Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors.

National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-ß-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

Rebamipide and Derivatives are Potent, Selective Inhibitors of Histidine Phosphatase Activity of the Suppressor of T Cell Receptor Signaling Proteins.

The suppressor of T cell receptor signaling (Sts) proteins are negative regulators of immune signaling. Genetic inactivation of these proteins leads to significant resistance to infection. From a 590,000 compound high-throughput screen, we identified the 2-(1H)-quinolinone derivative, rebamipide, as a putative inhibitor of Sts phosphatase activity. Rebamipide, and a small library of derivatives, are competitive, selective inhibitors of Sts-1 with IC50 values from low to submicromolar. SAR analysis indicates that the quinolinone, the acid, and the amide moieties are all essential for activity. A crystal structure confirmed the SAR and reveals key interactions between this class of compound and the protein. Although rebamipide has poor cell permeability, we demonstrated that a liposomal preparation can inactivate the phosphatase activity of Sts-1 in cells. These studies demonstrate that Sts-1 enzyme activity can be pharmacologically inactivated and provide foundational tools and insights for the development of immune-enhancing therapies that target the Sts proteins.

Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A(2).

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer.

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ∼10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways ( e.g., BRCA2 ). Personalized medicine approaches tailored toward patients' mutations would improve patients' outcome. To identify novel vulnerabilities of BRCA2 -deficient pancreatic cancer, we generated isogenic Brca2 -deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2 -deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2 -deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2 -deficient pancreatic cancer.

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer.

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ~10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients' mutations would improve patients' outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.

In Vitro and In Vivo Drug-Response Profiling Using Patient-Derived High-Grade Glioma.

BACKGROUND: Genomic profiling cannot solely predict the complexity of how tumor cells behave in their in vivo microenvironment and their susceptibility to therapies. The aim of the study was to establish a functional drug prediction model utilizing patient-derived GBM tumor samples for in vitro testing of drug efficacy followed by in vivo validation to overcome the disadvantages of a strict pharmacogenomics approach. METHODS: High-throughput in vitro pharmacologic testing of patient-derived GBM tumors cultured as 3D organoids offered a cost-effective, clinically and phenotypically relevant model, inclusive of tumor plasticity and stroma. RNAseq analysis supplemented this 128-compound screening to predict more efficacious and patient-specific drug combinations with additional tumor stemness evaluated using flow cytometry. In vivo PDX mouse models rapidly validated (50 days) and determined mutational influence alongside of drug efficacy. We present a representative GBM case of three tumors resected at initial presentation, at first recurrence without any treatment, and at a second recurrence following radiation and chemotherapy, all from the same patient. RESULTS: Molecular and in vitro screening helped identify effective drug targets against several pathways as well as synergistic drug combinations of cobimetinib and vemurafenib for this patient, supported in part by in vivo tumor growth assessment. Each tumor iteration showed significantly varying stemness and drug resistance. CONCLUSIONS: Our integrative model utilizing molecular, in vitro, and in vivo approaches provides direct evidence of a patient's tumor response drifting with treatment and time, as demonstrated by dynamic changes in their tumor profile, which may affect how one would address that drift pharmacologically.

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes.

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

Rapid deployment of inexpensive open-source orbital shakers in support of high-throughput screening.

Open-source projects continue to grow in popularity alongside open-source educational resources, software, and hardware tools. The impact of this increased availability of open-source technologies is that end users are empowered to have greater control over the tools that they work with. This trend extends in the life science laboratory space, where new open-source projects are routinely being published that allow users to build and modify scientific equipment specifically tailored to their needs, often at a reduced cost from equivalent commercial offerings. Recently, we identified a need for a compact orbital shaker that would be usable in temperature and humidity-controlled incubators to support the development and execution of a high-throughput suspension cell-based assay. Based on the requirements provided by staff biologists, an open-source project known as the DIYbio orbital shaker was identified on Thingiverse, then quickly prototyped and tested. The initial orbital shaker prototype based on the DIYbio design underwent an iterative prototyping and design process that proved to be straightforward due to the open-source nature of the project. The result of these efforts has been the successful initial deployment of ten shakers as of August 2021. This afforded us the scalability and efficacy needed to complete a large-scale screening campaign in less time and at less cost than if we purchased larger, less adaptable orbital shakers. Lessons learned from prototyping, modifying, validating, deploying and maintaining laboratory devices based on an open-source design in support of a full-scale drug discovery high-throughput screening effort are described within this manuscript.

