SLAS Discovery

Protein degradation mediated by PROteolysis TArgeting Chimeras (PROTACs) is a complex, dynamic, and multi-step process. The structural diversity of PROTACs, coupled with cellular heterogeneity, introduces considerable variability at each stage. Therefore, a straightforward yet comprehensive approach is essential to effectively dissect these mechanisms. The existing methods for evaluating PROTAC degradation are often time-consuming, labor-intensive, or expensive, and many capture only a partial view of the entire degradation process. Consequently, multiple complementary assays are typically required to obtain the complete picture. Here, we use commercially available Cyclin dependent Kinase 6 (CDK6) targeting PROTACs to demonstrate that a simple, single-step, no-wash, additive-free, GFP-based live-cell imaging approach can rapidly generate dynamic degradation metrics (Dmax, Rmax, TDmax, CDmax, DC50, Km, Tmax, Kdeg, Vmax, DT50, {tau} and TRmax) for comparing and ranking PROTACs within a controlled reporter system. The rapid assay turnaround time, excellent assay qualities and compatibility with high-throughput (HT) formats will facilitate quick hit identification and serve as a guiding assay for hit-to-lead optimization campaigns.

The dopamine D2 receptor is a principal target of antipsychotic and antiparkinsonian drugs, yet well-characterized fluorescent ligands for the D2 receptor remain scarce compared with its relative, the D3 receptor. Here we show that fluorescent ligands developed for the D3 subtype can be repurposed as tools for studying the D2 receptor pharmacology. Using a machine-learning-assisted, live-cell fluorescence microscopy assay, we screened twelve fluorescent probes and identified eight ligands that bound specifically to the D2 receptor at 10 nM. Four ligands exhibited classical saturable binding, yielding Kd in the nanomolar range, whereas the remaining probes displayed unsaturated, time-dependent binding profiles. Kinetic analyses revealed substantial differences in association and dissociation behavior, highlighting the importance of time-resolved measurements. Competition binding with dopamine and haloperidol yielded affinity estimates consistent with literature values. These results expand the toolkit of validated D2 receptor fluorescent probes and support cross-receptor repurposing as a strategy for fluorescent ligand development.

High-throughput enzymology enables quantitative characterization of enzyme function across hundreds to thousands of sequence variants and experimental conditions. Nevertheless, the scale and complexity of these datasets create substantial challenges for analysis and quality control. High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), for example, generates large microscopy datasets that must pass through multiple analytical stages, including image processing, initial-rate fitting, and kinetic modeling. Choices or errors made at any stage can propagate into the final kinetic parameters without being evident from fit statistics alone. Here, we provide a broadly applicable guide for analyzing high-throughput enzymology data from the HT-MEK platform, using Michaelis-Menten kinetics as a representative example. We first outline the conceptual workflow from raw fluorescence measurements to estimates of kcat, KM, and kcat/KM. We then discuss common experimental and analytical failure modes and present a framework for deciding when data should be refitted, filtered, qualified, or repeated. Finally, we provide a step-by-step workflow using Mercury, an open-source Python framework that integrates scalable HT-MEK data processing with traceable quality control and diagnostic visualization. This workflow preserves the connection between reported parameters and their underlying measurements and can be adapted to other kinetic models and biochemical assays.

Glycine transporter 1 (GlyT1) is a clinically investigated target whose conformational plasticity enables ligands to engage distinct extracellular and intracellular binding sites. However, methods to directly quantify intracellular binding remain limited and largely rely on radioligand-based approaches. Here, we report a NanoBRET-based binding assay to quantify intracellular GlyT1 target engagement in living cells using a fluorescent tracer derived from the GlyT1 inhibitor Cmpd1. With a structure-based approach followed by pharmacological characterization we identified a suitable meta-substituted exit vector for linker and fluorophore attachment while maintaining GlyT1 binding. A series of fluorescent probes was synthesized and evaluated, leading to the development of GlyT1-probe which displayed high-affinity binding and enabled robust NanoBRET measurements. Unlike the functional uptake assay, the NanoBRET assay distinguished intracellular from extracellular GlyT1 inhibitors and resolved structure-activity relationships at the intracellular binding site. This assay provides a quantitative approach to interrogate conformation-selective GlyT1 target engagement in living cells, facilitating the characterization of GlyT1 ligands targeting the intracellular site.

