SLAS Discovery

DNA-encoded library screening represents a significant advancement in the field of drug discovery. Its ability to rapidly and cost-effectively identify potential drug candidates from large compound libraries has the potential to revolutionize the way new medicines are discovered and developed. While the strategies for DEL screening and data analysis have improved over the years, data normalization remains an open challenge. Existing normalization methods can yield poor correlation for compounds with high read count, and they do not account for inherent sources of noise. To overcome these drawbacks, we have developed a robust normalization technique using an antibody fragment and a DNA-conjugated peptide as an internal control. This innovative approach allows for normalization between samples of different conditions and accounts for technical challenges that occur during screening. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/700605v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b04b91org.highwire.dtl.DTLVardef@1312295org.highwire.dtl.DTLVardef@d59713org.highwire.dtl.DTLVardef@b1786a_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisNormalization of DNA-encoded library selection data reduces bias and noise, enabling accurate identification of true binders and reliable enrichment analysis.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

The cellular thermal shift assay (CETSA) is an invaluable tool for target identification and validation in early drug discovery efforts. It relies on thermal melting curves to indicate drug binding and is typically performed in whole cells, cell lysates, or purified protein as validation of direct interaction. However, these approaches can result in disruption of the structural integrity of membrane proteins, hindering downstream analysis and drug-target engagement. Here, we describe the first application of CETSA in isolated mitochondria and show the effects of this approach on the analysis of the compound UK5099 and its known binding target, the mitochondrial pyruvate carrier (MPC), a mitochondrial inner membrane-localized protein complex. Our analysis supports a model in which the MPC must remain structurally intact for UK5099 binding. We demonstrate that the binding of UK5099 to the MPC is disrupted in whole cells and cell lysates, whereas isolating mitochondria maintains the binding interaction between drug and target observable using CETSA. These data suggest that isolating membrane-bound organelles through subcellular CETSA stabilizes membrane-bound proteins in their native conformation, allowing the identification of membrane-localized drug binding targets that might otherwise be missed.

The interaction between neuronal nitric oxide synthase (NOS1) and its adaptor protein CAPON (NOS1AP) plays a critical role in various neurological processes and has been implicated in cardiovascular and neuropsychiatric disorders. Disruption of this protein-protein interaction represents a potential therapeutic strategy, yet identifying small molecule inhibitors has been challenging. Here, we present the development and validation of a NanoBiT-based luminescence complementation assay optimized for high-throughput screening (HTS) of NOS1-NOS1AP interaction inhibitors. We engineered NOS1 and NOS1AP fusion proteins with HiBiT and LgBiT complementary subunits, respectively, and established stable CHO-K1 cell lines for robust signal generation. The assay demonstrated excellent performance characteristics with a signal-to-background ratio exceeding 240-fold, and was validated using TAT-GESV, a known peptide inhibitor that showed time- and dose-dependent inhibition. We successfully screened a diverse library of 10,240 compounds and identified 19 validated hits with IC50 values ranging from 2.54 to greater than 30 M, with the majority exhibiting IC50 values below 30 M. The top three compounds exhibited potent inhibitory activity with IC50 values of less than 5 M. This NanoBiT-based assay provides a reliable and efficient platform for discovering novel NOS1-NOS1AP interaction inhibitors and can be adapted for other protein-protein interaction studies.

Traditional nucleic acid extraction methods are costly, lengthy, and highly variable depending on the complexity of the sample matrix or the organism of interest. Workflows may exceed twenty steps, require separate kits for RNA and DNA, and demand expensive instrumentation, creating barriers to both speed and scalability. The AutolabTM HBH system addresses these limitations by using hyperbaric heating (HBH) to achieve temperatures above 100 {degrees}C in a sealed, pressurized environment through induction heating, enabling rapid lysis of diverse organisms and neutralization of macromolecular PCR inhibitors within minutes. The combination of extreme heat and HBH-optimized lyophilized reagents rapidly inactivates nucleases while preserving free nucleic acids. The workflow is streamlined to two steps: heating up to 1 mL of sample in the proprietary HBH bullet, followed by a brief centrifugation to pellet additives. The resulting supernatant is immediately compatible with real-time reverse transcription polymerase chain reaction (RT-PCR) and other downstream molecular assays. Here, we evaluate the systems broad compatibility with diverse sample buffers, matrices, and organisms. Comparative testing was conducted alongside Qiagen extraction methods to benchmark performance.