Screening for modulators of autism spectrum disorder using induced human neurons.

Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder. There are no drugs to treat the core symptoms. De novo mutations often play an important role in ASD and multiple high-risk loci have been identified in the last decade. These mutations range from copy number variants to small insertion/deletion and single nucleotide variants. Large-scale exome sequencing has identified over 100 risk genes that are associated with ASD. Both etiological heterogeneity and unavailability of human neurons remain major hurdles in understanding the pathophysiology of ASD and testing of new drug candidates. Hence, the most achievable and relevant model to screen for potential drugs is human neurons from inducible pluripotent stem cells (iPSCs), including those from individuals with genetic mutations. In this study, we tested stem cells from individuals carrying mutations in ADNP, FOXP1 or SHANK3. They were scaled and reprogrammed to glutamatergic neurons and assessed for the effects of their specific mutations on neurite outgrowth. High Content Analysis allowed us to observe phenotypic differences between ASD neurons compared to controls, in terms of neuron number, neurite number and neurite length per neuron. Further, neurons were derived from both patient derived and genetically modified iPSCs with DDX3X mutation which were tested against 5088 drug like compounds. We assessed individual compound effects on the induced neurons to determine if they elicited changes that would indicate neurite growth (neuroprotection) or, alternatively, reduce outgrowth and hence appear neurotoxic. This report includes all methods, phenotypic outcomes, and results for the largest ASD small molecule screening effort done to date.

Lead identification using 3D models of pancreatic cancer.

Recent technological advances have enabled 3D tissue culture models for fast and affordable HTS. We are no longer bound to 2D models for anti-cancer agent discovery, and it is clear that 3D tumor models provide more predictive data for translation of preclinical studies. In a previous study, we validated a microplate 3D spheroid-based technology for its compatibility with HTS automation. Small-scale screens using approved drugs have demonstrated that drug responses tend to differ between 2D and 3D cancer cell proliferation models. Here, we applied this 3D technology to the first ever large-scale screening effort completing HTS on over 150K molecules against primary pancreatic cancer cells. It is the first demonstration that a screening campaign of this magnitude using clinically relevant, ex-vivo 3D pancreatic tumor models established directly from biopsy, can be readily achieved in a fashion like traditional drug screen using 2D cell models. We identified four unique series of compounds with sub micromolar and even low nanomolar potency against a panel of patient derived pancreatic organoids. We also applied the 3D technology to test lead efficacy in autologous cancer associated fibroblasts and found a favorable profile for better efficacy in the cancer over wild type primary cells, an important milestone towards better leads. Importantly, the initial leads have been further validated in across multiple institutes with concordant outcomes. The work presented here represents the genesis of new small molecule leads found using 3D models of primary pancreas tumor cells.

Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance.

Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC(50) = 21 nM) and in situ (IC(50) = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance.

A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors.

Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS.

Identification of Potential Modulators of the RGS7/Gß5/R7BP Complex.

Regulators of G protein signaling (RGS) proteins serve as critical regulatory nodes to limit the lifetime and extent of signaling via G protein-coupled receptors (GPCRs). Previously, approaches to pharmacologically inhibit RGS activity have mostly focused on the inhibition of GTPase activity by interrupting the interaction of RGS proteins with the G proteins they regulate. However, several RGS proteins are also regulated by association with binding partners. A notable example is the mammalian RGS7 protein, which has prominent roles in metabolic control, vision, reward, and actions of opioid analgesics. In vivo, RGS7 exists in complex with the binding partners type 5 G protein ß subunit (Gß5) and R7 binding protein (R7BP), which control its stability and activity, respectively. Targeting the whole RGS7/Gß5/R7BP protein complex affords the opportunity to allosterically tune opioid receptor signaling following opioid engagement while potentially bypassing undesirable side effects. Hence, we implemented a novel strategy to pharmacologically target the interaction between RGS7/Gß5 and R7BP. To do so, we searched for protein complex inhibitors using a time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay that measures compound-mediated alterations in the FRET signal between RGS7/Gß5 and R7BP. We performed two HTS campaigns, each screening ~100,000 compounds from the Scripps Drug Discovery Library (SDDL). Each screen yielded more than 100 inhibitors, which will be described herein.

Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9).

We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme's active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.

Automating a Magnetic 3D Spheroid Model Technology for High-Throughput Screening.