Surface plasmon resonance (SPR) and biolayer interferometry (BLI) are well-established label-free analytical technologies used to determine biomolecular interaction rates (ka, kd) and equilibrium constants (KD). While assays historically rely on multi-cycle kinetics (MCK), this approach requires intermediate surface regeneration, risking sensor degradation and increasing reagent consumption. Single-cycle kinetics (SCK) avoids regeneration by sequentially injecting increasing analyte concentrations. However, implementing SCK workflows on biosensor platforms like the OctetRR8 BLI instrument (Sartorius) remains a challenge due to the lack of built-in or online data analysis tools. To address this, we introduce SCKAnalyzer, a free, user-friendly web application designed for processing and fitting SCK datasets. SCKAnalyzer accepts column-based text files and native raw BLI data (.frd format) from Sartorius instruments, performing data pre-processing and non-linear fitting to a 1:1 interaction model. Additionally, it offers advanced simulation capabilities that allow variable association and dissociation durations across sequential injection steps, a feature absent in existing platforms such as BiacoreTMSimul8. The performance of SCKAnalyzer was experimentally validated using a 3-step SCK method (SCK3) for DNA/RNA duplex formation on a Biacore T200, as well as 4-step (SCK4) and 5-step (SCK5) methods for DNA/DNA duplex formation on an OctetRR8 BLI instrument. Binding parameters (ka, kd, KD) derived from SCKAnalyzer showed excellent agreement with values from established proprietary softwares such as Biacore T200 Evaluation Software v3.2.1 and BIAevaluation v4.1.1. SCKAnalyzer provides a practical alternative to MCK, reducing probe consumption and extending SCK utility across biosensor platforms. SCKAnalyzer is available at sck.iecb.u-bordeaux.fr (source code: github.com/synthaze/sckanalyzer).

Numerous CRISPR-Cas systems have been developed for molecular detection of genetic elements exploiting the trans-cleavage activity of LbCas12a combined with short fluorescent probes. Alongside, a large variability of buffer conditions has been reported. However, how solution chemistry balances enzymatic turnover, signal stability, and detection performance have not been systematically defined. Here, we evaluated how the interplay between anions (chloride vs. acetate), additives, sequence probes, and ionic strength contributes to LbCas12a trans-cleavage activity. Buffer composition screening revealed that chloride-based buffers maintain a low background but slow catalytic rates, meanwhile acetate-based buffers accelerate enzymatic turnover with and without the target DNA sequence, increasing non-specific signals. Reducing agents and surfactants were the best enhancer combinations, improving cleavage performance by increasing ternary complex formation (Km). High salt concentrations only slightly improved the reaction kinetics. Finally, the 5FAM-TTATT-3BHQ-1 reporter showed the best balance between signal intensity and nonspecific background. Our findings indicate that LbCas12a tolerates a vast buffer composition landscape, maintaining robust trans-cleavage activity and detection performance. However, conditions that enhance catalytic activity simultaneously increase nonspecific probe cleavage and higher background, resulting in lower detection performance. Therefore, assay optimization should prioritize detection performance rather than maximal fluorescence output, where solution chemistry is a critical determinant.

The base excision repair pathway removes small base lesions from DNA and is initiated in humans by one of eleven DNA glycosylases with overlapping substrate specificities. Despite their central role in genome maintenance and transcription, systematic datasets describing small-molecule binders or inhibitors of DNA glycosylases are lacking. Here, through a collaborative effort across Scandinavia, we establish a high-throughput biochemical assay platform for human DNA glycosylases and use it to generate a systematic small-molecule screening dataset. A chemogenomic library was screened against nine glycosylases with assays of sufficient quality for hit identification, yielding primary screening and concentration-response data and identifying multiple previously unreported inhibitors. All primary and processed data, along with detailed assay protocols and metadata, are publicly available to support reuse in chemical biology, further compound optimization, assay development and comparative studies of DNA repair enzymes.

