Mus musculus is a suitable model organism for future preclinical studies and confirmed crucial structural selectivity against microbial off-target kinases.

Structure-based drug discovery is known to apply computational methods in a tiered hierarchy, with each layer narrowing the candidate set and refining the binding picture before committing to the next, more expensive step. We present a four-tiered computational benchmarking study evaluating five engines against a panel of 36 compounds targeting B-secretase 1 (BACE1), a validated Alzheimer's disease target with extensive co-crystal ground truth. This study evaluates Flexible Docking and Boltz2 Cofolding as the primary tier, followed by Ensemble Docking, and then Protein-Ligand MD with MM/PBSA and MM/GBSA post-processing. This is then concluded with Relative Binding Free Energy Perturbation (RevFEP) as the terminal refinement layer. Each method was benchmarked against the experimental binding free energies derived from the co-crystal structures spanning -7.85 to -11.35 kcal/mol. Our findings revealed that Flexible Docking reproduced the co-crystal binding mode for 35 of 36 ligands (97.2% within 2.0 A RMSD) but did not rank potency at this resolution. Boltz2 CoFolding provided an orthogonal structural cross-check with a receptor backbone RMSD of 0.293 A against the experimental co-crystal structure. Ensemble Docking identified the optimal receptor conformation for downstream FEP setup. MD with MM/GBSA decomposition identified van der Waals complementarity as the primary potency driver (Pearson r = +0.855, R2 = 0.732 on a 10-compound subset). RevFEP delivered the highest affinity correlation of any method (Pearson r = +0.662, R2 = 0.438, Spearman p = +0.624, mean absolute error 1.02 kcal/mol across all 36 ligands), resolving potency differences within a narrow 3.5 kcal/mol congeneric window that no other engine could discriminate. We characterize what each engine contributes independently and where RevFEP delivers signals no other engine achieves.

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.

We present BoltzMol-1, a small-molecule hit discovery pipeline, centered on an optimized version of Boltz-2, explicitly adapted for prospective discovery. Reliable hit discovery that generalizes across target classes (rather than only the well-characterized families that dominate existing ligand data) would broaden the range of biology accessible to small-molecule intervention and reduce reliance on resource-intensive high-throughput screening. Towards this goal, the system prioritizes compounds for rapid experimental validation by coupling model-driven ranking with streamlined procurement from commercial catalogs. To improve developability at the point of selection, we introduce a suite of ADMET models for kinetic solubility (logS), lipophilicity (logD), and Caco-2 permeability. These models act as an early triage layer, systematically filtering out compounds with unfavorable physicochemical and absorption properties prior to synthesis or purchase. Across a panel of ten targets (most with no representation in the underlying affinity training data) we observe strong prospective performance on challenging systems. Functional actives or binders were identified for 6 of 10 targets, despite modest experimental budgets of 28-96 compounds per target. These results include successes on receptors and enzymes traditionally considered difficult for structure- or ligand-based approaches. Collectively, this work establishes a practical framework for low-throughput, cost constrained discovery campaigns capable of delivering chemically tractable binders with favorable property profiles.

IntroductionIn this study, we developed a modular in vitro platform that integrates advanced polymer-drug conjugation chemistry with stepwise cytotoxicity screening in both 2D (monolayer) and 3D (spheroids) glioblastoma (GBM) models. Buparlisib was selected as the model therapeutic due to its well-characterised mechanism of action, high blood-brain barrier permeability, and relevance to PI3K-targeted therapy. MethodsTwo mechanistically distinct conjugation strategies were explored using N-(2-hydroxypropyl)methacrylamide-based copolymers. The first strategy was based on a redox-sensitive disulphide linkage designed for intracellular glutathione-triggered release, whereas the second used an azide-bearing derivative compatible with strain-promoted azide-alkyne cycloaddition. Drug release was assessed by high-performance liquid chromatography. Biological activity was systematically evaluated in U87MG, U118MG, and T98G cells under 2D conditions using a resazurin-based metabolic activity assay. Subsequently, the more promising disulphide-based formulations were assessed in 3D spheroids by metabolic activity measurements and live-cell monitoring of spheroid growth dynamics. ResultsFree Buparlisib showed the strongest inhibitory effect, while its modification and polymer conjugation reduced the apparent activity. Nevertheless, the disulphide-based derivative and polymer conjugate retained concentration-dependent activity, whereas the azide-based polymer conjugate showed minimal effects. Moreover, treatment responses differed between cell lines and between 2D and 3D models. DiscussionOverall, linker chemistry, cell-line-specific behaviour, and model dimensionality strongly influenced the biological performance of the polymeric Buparlisib formulations. The redox-sensitive polymer conjugate therefore represents the more promising strategy for further development.

