SLAS Discovery

Cytoskeletal plasticity is a defining feature of cancer progression, enabling tumor cells to adapt their morphology, mechanics, and migratory behavior during invasion and metastasis. Although actin filaments, microtubules, and intermediate filaments are known to cooperate in these processes, the molecular mechanisms coordinating their dynamics remain incompletely understood, particularly the role of post-translational modifications (PTMs). Here, we developed CytoFRET2, a multiparametric cytometry-based FRET platform that enables real-time and simultaneous monitoring of the dynamics of actin filaments, microtubules, and vimentin in living suspension cells. The system combines fluorescently tagged cytoskeletal reporters with spectral flow cytometry, allowing simultaneous high-content analysis of multiple cytoskeletal networks while overcoming autofluorescence and fluorescence interference from small molecules. Using spectral CytoFRET2, we screened a small library of epigenetic compounds targeting regulators of acetylation and methylation pathways. The screen revealed that inhibition of lysine deacetylases (KDACs) and sirtuins promoted stabilization of both microtubules and vimentin filaments, without impacting actin filament organization. In contrast, inhibition of lysine acetyltransferases (KATs), particularly with garcinol and anacardic acid, induced rapid vimentin disassembly. Mechanistically, the study reveals acetylation as a key post-translational modification regulating the dynamics of microtubules and vimentin filaments, with KAT inhibitors emerging as potent modulators of vimentin organization. Together, the findings establish spectral CytoFRET2 as a versatile platform for systematic investigation of cytoskeletal regulatory networks and therapeutic vulnerabilities in cancer.

High-content imaging produces thousands of morphological measurements per cell. Interpreting these measurements requires normalization to remove plate effects, statistical tests selected on the basis of data distribution, and control over false discoveries across many features tested at once. MorphoStat is an open-source Python pipeline that applies this sequence of steps automatically. Given a CSV file from CellProfiler or a compatible imaging platform, it removes low-quality wells, normalizes each plate against DMSO controls using a MAD-scaled z-score, routes each feature to a parametric or nonparametric test based on a distributional check, applies Benjamini-Hochberg correction, and writes out results and publication-ready figures. On the BBBC021 benchmark (MCF-7 breast-cancer cells, 632 wells, 473 features), MorphoStat recovered 12 of 13 known mechanism-of-action classes in principal component space, confirming that the normalization and statistical routing work as intended. The tool is available at https://github.com/Almunthir334/morphostat (DOI: 10.5281/zenodo.20354069) under the MIT license.

High-throughput screening (HTS) remains the cornerstone of early-phase small molecule discovery yet consistently underperforms against immunotherapy targets, yielding validated hit rates below 0.1%. Here we introduce HTS-Oracle v2, which features rigorous cross-validation that ensures honest performance estimates. HTS-Oracle v2 was trained and validated across four clinically significant immune checkpoint targets (CD28, ICOS, LAG-3, and TIGIT) achieving ROC-AUC values of 0.968, 0.969, 0.875, 0.928 respectively under rigorous cross-validation. For prospective experimental validation, HTS-Oracle v2 was applied to an 8,960-compound Enamine Protein Mimetic Library, selecting only 25 compounds per target for experimental testing using temperature-related intensity change (TRIC) technology, a 99.7% reduction in screening burden. HTS-Oracle v2 identified 4, 5, 4, and 6 validated binders from 25 prospectively selected compounds per target, corresponding to validated hit rates of 16%, 20%, 16%, and 24%, respectively. Notably, 67-80% of all experimentally confirmed hits across the full 8,960-compound library were captured within just 25 model-selected compounds per target. For CD28, this represents a 28-fold improvement over HTS-Oracle v1 (239x versus 8.4x), establishing HTS-Oracle v2 as an efficient platform for AI-guided prospective hit discovery across immunotherapy targets.

Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.