CRISPR-Cas12a-based diagnostics are promising for field detection yet are often constrained by two step workflows or laborious optimization of one-pot reactions. Herein, we present a universal one-pot RPA/CRISPR-Cas12a strategy using base-mismatched crRNA in the seed region (positions 1-6). Introducing of single-nucleotide mismatches (complementary or error) into crRNA suppresses cis-cleavage activity, enabling amplification and detection in a single tube without PAM re-design or costly modified crRNA. Molecular dynamics (MD) simulations indicated that moderate potential energy (PE) variation correlates with improved screening performance, providing guidance for the selection of effective mismatches. This strategy successfully converted established two-step assays for Burkholderia pseudomallei and Brucella into one-pot formats, achieving detection of targets at 10 copies/L within 30 min with good specificity. Lyophilized pellets supported field testing on lithium-ion battery-powered devices, and experiments with simulated soil samples confirm the robustness of the approach. Together, this cost-effective, adaptable strategy can streamline RPA-CRISPR diagnostics for infectious disease surveillance.

Neuropeptides regulate a wide range of physiological processes throughout the nervous and endocrine systems, yet their spatiotemporal dynamics in vivo remain poorly understood. While recently developed intensiometric sensors can detect neuropeptides, their use in vivo is often confounded by artifacts induced by hemodynamic changes, pH fluctuations, and motion. Here, we present a general strategy for engineering dual-excitation ratiometric (Ex-ratiometric) neuropeptide sensors by tuning the excited-state proton transfer properties of the fluorescent reporter. Dual excitation at 405 nm and 488 nm produces a ratiometric signal that intrinsically corrects fluorescence fluctuations unrelated to ligand binding. Using an AlphaFold3-guided in silico design approach combined with experimental validation, we developed a suite of Ex-ratiometric sensors. As a representative example, Ex-NTS2.0 enables robust detection of neurotensin (NTS) in vivo while remaining largely resistant to hemodynamics, pH, and motion-induced artifacts. Overall, these findings establish a scalable and mechanistically grounded platform for developing new Ex-ratiometric tools and provide a broadly applicable strategy for sensitive detection of neuropeptide dynamics in living systems.

The rapid expansion of mRNA-based medicines has driven demand for robust, high-resolution analytical methods capable of characterising critical quality attributes including sequence identity, 5[prime] capping efficiency, and poly(A) tail length and heterogeneity. Here, we present two complementary liquid chromatography-mass spectrometry based workflows for mRNA characterisation, both based on site-specific digestion with the endoribonuclease MazF from Escherichia coli. A middle-up approach, optimised via specific 5[prime]-ACA cleavage enables rapid characterisation via oligoribonucleotide mass mapping providing complete sequence coverage and simultaneous assessment of 5[prime]-capping efficiency and 3[prime]-poly(A) tail length and heterogeneity. This workflow was successfully applied to three different mRNA constructs, NLuc, eGFP and FLuc mRNAs, achieving sequence coverages of 100%, 87%, and 74%, respectively. A complementary bottom-up approach, employing less specific 5[prime]-ACX cleavage enables tandem mass spectrometry-based sequencing of shorter oligoribonucleotides and detailed characterisation via mRNA sequence mapping. Applied to SARS-CoV-2 Spike Protein mRNA, this method yielded 50% sequence coverage based on unique oligoribonucleotides with MazF alone, extended to 90% by combining MazF, partial RNase T1, and partial RNase U2 digests. Together, these workflows provide a flexible, orthogonal platform for both high-throughput quality assessment and in-depth primary sequence characterisation of mRNA vaccines and therapeutics.