Batch effects are recognized as major sources of technical confounding in high-throughput assays. However, their impact on organoid studies receives little attention in the literature. As organoids gain prominence as a class of emerging new approach methodologies (NAMs), consideration of batch variation will become increasingly important to ensure data reproducibility and accurate interpretation in pre-clinical and clinical studies. In this manuscript, we provide a practical description of our work in detecting, characterizing, and correcting batch effects in a prior published retrospective clinical colorectal cancer organoid drug-response study. We outline the workflow we employed, including exploratory diagnostics, experimental drift detection, and statistical adjustment. We detail the methods employed to evaluate batch effects, monitor longitudinal drift, and select approaches to remove technical artifacts, preserve biological signal and test for robustness. Our experience demonstrates that in even modestly sized studies, results can be adversely affected by insufficient consideration and attempts at ameliorating batch effects. By documenting the challenges we encountered and the solutions implemented within our study, we hope that we can provide a seminal practical reference for organoid researchers and enable increased discussion and adoption of robust batch-compensation practices in the organoid field, ensuring that the topic is more routinely addressed, improved, and eventually standardized.

The rapid engineering of high-affinity binding proteins, such as nanobodies and single-domain antibodies (sdAbs), is increasingly driven by cell-free, machine-learning-guided optimization. However, high-throughput, quantitative characterization of binding affinity remains a major bottleneck, particularly for proteins expressed in cell-free systems without purification. Here, we present High-Throughput FRET Affinity Screening Technique (HTFAST) for rapid affinity characterization of binders expressed directly in crude E. coli cell-free protein synthesis reactions. HTFAST leverages Forster resonance energy transfer (FRET) between fluorescent-protein-fused binders and dye-labeled antigens to enable real-time, quantitative measurement of equilibrium dissociation constants. We systematically optimized fluorophore pairs used and labeling parameters using the SpyTag003-SpyCatcher003 model system. Using donor-quenching and acceptor-emission FRET analyses, HTFAST reliably quantified nanomolar binding affinities in crude lysates for SpyTag003-SpyCatcher003 model system. We validated the platform for nanobodies by characterizing a CD4-binding nanobody, Nb457, and benchmarking multiple SARS-CoV-2 receptor-binding domain sdAbs, demonstrating HTFASTs ability to rank binding strengths across a range of affinities. Finally, we demonstrate that both binding partners can be expressed directly in CFPS, further streamlining screening workflows. Overall, HTFAST provides a scalable, quantitative, and cell-free-compatible approach for high-throughput affinity screening, well suited for DBTL campaigns aimed at accelerating the development of next-generation binding proteins.

Parkinsons disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) are progressive neurodegenerative disorders marked by the pathological aggregation of alpha-synuclein ([a]Syn). Despite significant research efforts, effective therapeutic interventions remain elusive due to limited understanding of the cellular effects of [a]Syn aggregation and propagation. This study presents the development of a scalable cellular seeding assay for screening small molecules targeting cellular [a]Syn seeded aggregation. By leveraging a fluorescent reporter of [a]Syn and phenotypic screening, the assay enables high-throughput evaluation of potential inhibitors in a cellular environment mimicking disease pathology. We evaluated three different Syn aggregation inhibitors tested in clinical trials for PD: Minzasolmin, Emrusolmin and EGCG and profiled gene expression using multiplexed single cell RNA sequencing in order to examine their distinct effects on cellular pathways associated with [a]Syn overexpression or seeded aggregation. Two cellular activities were prominently affected: lipid metabolism and rRNA processing. Notably, while EGCG effects were confined to cells with aggregated Syn, Minzasolmin and Emrusolmin also produced transcriptional changes in cells without aggregated Syn. Each of the compounds tested induced a partial reversal of transcriptional effects resulting from Syn seeded aggregation. We identified 391 genes that were no longer significantly differentially expressed upon addition of compound, relative to cells with seeded aggregation. This platform bridges phenotypic screening and molecular pathway analysis, providing insights into druggable pathways for synucleinopathies. The molecular signatures identified here can assist in testing and benchmarking future drug discovery leads.