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

The wide variety of physical and chemical properties in materials makes the study of unknown substances challenging. We have previously proposed a theoretical framework for agnostic material characterization based on using nucleic acid imprints of the materials and then analyzing material-specific patterns of derived sequences. Here we demonstrate an experimental and computational pipeline that can agnostically identify and distinguish varied materials based on DNA k-mer imprints and validate the ability of these imprints to distinguish closely related materials. This work lays the foundation for expansion of purely agnostic sensing technologies for the unbiased characterization and categorization of a much wider variety of biotic and abiotic materials.

The cytosolic histone deacetylase 6 (HDAC6) plays a key role not only in cancer but also in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) peripheral neuropathies. Pharmacological inhibition as well as genetic silencing of HDAC6 is able to rescue several defects. Therefore, developing pharmacological compounds targeting this enzyme is of crucial interest. Here, we report the design, synthesis, and characterization of Methyl Fluorescent Hydroxamic Acid (MeFluHyA), a novel fluorescent HDAC6-selective probe designed for cell imaging. By integrating a Cy5 fluorophore into a phenyl hydroxamic acid scaffold, MeFluHyA shows binding to the catalytically active CD2 domain of HDAC6 without significantly inhibiting its deacetylating function in the sub-micromolar range. Biochemical enzymatic activity assays confirmed its selectivity over other HDAC isoforms. Fluorescent imaging studies in HeLa cells demonstrated strong colocalization with HDAC6-eGFP and a commercial HDAC6 antibody. Competitive binding assays revealed that MeFluHyA effectively identifies known HDAC6 inhibitors, with reduced probe-binding serving as a readout for successful target engagement. MeFluHyA is a cell-permeable, fast and easy to use probe that can be added organism-independent, and its red-shifted fluorophore makes it compatible with multiplex, live and fixed imaging. Overall, we provide a novel tool for screening platforms aimed at identifying novel HDAC6-targeting inhibitors.

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.

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.

Digital bioassays enable precise single-molecule quantification but are difficult to adapt to point-of-care testing (POCT) because conventional protocols include off-chip complex processes for sample treatment and sealing, requiring hardware and workflow complexity. We present OASSIS (Open Aqueous two-phase Separation System for Integrated Single- molecule digital bioassay platform), an oil-free and open ATPS platform that localizes both targets and signals in femtoliter-scale dextran (DEX) droplets beneath a polyethylene glycol (PEG) phase. We integrated a CRISPR-Cas13a assay system with a novel, branched fluorescent reporter conjugated to a dextran-binding domain (DBD), which ensures signal retention within the DEX droplets after cleavage. Fluorescence recovery after photobleaching experiments confirmed this robust signal confinement. OASSIS not only performs amplification-free digital RNA detection but also enables serial sample introductions through its open-format architecture that progressively improve sensitivity: the limit of detection (LOD) improved from 1.08 fM (first introduction) to 0.34 fM (third introduction). Furthermore, OASSIS demonstrated specific detection and [~]10-fold enrichment from a complex, denaturant-treated nasopharyngeal swab matrix. Together, these results demonstrate that the open-format architecture of OASSIS provides a practical route toward sensitive, low-complexity POCT and clinical diagnostic applications.

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

Macrocyclic peptides have gained increased attention amid claims they are a "Goldilocks" therapeutic modality that can encode the selectivity of a biologic in a footprint close to that of a small molecule. Here we attempt to find a peptide that binds selectively to NRAS, sparing HRAS and KRAS, while accessing the cytosol via passive cell permeability. To do so, we combine subtractive affinity selection with mRNA display to identify Compound 1, an 11mer macrocyclic peptide which binds NRAS at a novel allosteric site between Helix 3 and Helix 4 of the GTPase domain. Compound 1 has total isoform selectivity and can be tuned to achieve activation-state selectivity with a single amino acid change. While it has preferential affinity for oncogenic NRAS-specific mutations, it does not inhibit NRAS function or achieve passive membrane permeability.