Lymphatic endothelial cell (LEC) function is strongly tied to transitions between button- and zipper-like junctions that control fluid flow into vessels. Published methods quantifying junction morphology are limited to single cell analysis or use few quantitative metrics to describe junctions across LEC monolayers. Therefore, a novel 3-step image analysis pipeline was developed to identify new metrics that quantify differences in button- and zipper-like junctions in LECs. The pipeline used Cellpose, CellProfiler, and Fiji to analyze VE-cadherin expression across lymphatic vessels within a 3D microfluidic chip and quantify VE-cadherin radial intensity, number of objects, object size, branchpoints, end-points, and junction thickness. Tumor necrosis factor (TNF)- exposure produced but-ton-like junctions, and unexposed cells were zippered. Radial intensity was heightened at the cell membrane in zipper-like junctions while perinuclear intensity increased for but-ton-like junctions. Increased branchpoints, endpoints, objects per cell, and junction thick-ness were associated with junction buttoning, while increased object size was associated with junction zippering. Principle component analysis results reinforced these trends and identified metric combinations that represented each junctional state. Overall, this semi-automated pipeline quantified differences in VE-cadherin junction morphology across LEC monolayers and generated new quantitative metrics to expand the tools available for studying junction morphology in lymphatic vasculature.

Constrained macrocyclic peptides can engage protein surfaces that resist both small molecules and biologics but producing them at library scale still depends on chemical synthesis and macrocyclisation workflows that are slow, specialised and hard to parallelise. We describe a recombinant pipeline for disulphide-stapled cyclic peptides, compatible with 96 well plates, that lowers this barrier. Peptide-encoding sequences are introduced by extension PCR and in vivo assembly (IVA) cloning at about $2 per variant, expressed as small ubiquitin-like modifier (SUMO) fusions secreted to the oxidising periplasm, where the intramolecular disulphide forms spontaneously, and purified by immobilised metal-affinity chromatography (IMAC) with analytical size-exclusion chromatography (SEC). The route from a list of designs to characterised material takes about one week, much of it unattended. Applied to a 60-design library across nine protein targets, the pipeline gave purified products for all 60 designs, at a median total soluble yield of approximately 135 micrograms per 6 mL culture (range 9 to 261) that varied by target. Ellman's assay across 38 designs showed disulphide-formation failure to be sequence-driven and uncorrelated with yield, detecting a failure mode invisible to chromatography. Surface plasmon resonance confirmed target binding and showed that the recombinant SUMO fusion constructs retained affinities comparable to their corresponding peptide-only forms. Complementary to general-purpose recombinant platforms, the pipeline enables higher-throughput disulphide macrocycle screening and is amenable to partially or fully automated workflows.

A broader understanding of RNA biology requires methods for visualizing transcript dynamics in native environments. Common imaging approaches rely on fluorescent probes that require excitation light and often depend on large aptamer arrays, limiting sensitivity and compatibility with many non-coding RNAs, particularly highly structured folds. To address these challenges, we developed a miniaturized bioluminescent platform for RNA imaging. This system employs a compact RNA tag that assembles luciferase fragments upon binding of monomeric RNA-binding proteins (RNA lanterns). Systematic optimization of the RNA component yielded a structured 24-nucleotide tag, representing greater than 3-fold reduction in size relative to the original C-11-B RNA tag, placing it among the smallest RNA imaging tags reported to date. The optimized tag enabled RNA-dependent bioluminescence detection in live cells, notably as a small, single insert within highly structured non-coding RNAs, including the MALAT1 ENE domain and a small HDV-like ribozyme. Collectively, this work establishes a miniaturized bioluminescent platform for imaging a broad range of RNA targets in living systems.

Fluorescent base analogues have proven to be valuable probes of RNA structure. Pyrrolocytosine (pC), an analogue of cytosine, exhibits highly environment-sensitive fluorescence within RNA and is strongly quenched in hydrogen-bonded contexts. In this work, we incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to determine if this quenching mechanism could allow for sensing of RNA-small molecule interactions. We utilized complementary fluorescence spectroscopy methods to under-stand the functional impacts and ligand-sensitivity of pC substitutions distal to and within the binding pocket. We found that pC substitution within a static structural region exhibits the expected ligand-independent fluorescence properties. When placed in the ligand binding pocket, pC exhibits a moderately decreased affinity for the ligands guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. We found that pC behaves as a turn-off sensor, with distinct changes in fluorescence upon G or Hx binding. These differences are dependent on the number of hydrogen bonds formed with pC and are resolvable in both time-dependent and steady-state fluorescence measurements. Our results show that pC can be used to study RNA-small molecule interactions and can discriminate between similar ligands even when they have identical binding modes.

