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.

Cell Painting is a popular assay for morphological profiling of multi-labeled 2D monolayer cell cultures used in a wide range of applications. Culturing cells in 3D has potential for higher physiological relevance, such as when studying effects of perturbations. Robust and scalable 3D models can be challenging to characterize through imaging - particularly because light has difficulty penetrating cell multilayers. We introduce a scalable method where the Cell Painting assay is combined with tissue-clearing and applied to 3D spheroids generated in a ULA microplate format. Multi-channel images are acquired using confocal microscopy, and cells can be segmented inside those spheroids allowing for relevant morphological features to be extracted. Our end-to-end analysis pipeline comprises cell segmentation, morphological feature extraction, and between-spheroids and within-spheroid normalization. We demonstrate the method using spheroids cultured from two colorectal cancer cell lines and successfully detect distinct phenotypic changes upon compound treatments, on both spheroid-level using maximum intensity projections and on single cell-level. We show that drugs group by mechanism of action, with biologically relevant clusters especially evident with single-cell data. Finally, we contrast our method to results from 2D Cell Painting and discover a different pattern in DNA damaging drugs in HCT116 colorectal cancer cells. This work lays the foundation for multi-channel image-based screening in 3D spheroids.

Metazoan cells signal to each other via direct contact between cell surface proteins (CSPs) and by interactions of CSP receptors with secreted ligands. CSP extracellular domain (ECD) interactions control organ development and physiology and are perturbed in disease states. However, because they cannot be accurately assessed using standard high-throughput screening techniques, they are underrepresented in protein interaction databases. Many ECD interactions are of low affinity, and their detection in vitro requires taking advantage of avidity effects, typically by multimerization of fusion proteins. Assays that test only one or a few interactions in each binding reaction are inadequate for global interactome screening. Here we describe a new multiplex method that uses purified dimeric ECD fusion proteins coupled to 60-mer nanoparticles as soluble prey, and the same dimers coupled to spectrally distinguishable fluorescent microspheres (beads) as bait. We add one prey to a mixture of up to 500 baits in a single well, then use a Luminex FLEXMAP 3D (FM3D) instrument to read out bait identity and prey binding. The FM3D measures the fluorescent dye ratio for each bead and simultaneously determines the amount of epitope-tagged prey bound to that bead. We use the method, denoted as the Multiplex Interactome Assay (MPIA), to analyze a proof-of-concept (PoC) set of 41 CSPs and secreted protens that is derived from larger collections examined in two interactome screens that used ELISA-based binding assays. By analyzing interactions among PoC proteins, we compared the MPIA with earlier screening methods. The MPIA has a dynamic range that is at least 30-fold greater than ELISA-based assays and appears to be more sensitive. By coupling the MPIA to an automated protein production and purification platform, we hope to be able to conduct a screen for interactions among thousands of human CSPs and secreted ligands.

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

Morphological profiling with the Cell Painting assay has emerged as a promising method in drug discovery research. The assay captures morphological changes across various cellular compartments enabling the rapid identification of the effect of compounds. We present a comprehensive morphological profiling dataset using the carefully curated and well-annotated EU-OPENSCREEN Bioactive Compound Set. Our profiling dataset was generated across multiple imaging sites with high-throughput confocal microscopes using the Hep G2 as well as the U2 OS cell line. We employed an extensive assay optimization process to achieve high data quality across the different imaging sites. An analysis of the four replicates validates the robustness of the generated data. We compare morphological features of the different cell lines and map the profiles to activity, toxicity, and basic compound targets to further describe the dataset as well as to demonstrate the potential of this dataset to be used for mechanism of action exploration.

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

High-throughput screening (HTS) of proteins has a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimisation, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high throughput protein expression and screening methods, be that for expression, activity, stability, or binding as says, include the required yield, reproducibility, solubility, stability, purity and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce sufficient quantity of protein of appropriate purity. This limits the type of assay, and number of protein variants that can be simultaneously tested in an experiment. Here we describe a HTS protocol that allows the overnight expression, export and assay of recombinant proteins from E. coli cells in the same multi-well plate tube. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture media. The resulting protein is of sufficient purity and yield that in can be used directly in plate-based enzymatic assays without additional purification. This simple single plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications.

By combining the radial migration assay with injection molded gaskets and a rigid fixture, we have developed a more reliable and sensitive method for measuring radial cell migration. This method is well adapted for use on high throughput automated imaging systems. The use of injection molded gaskets enables low cost replacement of cell-wetted components. Furthermore, the design enables secondary placement of attractants and co-cultures. This device and high-throughput application permit the use of therapeutic screening to evaluate phenotypic responses e.g. cancer cell migration. This approach is orthogonal to other 2D cell migration applications such as scratch wound assays, although here we offer a non-invasive, high-throughput device which is currently not commercially available. Collectively, we have designed a systematic, reliable, high-throughput application to monitor phenotypic responses to chemotherapeutic screens, genetic alterations (e.g. RNAi; CRISPR; others), supplemental regiments, and other approaches offering a reliable methodology to survey unbiased and non-invasive cell migration.