Luminescence is a powerful method for detecting trace analytes and monitoring biological processes. However, most bioluminescence reagents, including luciferase and its substrates, are sensitive to temperature, limiting their useable shelf lives, and resulting in inconsistent performance. Enhancing the stability of these reagents could improve data quality, simplify workflows, and address cold chain storage issues. In this study, we demonstrate the application of the platform stabilization technology, capillary-assisted vitrification (CAV), as a tool to stabilize different luciferases and their substrates, and the application of the stabilized reagents in both in vitro and in vivo bioluminescent assays. We demonstrate that CAV-stabilized reagents can be stored and shipped ambiently, maintain consistent performance over time, and are suitable for use in cell viability quantification, tumor monitoring, in vivo imaging, microbial detection, and immunoassays. Additionally, different reagents can be co-formulated to make ready-to-use assay kits that can also be shipped and stored ambiently. Our results demonstrate that CAV stabilization is a viable alternative to traditional storage methods, with broad potential to improve bioluminescence workflows.

Antimicrobial resistant infections present a growing threat to public health and were associated with or directly caused 6 million deaths globally in 2021. This huge death toll highlights the need for novel strategies to address AMR infections. Interfering with the regulation of resistance mechanisms could provide an alternative approach to treat drug-resistant infections. PhoQ, a sensor histidine kinase ubiquitous amongst gram-negative bacteria, regulates several virulence factors, as well as resistance to outer membrane-targeting antibiotics, making it an attractive target for adjuvant therapy development. However, the identification of potent small molecule inhibitors is limited by the assays available for in vitro assessment of binding and activity inhibition in PhoQ. Thus, we sought to investigate the use of a fluorescence-based assay to evaluate enzymatic activity, as well as a thermal shift assay to assess inhibitor-protein binding in PhoQ. Together, these newly implemented protocols are valuable contributors to the toolbox of methods available for the development of PhoQ-targeted inhibitors to block this major contributor to antimicrobial resistance.

To explore adapting qPCR systems for end-point nucleic acid quantification using dyes such as SYTO-9, we quantified serial dilutions of DNA and RNA standards in the range of 0.75 - 200 ng/{micro}l on 384-well qPCR devices. SYTO-9 fluorescence was successfully measured using standard SYBR Green settings. Blank-subtracted relative SYTO-9 signal showed a logarithmic dependence on DNA/RNA concentration (R2 > 0.95). Measurements were highly stable with different incubation times, temperatures of up to 95{degrees}C, and photobleaching. The described approach is a valuable QC option for high-throughput DNA/RNA isolations and could be adapted to additional fluorometric assays beyond nucleic acids.

High-risk neuroblastoma patients face poor survival despite intensive treatment. Drug testing using patient-derived models can support therapy prioritization for precision medicine and drug development. Models incorporating tumour microenvironmental components, such as co-cultures and short-term cultured patient samples containing substantial non-malignant cell fractions, could better recapitulate microenvironment-dependent drug responses. However, conventional viability assays measure the combined signal from all viable cells in a well and therefore cannot determine tumour-specific drug responses. Here, we establish a microscopy-based readout to quantify cell-type-specific viability in two complementary settings: neuroblastoma-PBMC co-cultures and freshly dissociated patient tumour samples. In the co-cultures, PBMCs were pre-labelled with a cell-tracking dye, and Calcein staining was used to independently quantify the viability of tumour cells and PBMCs in the same well. The Calcein-based viability readout correlated strongly with conventional CellTiter-Glo measurements and was compatible with automated high-throughput drug screening. The imaging workflow enabled identification of compounds with differential efficacy in co-culture versus monoculture and distinguished tumour-specific effects from PBMC toxicity. The microscopy-based viability readout was further adapted to short-term cultured patient samples. Neuroblastoma tumour cells were distinguished from the non-malignant cells using a combination of tumour-specific surface markers NCAM, L1CAM and B7H3. This enabled determination of tumour fractions and measurement of tumour-specific drug responses. Tumour fractions varied substantially between patient samples, highlighting the importance of tumour-specific viability measurements. Together, the microscopy-based viability readout for co-cultures and patient samples enables scalable assessment of tumour-specific drug responses.