Non-small-cell lung cancer (NSCLC) diagnosis is challenged owing to the need for reliable, non-invasive biomarkers. Circulating cell-free microRNAs (ccf-miRNAs) are promising candidates, but their low levels and short sequences in blood complicate detection. In this study, a novel nano-hybrid fluorescent array was developed to enable rapid and sensitive identification of NSCLC-related ccf-miRNAs. The array consists of poly(T)-modified fluorescent nano-polystyrene beads for capturing target miRNAs, and fluorophore-labeled, sequence-specific locked nucleic acid (LNA) probes for their detection. This design forms a sandwich structure with the target miRNA. Nano cytometry and fluorescence microscopy assessed both the capture and specificity of detection. The assay was tested on plasma samples for miR-16-5p and U6, evaluating selectivity, sensitivity, and reproducibility. The results showed that the poly(T)-modified beads efficiently captured the target miRNAs, and the LNA probes accurately distinguished the sequences. The assay enabled direct detection of miR-16-5p and U6 from plasma without amplification, demonstrating high selectivity, sensitivity, and reproducibility. Combining enrichment with nano-polystyrene beads and sequence-specific LNA probes addressed the main challenges of ccf-miRNA detection, namely their low abundance and short sequence length. As ccf-miRNAs are linked to NSCLC progression, this method could assist in early diagnosis, disease monitoring, and assessment of treatment response. The target-independent nature of the poly(T) capture layer allows for easy calibration to new miRNAs by changing the LNA probes, rendering it suitable for multiplex detection in clinical settings. However, further preclinical and clinical studies are needed before adoption in routine practice. This nano-hybrid fluorescent array presents a rapid and reliable approach for detecting low-abundance ccf-miRNAs in plasma. Its encouraging performance and flexible design show potential for future application in NSCLC diagnosis and monitoring, subject to additional confirmation.

Context: Cytological specimens allow minimally invasive, repeated sampling for diagnosis and molecular testing. However, cytology biobanking protocols have primarily been optimized for morphology, and the effects of preanalytical handling and storage on intracellular proteins remain unclear. Objective: To assess how fixation, staining, storage temperature, and duration affect intracellular protein preservation in cytological specimens and to identify conditions suitable for in situ proximity ligation assay (isPLA). Design: Human cancer cell lines were processed under different preanalytical conditions. Intracellular proteins and protein protein interactions were assessed by isPLA, fluorescence microscopy, and quantitative image analysis. Slides were stored at room temperature or minus 20 Celsius, with or without MayGrunwaldGiemsa (MGG) staining. Results: Storage temperature had the greatest effect on protein signal. Unstained slides stored at minus 20 Celsius retained strong signals for up to 24 months, whereas room temperature storage reduced detectable protein interactions by approximately 95%. MGG staining further reduced signal intensity during prolonged storage. Nevertheless, stained slides remained suitable for isPLA when staining, microscopic evaluation, destaining, and antibody labeling were completed on the same day. Prolonged room temperature storage of stained slides caused loss of antigenicity, increased autofluorescence, and false negative findings. Conclusions: Low temperature storage and standardized preanalytical handling preserve intracellular protein signals in cytological specimens. These conditions support reliable single cell protein profiling of archived samples and their use in biomarker research and translational pathology.