Advances in the generation of proteins in silico has enabled the efficient design of such that can bind to a specified target. Here, we demonstrate the use of a fluorescently-labelled de novo-designed protein to bind its target in situ and be imaged using fluorescence microscopy, a widely used experimental technique that typically relies on antibodies or similar evolutionary derived binders to identify the presence and location of targets in their native environment. Our de novo-designed protein binds the C-terminal domain M10 (Ig-169) of the giant muscle protein titin, which spans half a sarcomere, the basic contractile unit of striated muscle. M10 antibodies suitable for fluorescence microscopy are unavailable. Confocal microscopy of muscle sections shows the binder localises to the M-band of the sarcomere - where M10 is found - and fails to label muscle in competition experiments and in mutant muscle where M10 is absent. These results demonstrate the utility of de novo-designed proteins in immunostaining-like experiments and suggest a future where targets can be routinely identified in complex biological samples by in silico-generated binders. Such an approach avoids the need to generate antibodies or similar binders either in vivo or in vitro, which can have technical, financial and ethical challenges.

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene, encoding an expanded polyglutamine tract in the huntingtin (HTT) protein. The pathogenic CAG repeat of HTT is unstable and undergoes progressive somatic expansion in specific brain cells and peripheral tissues throughout life. Genes involved in DNA mismatch repair pathways, which promote repeat expansion, have been identified as genetic modifiers of the disease. Consequently, the rate of CAG repeat expansion is a key determinant driving the age of onset and disease progression. As the CAG repeat expands, alternative processing of HTT pre-mRNA increasingly favours production of the HTT1a transcript, which encodes the highly pathogenic and aggregation-prone HTT1a protein. This process provides a mechanistic link between CAG repeat expansion and disease pathogenesis, as increased HTT1a production accelerates HTT aggregation and neuronal dysfunction. HTT1a has previously been detected in Huntingtons disease mouse models by using immunoprecipitation coupled with western blotting, homogeneous time-resolved fluorescence (HTRF) and Meso Scale Discovery (MSD) bioassays, and immunohistochemistry. These approaches were developed using MW8, a neoepitope antibody that specifically recognizes the C-terminus of HTT1a. MW8 is a relatively weak antibody with limited detection sensitivity. To generate more robust HTT1a-specific reagents, two novel recombinant antibodies, 1B12 and 11G2, have been developed for evaluation. Using an allelic series of knock-in (HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175) mice, alongside transgenic YAC128 and N171-82Q models, we extensively evaluated and compared the performance of MW8, 1B12 and 11G2. We demonstrate that 1B12 and 11G2 function as HTT1a-specific neoepitope antibodies by immunoprecipitation with western blotting, and by immunohistochemistry. To enhance HTT1a detection using HTRF and MSD technology platforms, we further evaluated the performance of 1B12 and 11G2 in HTT bioassays using cortical lysates from zQ175 and YAC128 mice. In zQ175 mice, enhanced detection of aggregated HTT1a by HTRF and MSD revealed that HTT fragments longer than HTT1a can be incorporated into HTT1a-containing aggregates. The most sensitive assays were subsequently applied across the allelic series of knock-in mice to assess the effect of polyglutamine length on bioassay performance. For optimal sensitivity, we recommend the preferential use of 1B12 for HTRF assays and 11G2 for MSD assays. Collectively, these findings establish 1B12 and 11G2 as robust antibodies to reliably detect and track HTT1a pathology in vivo and promotes the replacement of previously used MW8-based experimental approaches. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/708805v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@16d10faorg.highwire.dtl.DTLVardef@1759f41org.highwire.dtl.DTLVardef@12a8c21org.highwire.dtl.DTLVardef@55fe6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Osborne et al. used Huntingtons disease mouse models to evaluate and compare the performance of HTT1a-specific neoepitope antibodies by using immunoprecipitation with western blotting, bioassays, and immunohistochemistry. In contrast to MW8, they establish that 1B12 and 11G2 are robust antibodies to reliably detect and track HTT1a pathology in vivo.

