Recent Publications:

Journal of Medicinal Chemistry	Identification and Optimization of Inhibitors of Trypanosomal Cysteine Proteases: Cruzain, Rhodesain, and TbCatB [ PDF ] Mott BT, Ferreira RS, Simeonov A, Jadhav A, Ang KK, Leister W, Shen M, Silveira JT, Doyle PS, Arkin MR, McKerrow JH, Inglese J, Austin CP, Thomas CJ, Shoichet BK, Maloney DJ Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas’ disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival: cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a cocrystal was obtained of our most potent analogue bound to cruzain.
Journal of Medicinal Chemistry	Quantitative Analyses of Aggregation, Autofluorescence, and Reactivity Artifacts in a Screen for Inhibitors of a Thiol Protease [ PDF ] Jadhav A, Ferreira RS, Klumpp C, Mott BT, Austin CP, Inglese J, Thomas CJ, Maloney DJ, Shoichet BK, Simeonov A The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of nonspecific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds, and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.5% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.
Journal of Medicinal Chemistry	Discovery of a 2,4-Diamino-7-aminoalkoxyquinazoline as a Potent and Selective Inhibitor of Histone Lysine Methyltransferase G9a [ PDF ] Liu F, Chen X, Allali-Hassani A, Quinn AM, Wasney GA, Dong A, Barsyte D, Kozieradzki I, Senisterra G, Chau I, Siarheyeva A, Kireev DB, Jadhav A, Herold JM, Frye SV, Arrowsmith CH, Brown PJ, Simeonov A, Vedadi M, Jin J SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of Compound 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the G9a-Compound8 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. The cocrystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. Compound 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling.
Molecular BioSystems	A strategy to discover inhibitors of Bacillus subtilis surfactin-type phosphopantetheinyl transferase [ PDF ] Yasgar A, Foley T, Jadhav A, Inglese J, Burkart M, Simeonov A Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and non-ribosomal peptide synthases of prokaryotes and have been implicated in the activation of a variety of pathogen-associated virulence factors. As such, inhibitors of this enzyme class represent enticing leads for antibiotic development and can serve as tools in studies of bacterial metabolism. Currently, no small molecule inhibitors of Sfp-PPTase are known, highlighting the need for efficient methods for PPTase inhibitor identification and development. Herein, we present the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of Sfp-PPTase using the substrate combination of rhodamine-labeled coenzyme A and Black Hole Quencher-2 labeled consensus acceptor peptide YbbR. Upon PPTase-catalyzed transfer of the rhodamine-labeled phosphopantetheinyl arm onto the acceptor peptide, the fluorescent donor and quencher are covalently joined and the fluorescence signal is reduced. This assay was miniaturized to a low 4 L volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC1280). Top inhibitors identified by the screen were further characterized in secondary assays, including protein phosphopantetheinylation detected by gel electrophoresis. The present assay enables the screening of large compound libraries against Sfp-PPTase in a robust and automated fashion and is applicable to designing assays for related transferase enzymes.
Molecular BioSystems	A miniaturized screen for inhibitors of Jumonji histone demethylases [ PDF ] Sakurai M, Rose NRR, Schultz L, Quinn AM, Jadhav A, Ng SS, Oppermann U, Schofield CS, Simeonov A 2-Oxoglutarate- and Fe(II)-dependent oxygenases are a major class of N-methyl lysine demethylases that are involved in epigenetic regulation. Assays suitable for implementation in a high-throughput manner have been lacking for these enzymes. Here, we describe the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of JMJD2E using a formaldehyde dehydrogenase-coupled reaction with real-time fluorescence detection. Reactant compatibility studies resulted in simplification of the assay scheme to the mixing of two reagent solutions, both of which were stable overnight. The assay was miniaturized to a 4 L volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC1280). Inhibitors identified by the screen were further characterized in secondary assays including FDH counterscreen and demethylation assays that monitored demethylation by MALDI-TOF MS. The assay developed here will enable the screening of large compound libraries against the Jumonji demethylases in a robust and automated fashion.
Current Topics in Medicinal Chemistry	The Pilot Phase of the NIH Chemical Genomics Center. [ PDF ] Thomas CJ, Auld DS, Huang R, Huang W, Jadhav A, Johnson RL, Leister W, Maloney DJ, Marugan JJ, Michael S, Simeonov A, Southall N, Xia M, Zheng W, Inglese J, Austin CP. The NIH Chemical Genomics Center (NCGC) was the inaugural center of the Molecular Libraries and Screening Center Network (MLSCN). Along with the nine other research centers of the MLSCN, the NCGC was established with a primary goal of bringing industrial technology and experience to empower the scientific community with small molecule compounds for use in their research. We intend this review to serve as 1) an introduction to the NCGC standard operating procedures, 2) an overview of several of the lessons learned during the pilot phase and 3) a review of several of the innovative discoveries reported during the pilot phase of the MLSCN.