Microdroplets are compartments made in the laboratory that allow the miniaturisation of chemical and biological experiments to the femto- to picolitre scale, replacing the classical test tube with a droplet. Ideally containment of the contents of individual droplets would be perfect, but in reality this situation rarely occurs. Instead the leaking of molecules even from intact droplets presents a challenge to the success of miniaturisation and must be assessed on a case-by-case basis. We now present a new method for quantitative determination of leakage: a sheath fluid-free flow cytometer (Guava EasyCyte) is used to directly determine the fluorescence of water-in-oil droplets as a function of time. We validate this method by demonstrating that this assessment of leakage provides a framework for experimental improvements that reduce the leakage of two widely used fluorophores. A 40-fold better retention compared to current protocols is achieved for resorufin with an optimized mix (oil: FC-70, surfactant: 0.1% w/w AZ900C, additive: 1% BSA) to maintain useful retention for up to 130 hours. Likewise leakage of the fluorophore methylumbelliferone is reduced by 75-fold. The availability of a method to quantitate leakage quickly for a variety of experimental conditions will facilitate future applications of droplet-based experiments (e.g. in directed evolution or diagnostics), aid miniaturisation of lab-scale assays into this format, and improve the degrees of freedom in setting up such ultrahigh-throughput experiments.

Small molecule-protein interactions underpin many biological functions and play an integral role in the treatment and prevention of several human diseases. These interactions can be key to understanding the mechanism of action of these compounds. Previous methods of determining protein-protein or protein-antibody interactions have been well established; however, the use of BLI in antiviral discovery is a promising and relatively new avenue. The high-throughput nature of this method in tandem with its pM sensitivity allows for quick and seamless identification of hit compounds. Here we discuss ways to overcome common pitfalls that can occur while using BLI such as nonspecific binding (NSB) and ligand drift while offering possible solutions. Characterizing small molecule-protein interactions is not trivial and optimizing the experimental conditions is imperative. To address this gap in knowledge, we present optimized BLI protocols for the study of three cases of protein-small molecule interactions: PF74 or Lenacapavir (LEN) with HIV-1 capsid protein (CA), and Nirmatrelvir (NIR) with SARS-CoV-2 Mpro. LEN and NIR are of particular interest because they are clinically relevant, and PF74, a well-studied control, was the first compound reported to target the LEN binding site. We demonstrate that BLI can be a powerful and effective tool in calculating the binding affinities between a protein and small molecule. These newly designed methods enabled calculation of KD values, the affinity between ligand and analyte, ranging from the micro to the sub-nanomolar range for CA binding events and confirmed the covalent interaction between NIR and Mpro. These protocols will facilitate efficient testing of new antivirals or derivatives in a high- throughput format. SummaryBio-Layer Interferometry (BLI) is a multifunctional technology that is used to determine valuable information on real-time kinetics including association and dissociation. Optimizing experimental conditions to acquire data about protein-ligand interactions can be challenging. We provide three example methods of collecting binding data that characterize how viral proteins interact with antivirals.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by dystrophin deficiency. Effective methods for drug discovery for the treatment of DMD requires systems to be physiologically relevant, scalable, and effective. To this end, the Myoscreen platform offers a scalable and physiologically relevant system for generating and characterizing patient-derived myotubes. Morphological profiling is a powerful technique involving the simultaneous measurement of hundreds of morphological parameters from fluorescence microscopy images and using machine learning to predict cellular activity. Here, we describe combining the Myoscreen platform and high dimensional morphological profiling to accurately predict a phenotype associated with the lack of Dystrophin expression in patient derived myotubes. Using this methodology, we evaluated a series of Dystrophin-associated protein complex (DAPC) candidates and identified that the combination of Utrophin and - Sarcoglycan yielded highest morphological differences between DMD and non-DMD donors. Finally, we validated this methodology by knocking down Dystrophin expression in non-DMD cells as well as introducing Dystrophin expression in DMD cells. Knocking down Dystrophin in non- DMD cells shifted their morphological profile to one that is similar to DMD cells while introducing Dystrophin in DMD cells shifted their morphological profile towards non-DMD cells. In conclusion, we have developed a platform that accurately predicts the DMD disease phenotype in a disease relevant cell type. Ultimately this platform may have wide applications in the drug development process include identification of disease modifier genes, screening of novel therapeutic moieties, and as a potency assay for future therapeutics.

Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counter-selection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counter-selection parameters. As a result, counter-selection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel, while also eliminating the need for counter-selection. We employ a platform based on a modified benchtop sequencer to conduct a massively-parallel aptamer screening process that enables the selection of highly-specific aptamers against multiple structurally similar molecules in a single experiment, without any counter-selection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes, and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes. Significance statementAptamers offer the exciting potential to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has been proven challenging using the conventional process of counter-selection. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers towards a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counter-selection. As exemplars, we generated aptamers with high affinity and specificity towards three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.

-Synuclein seed amplification assays are a promising diagnostic tool for synucleinopathies such as Parkinsons disease and multiple system atrophy. Standardized conditions are required to ensure a high degree of inter- and intra-laboratory reproducibility when performing these assays. A significant issue that hinders the utility of seed amplification assays is the de novo aggregation propensity of the -synuclein substrate as well as inter-batch heterogeneity. While much work has focused on determining appropriate seed amplification assay buffer compositions as well as the type and amount of seed used, a robust comparison of -synuclein substrate purification methods has not been reported. We therefore compared the utility of recombinant -synuclein purified using four different methods as seed amplification assay substrates across two laboratories. Osmotic shock-purified -synuclein monomer substrate showed the lowest propensity for de novo aggregation, which translated into being the best substrate for seed amplification assay reactions seeded with -synuclein preformed fibrils or patient brain homogenates. Furthermore, osmotic shock -synuclein monomer showed the best inter-batch reproducibility compared to all other substrates tested. As -synuclein seed amplification assays continue to evolve and move towards adoption in the clinical realm, this work showcases the vital importance of standardizing the production and characterization of recombinant -synuclein substrate. We encourage the widespread adoption of osmotic shock -synuclein monomer as the universal substrate for seed amplification assays to maximize intra- and inter-laboratory reproducibility.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

Inhibition of the target of rapamycin (TOR/mTOR) protein kinase by the drug rapamycin extends lifespan and healthspan across diverse species. However, rapamycin has potential off-target and side effects that warrant the discovery of additional TOR inhibitors. TOR was initially discovered in Saccharomyces cerevisiae (yeast) which contains two TOR paralogs, TOR1 and TOR2. Yeast lacking functional Tor1 are viable but are hypersensitive to growth inhibition by TORC1 inhibitors, which is a property of yeast that can be exploited to identify TOR inhibitors. Additionally, yeast lacking FK506-sensitive proline rotamase (FPR1) or containing a tor1-1 allele (a mutation in the Fpr1-rapamycin binding domain of Tor1) are robustly and selectively resistant to rapamycin and analogs that allosterically inhibit TOR activity via an FPR1-dependent mechanism. To facilitate the identification of TOR inhibitors, we generated a panel of yeast strains with mutations in TOR pathway genes combined with the removal of 12 additional genes involved in drug efflux. This creates a drug sensitive strain background that can sensitively and effectively identify TOR inhibitors. In a wildtype yeast strain background, 25 {micro}M of Torin1 and 100 {micro}M of GSK2126458 (omipalisib) are necessary to observe TOR1-dependent growth inhibition by these known TOR inhibitors. In contrast, 100 nM Torin1 and 500 nM GSK2126458 (omipalisib) are sufficient to identify TOR1-dependent growth inhibition in the drug sensitized background. This represents a 200-fold and 250-fold increase in detection sensitivity for Torin1 and GSK2126458, respectively. Additionally, for the TOR inhibitor AZD8055, the drug sensitive system resolves that the compound results in TOR1-dependent growth sensitivity at 100 {micro}M, whereas no growth inhibition is observed in a wildtype yeast strain background. Our platform also identifies the caffeine analog aminophylline as a TOR1-dependent growth inhibitor via selective tor1 growth sensitivity. We also tested nebivolol, isoliquiritigenin, canagliflozin, withaferin A, ganoderic acid A, and taurine, and found no evidence for TOR inhibition using our yeast growth-based model. Our results demonstrate that this system is highly effective at identifying compounds that inhibit the TOR pathway. It offers a rapid, cost-efficient, and sensitive tool for drug discovery, with the potential to expedite the identification of new TOR inhibitors that could serve as geroprotective and/or anti-cancer agents.

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.

Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico, this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro. To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators.

Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al, Cell Physiol Biochem 2019;53:656-86). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H that can be used for melanoma therapy and research. ResultsWe established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. All plate-based QC parameters were acceptable: %CV = 6.7{+/-}0.3, S/B = 21{+/-}2.1, Z = 0.75{+/-}0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. ConclusionsWe developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50 values determined at a single point in time, because the same IC50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC50 values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanism E + I {rightleftharpoons} E{middle dot}I [-&gt;] EI, the two IC50 values alone can be used to estimate both the inhibition constant (Ki) as a measure of binding affinity and the inactivation rate constant (kinact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanism E + I [-&gt;] EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (kinact/Ki) from a single experimental value of IC50. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.