MAPK-activated protein kinase 2 (MAPKAPK2, MK2) is a downstream substrate of p38 MAPK that regulates inflammatory signaling and cytokine synthesis, making it an attractive therapeutic target. Although several crystal structures of MAPKAPK2 with ATP-competitive inhibitors have supported drug discovery, structural elucidation of selective non-ATP-competitive inhibitors has remained challenging. MK-25 is a non-ATP-competitive furan-2-carboxamide MAPKAPK2 inhibitor developed at Merck to overcome the selectivity and cellular-efficacy limitations of ATP-competitive chemotypes. Here, we show through isothermal titration calorimetry that MK-25 binds the activated MAPKAPK2, consistent with state-dependent allosteric binding. To determine the binding mode of MK-25, we solved a 2.8 A resolution crystal structure of an activated MAPKAPK2 construct in ternary complex with the ATP-site inhibitor PF-3644022 and a bromo-analogue of MK-25. The ligand occupies a pocket in the C-lobe overlapping the protein-substrate recognition region and formed through rearrangements of D and G helices and the activation segment. These interactions are consistent with the structure-activity relationships reported for the furan-2-carboxamide chemotype and identify a ligandable allosteric pocket that may facilitate future structure-guided design of selective, non-ATP-competitive MAPKAPK2 inhibitors.

Malaria kills {approx}600,000 people annually despite recently deployed vaccines. The parasite's blood-stage invasion of erythrocytes, driven by the essential, non-redundant interaction between Plasmodium falciparum RH5 and erythrocyte basigin, remains a leading therapeutic target. Here, we designed de novo mini-proteins (minibinders) to occlude this interface. From {approx}1,500 computationally generated designs, we synthesized 18 candidates ranging from 9.3 to 16.9 kDa which were easily expressed in E. coli as soluble, well-folded proteins. Of these, 7 minibinders blocked blood-stage replication of P. falciparum asexual stage parasites with greater potency than {approx}300nM IC50. Three potent candidate minibinders, mb-5, mb-7, and mb-21, were confirmed to bind RH5 via SPR and BLI and block the merozoite-to-ring transition in stage-synchronized parasite progression assays, with CryoEM of minibinder-RH5 complex confirming the predicted binding at RH5-basigin interface. Although pharmacological challenges remain, our data demonstrate that potent inhibitors of parasite growth, rivaling existing antimalarials, can be designed rather than discovered via extensive screening. Our data highlight the potential for artificial intelligence to change the development landscape of new antimalarial therapies.

Reactivation of telomerase confers immortality to approximately 90% of human tumors by enabling continuous elongation of the DNA at chromosome ends, or telomeres. The telomerase catalytic subunit TERT adds TTAGGG repeats using a portion of the telomerase RNA component hTR as a template. Because telomerase is inactive in most normal somatic cells, it remains an attractive therapeutic target; however, no telomerase inhibitor has yet demonstrated robust clinical efficacy with acceptable safety. Here we evaluate thiomorpholino oligonucleotides (TMOs) as a new class of antisense oligonucleotides targeting the template region of hTR. TMOs incorporate morpholino rings and phosphorothioate linkages, which enhance nuclease resistance, RNA binding and nuclear uptake. Two anti-hTR TMOs inhibited telomerase activity in vitro with an IC50 below 1 nM, whereas two control TMOs were at least 100-fold less active. HeLa cells treated with anti-hTR TMOs showed progressive telomere shortening, detectable after one week of treatment. Growth inhibition was observed after substantial telomere erosion, and both telomere length and proliferation recovered upon withdrawal of TMOs. These findings establish TMOs as a promising new chemistry for telomerase-targeted therapeutics.

Although use of AI-based tools has gained ground in molecular biology research, their reliability remains a matter of debate. Here, we combined AlphaFold 3 modelling with microscale thermophoresis to investigate the interactions between glucan-binding protein C and the biofilm-inhibiting aptamer and its five derivatives. Structural modelling identified a binding motif on the aptamer and suggested a mechanism of action; however, the predictions were only partially supported by the experimental results. The AlphaFold generated confidence metrics did not correlate directly with the measured binding affinities, indicating that confidence scores can help identify plausible interaction interfaces, but they are not reliable predictors of binding strength. These results highlight the importance of integrating structural prediction with biophysical validation to dissect aptamer-protein recognition.