Mimiviral polymerase X, mvPolX, is a repair polymerase that is involved in base excision repair (BER) and carries out the gap-filling function in double-stranded DNA (dsDNA). We demonstrate a sensitive and sequence-specific DNA detection method using this polymerase. mvPolX begins polymerizing DNA from the 3 end of a gap, displacing the downstream nucleotides without exonuclease activity. Our detection method is built on this activity of mvPolX. We designed a probe molecule consisting of a partial dsDNA with a 3 over-hang region complementary to the target DNA to be detected. The probe has a fluorophore-quencher (FAM-BHQ1) tag to facilitate detection upon strand removal. Binding of the probe to the complementary target forms a dsDNA with a single nucleotide gap in one strand. mvPolX binds this gap region and begins polymerisation eventually displacing the quencher strand leading to an increase in fluorescence. Proof-of-concept has been established using a synthetic 19 bp target DNA sequence. The method is specific and did not show any strand displacement when a single or double mismatched nucleotide at the 3 end of the target DNA was used. To demonstrate this molecular assay, we used M13 phage as our target. Asymmetric PCR (aPCR) was used to obtain single-stranded target DNA (158 bases) from M13 genomic DNA, which was directly used in the assay as target. The combination of aPCR and mvPolX assay can detect as low as 10 copies of genomic DNA. The enzymatic reaction is fast, requiring only 15 min of incubation with mvPolX at 30 {degrees}C. We have further demonstrated the efficiency of the assay in presence of multiple non-target DNA by detecting the target DNA from M13 phage spiked lakewater samples.

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, yet the factors governing reaction longevity remain poorly understood despite their importance for high-throughput applications. Here, the three principal determinants of CFPS performance--DNA template design, reaction composition, and lysate genotype--were systematically optimized to extend reaction lifetime in a 384-well plate format. Different energy regeneration systems were evaluated through real-time pH monitoring and metabolomic analyses to identify the metabolic constraints limiting prolonged protein synthesis. Lysates prepared from engineered Escherichia coli BL21(DE3) strains were further examined to assess the contributions of DNA, RNA, and amino acid stabilization. Systematic optimization of amino acid, nucleoside triphosphate, polyethylene glycol, and lysate concentrations identified DNA template stability and amino acid preservation as the primary factors sustaining CFPS activity. Combining these improvements yielded reactions that remained productive for >14 h and produced 567 {+/-} 64 g mL-1 active deGFP. These findings establish practical strategies for extending CFPS lifetime and improving high-throughput cell-free platforms.

Antimicrobial resistance and tolerance pose escalating global health threats, necessitating reproducible and accessible tools for antimicrobial susceptibility testing (AST). While disk diffusion assays (DDAs) and Epsilometer tests (Etests) are widely used, there are limited open-source tools to analyze them. We present J-AST, a free, open-source, web-based platform for analyzing both DDAs and Etests. It provides automated and interactive annotation of regions of interest and metadata management, and quantifies microbial resistance and tolerance. J-AST outputs correlate strongly with those of existing tools, and equivalent DDA and Etest results correlate strongly with each other. The automated MIC detection achieved >90% agreement with manual readouts. J-AST is deployable both as desktop software and cloud service, unifies automated analysis with interactive review, and advances both fundamental research and clinical AST workflows.

Accurate measurement of protein aggregation is essential for studying neurodegenerative diseases. The standard ThT assay reports on amyloid formation but is blind to early oligomers and is prone to interference. We describe Q-DOAS, a plate-reader assay that quantifies protein self-assembly in real time via proximity-quenching of a single, site-specifically conjugated dye (BODIPY-TMR). Using mutant Huntingtin-exon 1 (mHTT-Ex1) and -Synuclein A53T, we show Q-DOAS detects pre-amyloid oligomers, yielding quantitative kinetic data compatible with mechanistic analysis. We demonstrate its utility to dissect mutational effects, screen for protein and small-molecule inhibitors, and quantify amyloid seeding activity in cellular and mouse models of Huntingtons disease. Q-DOAS also detects seeds in cerebrospinal fluid from Parkinsons disease patients without amplification. Q-DOAS provides a sensitive, robust, and scalable tool for studying the earliest events in amyloid pathologies and for advancing therapeutic development.