Affordable and physiologically relevant three-dimensional (3D) cell-based assays used in high-throughput screening (HTS) are on the rise in early drug discovery. These technologies have been aided by the recent adaptation of novel microplate treatments and spheroid culturing techniques. One such technology involves the use of nanoparticle (NanoShuttle-PL) labeled cells and custom magnetic drives to assist in cell aggregation to ensure rapid 3D structure formation after the cells have been dispensed into microtiter plates. Transitioning this technology from a low-throughput manual benchtop application, as previously published by our lab, into a robotically enabled format achieves orders of magnitude greater throughput but required the development of specialized support hardware. This effort included in-house development, fabrication, and testing of ancillary devices that assist robotic handing and high-precision placement of microtiter plates into an incubator embedded with magnetic drives. Utilizing a "rapid prototyping" approach facilitated by cloud-based computer-aided design software, we built the necessary components using hobby-grade 3D printers with turnaround times that rival those of traditional manufacturing/development practices at a substantially reduced cost. This approach culminated in a first-in-class HTS-compatible 3D system in which we have coupled 3D bioprinting to a fully automated HTS robotic platform utilizing our novel magnetic incubator shelf assemblies.

The Scripps Molecular Screening Center and Translational Research Institute.

The Scripps Research Molecular Screening Center (SRMSC) was founded in 2004 and comprises more than $22 million of specialized automation. As part of the Translational Research Institute (TRI), it comprises early drug discovery labs and medicinal chemistry. Together with Scripps Research at the La Jolla, California, campus, this represents one of the most competitive academic industrial screening centers worldwide. The SRMSC uses automated platforms, one a screening cell and the other a cherry-picking platform. Matched technologies are available throughout Scripps to allow scientists to develop assays and prepare them for automated screening. The library comprises more than 1 million drug-like compounds, including a proprietary collection of >665,000 molecules. Internal chemistry has included ~40,000 unique compounds that are not found elsewhere. These collections are screened against a myriad of disease targets, including cell-based and biochemical assays that are provided by Scripps faculty or from global investigators. Scripps has proven competence in all detection formats, including high-content analysis, fluorescence, bioluminescence resonance energy transfer (BRET), time-resolved fluorescence resonance energy transfer (TR-FRET), fluorescence polarization (FP), luminescence, absorbance, AlphaScreen, and Ca++ signaling. These technologies are applied to NIH-derived collaborations as well as biotech and pharma initiatives. The SRMSC and TRI are recognized for discovering multiple leads, including Ozanimod.

Identification of Novel, Structurally Diverse, Small Molecule Modulators of GPR119.

GPR119 drug discovery efforts in the pharmaceutical industry for the treatment of type 2 diabetes mellitus (T2DM) and obesity, were initiated based on its restricted distribution in pancreas and GI tract, and its possible role in glucose homeostasis. While a number of lead series have emerged, the pharmacological endpoints they provide have not been clear. In particular, many lead series have demonstrated loss of efficacy and significant toxic side effects. Thus, we sought to identify novel, potent, positive modulators of GPR119. In this study, we have successfully developed and optimized a high-throughput screening strategy to identify GPR119 modulators using a live cell assay format that utilizes a cyclic nucleotide-gated channel as a biosensor for cAMP production. Our high-throughput screening (HTS) approach is unique to that of previous HTS approaches targeting this receptor, as changes in cAMP were measured both in the presence and absence of an EC10 of the endogenous ligand, oleoylethanolamide, enabling detection of both agonists and potential allosteric modulators in a single assay. From these efforts, we have identified positive modulators of GPR119 with similar as well as unique scaffolds compared to existing compounds and similar as well as unique signaling properties. Our compounds will not only serve as novel molecular probes to better understand GPR119 pleiotropic signaling and the underlying physiological consequences of receptor activation, but are also well-suited for translation as potential therapeutic agents.

A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. Thus, significant efforts have been directed at identifying downstream targets on which RAS is dependent. These efforts have proven challenging, in part due to confounding factors such as reliance on two-dimensional adherent monolayer cell cultures that inadequately recapitulate the physiologic context to which cells are exposed in vivo. To overcome these issues, we implemented a high-throughput screening (HTS) approach using a spheroid-based 3-dimensional culture format, thought to more closely reflect conditions experienced by cells in vivo. Using isogenic cell pairs, differing in the status of KRAS, we identified Proscillaridin A as a selective inhibitor of cells harboring the oncogenic KRasG12V allele. Significantly, the identification of Proscillaridin A was facilitated by the 3D screening platform and would not have been discovered employing standard 2D culturing methods.

Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles.

There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.