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@982d1forg.highwire.dtl.DTLVardef@ba86f2org.highwire.dtl.DTLVardef@1f19f2borg.highwire.dtl.DTLVardef@8e807_HPS_FORMAT_FIGEXP M_FIG C_FIG

The rapid emergence of SARS-CoV-2 variants that evade neutralizing antibodies underscores the need for next-generation antiviral biologics that combine molecular precision with scalable, cost-effective manufacturing. Computationally designed miniproteins targeting the receptor-binding domain (RBD) of the spike protein offer a compelling alternative to monoclonal antibodies due to their small size, high thermal stability, and compatibility with microbial expression systems. Here we report the end-to-end development and cGMP production of IPD-52520, a de novo antiviral miniprotein, using an optimized E. coli platform. Two miniprotein candidates, a homotrimeric construct (Trimer is referred to as IPD-52520, 17 kDa) and a tandem fusion (Daisy is referred to as IPD-52521, 25 kDa), were evaluated in parallel through systematic optimization of strain selection, media composition, fed-batch fermentation, inclusion-body solubilization, refolding, and chromatographic purification. The Trimer was downselected as the lead molecule based on superior preclinical efficacy, favorable pharmacokinetic properties, and higher volumetric manufacturing yields. The optimized process delivers approximately 2 g/L of purified protein at greater than 90% purity. Scale-up from 5 L to 50 L under cGMP conditions demonstrated excellent batch-to-batch reproducibility across six independent batches, supporting nonclinical and Phase 1 clinical supply. Comprehensive biophysical characterization confirmed a well-folded, predominantly alpha-helical trimer (Tm = 73.4 {degrees}C; polydispersity = 1.005) with an intact primary structure and strong target-binding affinity (KD < 1 pM). Real-time stability studies indicate that the drug substance is stable at 2-8 {degrees}C for at least 12 months, with ongoing stability studies. These results demonstrate the feasibility of translating computationally designed antiviral miniproteins into manufacturable biologics and provide a platform applicable to rapid-response therapeutics against current and future pandemic threats.

Pyrrolizidine alkaloids (PA) are well-known and widespread natural products from plants, which have also been identified in several different bacteria. In the latter case, the core structure is constructed by a non-ribosomal peptide synthetase (NRPS), which then undergoes oxidative ring contraction catalyzed by a Baeyer-Villiger monooxygenase. By deploying various NRPS engineering strategies, we have successfully generated five novel peptides carrying the unusual PA moiety at their C-terminus. Nonetheless, efforts to obtain a larger library of PAs were unsuccessful. Combined computational modelling and docking experiments suggest that this failure stems from the strict specificity of the thioesterase (TE) domain at the end of the NRPS, which discriminates against peptides carrying more than two amino acids. Our work thus suggests protein design strategies by which this intrinsic limitation to NRPS engineering may be overcome in future.

Discovering small molecules targeting proteins represents a major effort in drug development. RNA, however, as a class of macromolecule that carrying out important regulatory roles in the cell as drug target, only received attention recently. Although several methods have been proposed, an easy to operate, fast and robust method is still lacking. We designed a generic florescence screening method by fusing the target RNA with a florescent aptamer (fusion RNA) and then carried out screening using high-throughput format (Fluorescent Aptamer Screening, FAS). In this work, we chose SL5 on SARS-Cov-2 5’UTR as the test target. SL5 is a conserved motif across several corona virus family members whose core is not prone to mutation. We screened 9528 compounds, successfully identified four molecules (Sertraline (hydrochloride), Samuraciclib (hydrochloride), Minocycline (hydrochloride), JG-98 bind direct to the full-length SL5 at micromolar or higher affinity. The design of FAS could be easily adapted to structured RNA motifs without prior knowledge of its 3D structural information. In addition, this work showed the possibility of developing generic drugs for RNA virus by targeting the conserved viral RNA genome and paved a new way for the discovery of small molecule drugs in combating human diseases.