Extracellular adenosine triphosphate (eATP) is a mediator of purinergic signalling in the airways, implicated in mucociliary function, inflammation, and cough via activation of P2X3 receptors. Elevated airway eATP has been associated with multiple respiratory diseases, yet reliable measurement of eATP remains challenging due to its rapid enzymatic degradation and confounding contributions from intracellular ATP. Here, we describe an optimized, microwell plate-based luminescence method for quantifying eATP from human airway epithelial cell cultures and bronchoalveolar lavage (BAL) fluid with enhanced signal stability. Using a commercially available ATP detection assay with a prolonged luminescence half-life, we introduced a simple 0.45 {micro}m syringe filtration step to remove cells and thereby isolate extracellular ATP. This approach demonstrated ATP specificity via apyrase degradation, and provided a linear detection range from 5 nM to 5 {micro}M. Addition of ATP stabilization buffer preserved ATP levels in cell culture media for at least 4 hours at 4 {degrees}C and in human BAL samples for at least 6 weeks at -80{degrees}C. Applying this method to primary human bronchial epithelial cells revealed detectable eATP release, with preferential secretion at the apical surface under air-liquid interface conditions. Collectively, this optimized assay enables robust, high-throughput, and time-flexible quantification of eATP in both experimental and clinical airway samples. These methods support improved investigation of purinergic signalling in airway health and disease and may facilitate biomarker development relevant to eATP in the airways.

Compounds that bind to the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) main protease (MPro) often produce biphasic concentration-response curves (CRCs) in biochemical assays; low concentrations activate the enzyme and high concentrations inhibit it. This biphasic behavior complicates data analysis. Here, we compare three approaches to data analysis: fitting the Hill equation to the activation phase, fitting it to the inhibition phase, and fitting an enzyme kinetics model that incorporates dimerization and ligand binding to the complete CRC. In the latter case, cellular efficacy is predicted by extrapolating the model to high enzyme concentrations. For compounds in our drug lead series, all three procedures yield inhibitory concentrations that are correlated with live-virus antiviral assays. The latter procedure provides the most accurate forecast of cellular efficacy rank. These data analysis procedures may be valuable for antiviral drug discovery against MERS-CoV MPro and other enzymes with similar kinetics.

Prokaryotic transcription factors (TFs) are used as small molecule biosensors with broad applications in biotechnology, yet only a small fraction from microbial genomes have been characterized. To address this gap, we recently described the bioinformatic method Ligify, which leverages information from genome context and enzyme reaction databases to predict a TFs cognate effector molecule. Here we report Ligify 2.0, a modern web server for Ligify predictions. We systematically evaluate 10,965 small molecules within the Rhea enzyme reaction database for associations to TFs, ultimately generating 13,435 hypothetical interactions between 1,362 small molecules and 3,164 TFs. We then develop an interactive web server (https://ligify.groov.bio) to search and visualize prediction data. Each TF sensor page includes visualizations for chemical ligand structures, interactive TF protein structures, and genome context. Pages also include metadata links, predicted promoter sequences, prediction confidence metrics, and references to relevant literature. A plasmid builder tool enables users to generate custom biosensor circuit designs. Finally, we provide case studies using Ligify 2.0 to identify two TFs from the pathogens Escherichia coli O157:H7 and Mycobacterium abscessus responsive to 4-hydroxybenzoate and Pseudomonas Quinolone Signal, respectively. The Ligify web server aims to facilitate the systematic characterization of biosensors for chemical-control of biological systems. Key pointsO_LILigify 2.0 contains >13,000 predicted transcription factor-small molecule interactions C_LIO_LIA rich web interface provides interactive visualizations and a plasmid design tool C_LIO_LIPredicted ligands for regulators from pathogenic bacteria are experimentally validated C_LI Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/683484v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1afa575org.highwire.dtl.DTLVardef@97c811org.highwire.dtl.DTLVardef@cfdb93org.highwire.dtl.DTLVardef@58977d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fluorescent reporters provide a useful tool for studying degron motifs. Fusing a degron of interest to a fluorescent protein allows to accurately track protein levels overtime to characterise the degradation kinetics of studied degrons. Here we describe a rapid and simple method to study degron peptides in Escherichia coli using plasmid-encoded eGFP-degron fusion constructs. The described methods provide an accessible workflow to evaluate degrons. We provide protocols for generation of pBAD plasmids encoding the studied constructs and two different methods for evaluating degrons - an end-point fluorescence measurement on agar plates and a kinetic measurement in liquid cultures in a 96-well format for high-throughput degron studies.

New approaches to mitigate the reduction of crop yields by plant parasitic nematodes are needed in the face of increasing concerns of the impact of nematicides on precious ecosystems. One approach is to target receptors in the parasitic nematode that are vital for their survival that less widely expressed in non-target organisms. Nematodes express a phylogenetically restricted 5-HT-gated chloride channel, MOD-1, activation of which causes paralysis in Caenorhabditis elegans. We show that MOD-1 is expressed in the motor nervous system of the plant parasitic nematode Globodera pallida and its functional characterisation is validated by 5-HT activation when reconstituted in Xenopus laevis oocytes. To evaluate MOD-1 as a nematicide target we utilised a previously described platform called PhaGeM4 for PharmacoGenetic targeting of M4 neurone in which MOD-1 is expressed in transgenic C. elegans and nematode development in the face of MOD-1 chemical modulation is tracked. We screened Pathogen Box, a chemical library of 400 diverse drug-like molecules, using PhaGeM4. This identified 10 putative hits for C. elegans MOD-1. These hits were pursued through a sequential, iterative pipeline encompassing mod-1 dependent C. elegans motility and G.pallida motility assays in combination with pharmacological interrogation of G. pallida mod-1 in PhaGeM4. This approach highlights 3 compounds with a mod-1 dependent action (quipazine, our benchmark compound; MMV687251, a vancomycin-like compound; MMV688774, an antifungal with common name posaconazole) and one compound that acts through an undetermined target (MMV002816, also known as the antifilarial drug, diethylcarbamazine). Each of these compounds had a significant inhibitory effect on G. pallida J2 root invasion. Overall, this lends confidence that the PhaGeM4 screening platform can delivery new chemical leads for crop protection and highlights four new chemistries of interest. More generally, this approach could be applied to other ligand-gated ion channels of interest as targets.

BackgroundThe clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding specificity, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. ResultsWe have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R2 = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 minutes, a significant improvement over traditional methods. ConclusionsSE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The methods compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.

The expansion of short tandem repeats is a feature of over 60 different human diseases. Ongoing somatic instability throughout a patients lifetime can influence disease progression and has emerged as a therapeutic target. Understanding its mechanism is essential for the identification of both drug targets and therapeutic interventions. A major obstacle towards this translational goal has been to measure changes in repeat size distribution in a timely manner. To address this, here we present Single Clone-based Instability Assay (SCIA), a streamlined experimental design that saves weeks in assessing the effect of a gene knockout on repeat instability. The approach avoids bulk cultures and does not require a reporter cell line. It uses targeted long-read sequencing as a readout for repeat instability. We have validated the approach using FAN1, PMS1, and MLH1 knockouts in HEK293-derived cells. We provide a visualization software that generates delta plots, extracts the instability frequency, the bias towards expansion or contraction, and the average size of the changes. Using SCIA, we find that although FAN1 knockout clones showed increased frequency of expansions, the size of the expansions were smaller. This highlights the wealth of information that can be extracted and the potential for novel insights into the mechanism of repeat instability.