EIF2alpha kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2alpha-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-delta-KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-delta-KI and of its complex with ATP at 2.1 & 2.5 Angstrom resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-delta-KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

The inhibition of A{beta}42 ({beta}-amyloid) fibril formation is a key therapeutic strategy in Alzheimer's disease research. Paddlewheel diruthenium complexes have shown promising activity against A{beta}42 aggregation and preformed fibril disaggregation, yet their molecular mode of action remains poorly understood. In this work, we perform atomistic simulations to explore how charge modulation influences the interactions of three analogous paddlewheel diruthenium complexes, the parent neutral complex [Ru2Cl(D-p-FPhF)(O2CCH3)3], and its anionic [Ru2Cl2(D-p-FPhF)(O2CCH3)3]- and cationic [Ru2(D-p-FPhF)(O2CCH3)3]+ counterparts (D-p-FPhF- is the N,N' -bis(4-fluorophenyl)formamidinato ligand) with A{beta}42. Our results indicate that electrostatic tuning governs binding affinity and the extent of interaction across the A{beta}42 fibril surface. As the complexes' charge changes from -1 to +1, the interaction pattern shifts from localized contacts to widespread, multi-site engagement encompassing key charged, aromatic, and hydrophobic regions of A{beta}42. This enhanced binding correlates with longer-lived, thermodynamically stable interactions at the fibril interface, which effectively lower the free energy penalty for fibril disassembly. Overall, our findings propose a mechanism in which charge-dependent activation through ligand exchange enhances fibril recognition and promotes disruptive binding modes, demonstrating the potential of charge-tunable diruthenium complexes as therapeutic modulators of A{beta}42 fibril stability.

Background: Medulloblastoma (MB) is the most common malignant brain tumour in children, and aggressive subgroups are frequently driven by the oncoproteins MYC or MYCN. Direct therapeutic targeting of MYC/MYCN has been challenging because of their intrinsically disordered protein structures. The aim of this study was to determine whether novel SE486-11 analogues (UNSW-SCs) can therapeutically target MYC/MYCN-driven MB. Methods: The anticancer activity of UNSW-SCs was assessed in MB cell lines with differential MYC/MYCN expression. Target engagement was evaluated using surface plasmon resonance and drug affinity responsive target stability assays. Blood-brain barrier penetration, MYC/MYCN protein degradation, cell cycle effects, apoptosis, DNA damage, and synergy with histone deacetylase (HDAC) inhibitors were examined. Therapeutic efficacy was evaluated in murine models of MYC- and MYCN-driven human MB. Results: UNSW-SCs showed potent anticancer activity, with preferential selectivity toward MB cells expressing high MYC/MYCN levels and IC50 values ranging from 0.22 to 1.18 M. The lead molecule, UNSW-SC-22, directly bound MYC, crossed the blood-brain barrier, and achieved a brain-to-plasma ratio of 1.44 at peak concentrations. UNSW-SC-22 induced MYC/MYCN-dependent cytotoxicity associated with enhanced proteasomal degradation, cell cycle arrest, apoptosis, and DNA damage. Combined treatment with HDAC inhibitors further reduced MYC/MYCN protein levels, increased DNA damage, and enhanced apoptosis. In vivo, UNSW-SC-22, either alone or with entinostat, significantly suppressed intracranial tumour growth and prolonged survival. Conclusions: UNSW-SC-22 is a brain-penetrant MYC/MYCN-targeting molecule with potent preclinical activity in MYC/MYCN-driven MB, supporting its development as a monotherapy or combination strategy with HDAC inhibition.