Specific drug metabolism rates are defined by the constituency of the cytochrome P450 (CYP) genome, including polymorphic changes in any of 200+ CYP genes. An example is CYP2C19, where associations of gene polymorphisms with variability in certain drug metabolism rates have been linked to inter-individual and inter-ethnic differences in therapeutic outcomes. While pharmacogenomic screening for these variants prior to drug and dosage prescription has well-defined links to better treatment outcomes, current implementation is limited to complex and costly variant-probing and DNA sequencing protocols, which have limited availability in clinical laboratories, leading to slow turnaround times, impacting effective clinical intervention. Here we describe a novel, cost-effective, multiplex genotyping approach to screening CYP2C19 variants. Fluorescence nested allele-specific (FAS) PCR was used with primers to detect CYP2C19 variants of interest in specific hot spots, including the Tier 1 haplotypes identified by the Association for Molecular Pathology (AMP): CYP2C19*2, *3, and *17. The presence/absence of wild-type and mutant alleles were identified independently as haplotypes, and in a multiplex reaction as diplotypes representing the 10 possible genotype combinations/profiles. FAS-PCR achieved the same genotype calls as a pyrosequencing protocol optimized for validating genotypes, but with a simpler and more sensitive interface. The FAS-PCR method correctly identified the genotypes of both synthesized DNA and a human genomic DNA standard. Uniquely, the FAS-PCR protocol generates patterns using one fluorescently-labeled primer irrespective of the number of variants targeted, establishing it as considerably more cost-effective than other allele-specific PCR-based techniques that involve labeling both the forward and reverse primers.

Developing novel drugs against membrane proteins is a major challenge in drug discovery due to the difficulty of stabilizing these targets for high-throughput screenings. Pannexin 1 (PANX1) is a membrane channel protein involved in various physiological and pathological processes, making it a promising target for drug discovery. However, efforts to develop PANX1-targeting therapeutics have been hindered by the inherent challenges of stabilizing the protein channel and conducting effective pharmacological screening. Here, we report a proof-of-concept workflow that integrates the Salipro lipid nanoparticle platform with DNA-Encoded Library screenings in a detergent-free format. In this case study, the Salipro DirectMX method was used to generate functional PANX1 nanoparticles for drug discovery and characterisation. Using a high-stringency selection strategy and computational approaches, we identified a specific set of candidate compounds with selective PANX1 enrichment. Surface Plasmon Resonance analysis confirmed the identification of hit compounds. Cryo-Electron Microscopy of the Salipro-PANX1-Compound complex provided structural insights into a potential compound binding site. Electrophysiological recordings in PANX1-expressing Xenopus laevis oocytes demonstrated dose-dependent inhibition of PANX1-mediated ion conductance by the compounds. These findings establish a robust workflow for ligand discovery against challenging membrane protein targets and provide novel chemical starting points for the development of PANX1 modulators.

Antimicrobial resistance remains a global existential threat. Given that antimicrobial therapy commonly starts before pathogen identification, rapid and scalable methods capable of determining effective antimicrobial compounds are needed. In this paper, we demonstrate a 2 x 2 array of parallelised microscopes that uses low numerical aperture (NA=0.25) detection optics and LED excitation to determine bacterial viability based on their fluorescence response to an electrical stimulus. Following a 2-hour incubation, the fluorescent viability readout requires less than one minute. We use K-means clustering to classify pixels in a time lapse sequence of widefield fluorescence images and extract changes seen within bacterial clusters. We demonstrate sufficient sensitivity to measure fluorescence changes after electrical stimulation in a bacterial monolayer. To capture these subtle fluorescence changes at high signal-to-background ratios, we place a limit on the minimum optical density of the bacterial sample. This novel approach is scalable to 96-well formats using a suitable consumable electrode array.