The cysteine to serine mutation at residue 481 of Bruton's tyrosine kinase (BTK) is the most common mechanism of clinical resistance against ibrutinib for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia. We report small molecule ligands containing chiral {beta}-lactone electrophiles to address this challenge. The asymmetric warhead enabled stereoselective covalent modification of wild-type and ibrutinib-resistant mutant BTK(C481S) through distinct sites of reactivity. Building on these findings, we developed kinase-directed {beta}-lactone probes and demonstrated that individual enantiomers preferentially engage distinct subsets of the kinome. These studies establish {beta}-lactones as stereochemically encodable covalent warheads whose stereochemistry can serve as a selectivity filter in covalent drug discovery.

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

Inducible nitric oxide synthase (iNOS) is frequently overexpressed in inflammatory disorders and solid tumors, where sustained nitric oxide (NO) production promotes angiogenesis, tumor progression, and resistance to therapy. Despite promising preclinical results, the clinical translation of iNOS inhibitors remains limited by poor tumor selectivity, rapid systemic clearance, and off-target toxicities. To address these challenges, we developed a protease-responsive polymeric iNOS-inhibiting prodrug (ProCIP) designed for localized activation within protease-rich pathological microenvironments. ProCIP was synthesized from poly(ethylene glycol)-poly(L-glutamate) and functionalized with amidine-based iNOS inhibitory moieties. The resulting cationic polymer readily formed nanoscale polyionic complexes with anionic polymers or molecules. In cell-free assays, enzymatic activation of ProCIP resulted in a significant reduction in iNOS activity, whereas non-activated nanoparticles showed minimal inhibition. Cellular studies confirmed efficient nanoparticle uptake by RAW264.7 macrophages and revealed a significant reduction in intracellular NO levels in lipopolysaccharide-stimulated cells. These findings demonstrate that ProCIP enables protease-triggered iNOS inhibition and localized NO regulation, offering a promising strategy for improving the safety and efficacy of iNOS-targeted therapies in cancer and other inflammatory diseases.

Ubiquitin signalling is mediated by structurally distinct polyubiquitin chains that encode discrete cellular functions. Progress in deciphering this ubiquitin code, particularly for the less abundant atypical chain types, has been hindered by limited availability of versatile chain type-specific affinity reagents. Here, we demonstrate that human single-domain antibodies (sdAbs) provide a versatile scaffold for the generation of ubiquitin linkage-specific binders. Using phage display and synthetic human sdAb libraries, we identified 2A6, an sdAb that specifically recognises methionine-1 (M1)-linked ubiquitin chains. To our knowledge, 2A6 represents the first reported sdAb with specificity for a defined homotypic ubiquitin chain linkage. 2A6 bound M1-linked ubiquitin chains with nanomolar affinity and was specific for M1-linked chains at the level of both diubiquitin and long polyubiquitin chains. AlphaFold3 modelling, supported by saturation mutagenesis, predicted that 2A6 recognises the proximal and distal ubiquitin moieties together with the region near the M1 linkage. Functionally, 2A6 enabled specific detection and enrichment of M1-linked ubiquitin across multiple applications, including ELISA, immunoblotting, immunoprecipitation under semi-denaturing conditions, substrate ubiquitination analysis, and immunofluorescence microscopy. The sdAb can be readily produced in E. coli from a single expression plasmid, providing a tractable, cost-effective and versatile reagent for investigating M1-linked ubiquitin signalling. Our work establishes sdAbs as a versatile scaffold for ubiquitin linkage-specific affinity reagents, providing a framework for the development of analogous binders specifically targeting additional ubiquitin linkages or architectures.

Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3Dpol) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3Dpol, including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3Dpol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3Dpol, including hit-to-lead compound 727590, which displayed an IC50 value of 25 M and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737532v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14a54a6org.highwire.dtl.DTLVardef@fb6621org.highwire.dtl.DTLVardef@ee2e2aorg.highwire.dtl.DTLVardef@118f91d_HPS_FORMAT_FIGEXP M_FIG Created with biorender.com and PyMOL Molecular Graphics System, version 2.5.0. Schrodinger, LLC. C_FIG