Toxicological Sciences	Weighted Feature Significance (WFS): A Simple, Interpretable Model of Compound Toxicity Based on the Statistical Enrichment of Structural Features. [ PDF ] Huang R, Southall N, Xia M, Cho MH, Jadhav A, Nguyen DT, Inglese J, Tice RR, Austin CP In support of the U.S. Tox21 program, we have developed a simple and chemically intuitive model we call Weighted Feature Significance (WFS) to predict the toxicological activity of compounds, based on the statistical enrichment of structural features in toxic compounds. We trained and tested the model on: (1) data from quantitative high-throughput screening (qHTS) cytotoxicity and caspase activation assays conducted at the NIH Chemical Genomics Center (NCGC), (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program (NTP), and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances (RTECS). Enrichments of structural features in toxic compounds are evaluated for their statistical significance and compiled into a simple additive model of toxicity, and then used to score new compounds for potential toxicity. The predictive power of the model for cytotoxicity was validated using an independent set of compounds from the U.S. Environmental Protection Agency (EPA) tested also at the NCGC. We compared the performance of our WFS approach with classical classification methods such as Naïve Bayesian clustering and support vector machines. In most test cases, WFS showed similar or slightly better predictive power, especially in the prediction of hepatotoxic compounds, where WFS appeared to have the best performance among the three methods. The new algorithm has the important advantages of simplicity, power, interpretability, and ease of implementation.
PLoS One	A High-Throughput Approach for Identification of Novel General Anesthetics [ PDF ] Lea WA, Xi J, Jadhav A, Lu L, Austin CP, Simeonov A, Eckenhoff RG Anesthetic development has been a largely empirical process. Recently, we described a GABAergic mimetic model system for anesthetic binding, based on apoferritin and an environment-sensitive fluorescent probe. Here, a competition assay based on 1-aminoanthracene and apoferritin has been taken to a high throughput screening level, and validated using the LOPAC1280 library of drug-like compounds. A raw hit rate of ~15% was reduced through the use of computational filters to yield an overall hit rate of ~1%. These hits were validated using isothermal titration calorimetry. The success of this initial screen and computational triage provides feasibility to undergo a large scale campaign to discover novel general anesthetics.
Journal of Medicinal Chemistry	Structure Mechanism Insights and the Role of Nitric Oxide Donation Guide the Development of Oxadiazole-2-Oxides as Therapeutic Agents against Schistosomiasis [ PDF ] Rai G, Sayed AA, Lea WA, Luecke HF, Chakrapani H, Prast-Nielsen S, Jadhav A, Leister W, Shen M, Inglese J, Austin CP, Keefer L, Arner ES, Simeonov A, Maloney DJ, Williams DL, Thomas CJ Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.
Molecular Biosystems	A quantitative high-throughput screen for modulators of IL-6 signaling: a model for interrogating biological networks using chemical libraries. [ PDF ] Johnson RL, Huang R, Jadhav A, Southall N, Wichterman J, MacArthur R, Xia M, Bi K, Printen J, Austin CP, Inglese J. Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK-STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11 693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 microM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and non-selective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK-STAT activation.
PLoS ONE	Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1 Simeonov AS, Kulkarni A, Dorjsuren D, Jadhav A, Shen M, McNeill DR, Austin CP, Wilson DM APE1 is the major nuclease for excising abasic (AP) sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.
Molecular Cancer Therapeutics	Identification of phosphotyrosine mimetic inhibitors of Human Tyrosyl-DNA Phosphodiesterase I by a novel AlphaScreen high-throughput assay. Marchand C, Lea W, Jadhav A, Dexheimer T, Austin CP, Inglese J, Pommier Y, Simeonov A. Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves topoisomerase I (Top1)-DNA adducts accumulated from natural DNA damage, as well as from the action of certain anticancer drugs. Tdp1 catalyzes the hydrolysis of the phosphodiester bond between the catalytic tyrosine residue of Top1 and the DNA 3’ phosphate. Only a limited number of weak inhibitors have been reported for Tdp1, and there is an unmet need to identify novel chemotypes through screening of chemical libraries. Herein we present an easily configured, highly-miniaturized, and robust Tdp1 assay utilizing the AlphaScreen technology. Uninhibited enzyme reaction is associated with low signal while inhibition leads to a gain of signal, making the present assay format especially attractive for automated large-collection high-throughput screening. We report the identification and initial characterization of four previously-unreported inhibitors of Tdp1. Among them, suramin, NF449 and methyl-3,4-dephostatin are phosphotyrosine mimetics that may act as Tdp1 substrate decoys. We also report a novel biochemical assay using the SCAN1 Tdp1 mutant to study the mechanism of action of methyl-3,4 dephostatin.
Combinatorial Chemistry & High Throughput Screening	Optimization and Validation of Two Miniaturized Glucocerebrosidase Enzyme Assays for High Throughput Screening Urban DJ, Zheng W, Goker-Alpan O, Jadhav A, LaMarca ME, Inglese J, Sidransky E, Austin CP Glucocerebrosidase (GC) catalyzes the hydrolysis of ß-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl ß-D-glucopyranoside and resorufin ß-D-glucopyranoside, have Km values of 768 µM and 33 µM, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value.
ASSAY and Drug Development Technologies	A robotic platform for quantitative high-throughput screening. Michael S, Auld D, Klumpp C, Jadhav A, Zheng W, Thorne N, Austin CP, Inglese J, Simeonov A. High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation.
ASSAY and Drug Development Technologies	A 1,536-Well-Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase. Lea WA, Jadhav A, Rai G, Sayed AA, Cass CL, Inglese J, Williams DL, Austin CP, Simeonov A. Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.
PLoS Neglected Tropical Diseases	Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade. Simeonov S, Jadhav A, Sayed AA, Wang Y, Nelson ME, Inglese J, Williams DL, Austin CP. Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principle components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to a glutathione intermediate via a TGR-catalyzed reaction and then to hydrogen peroxide via Prx-catalyzed step. A fully-automated qHTS experiment (Inglese et al, PNAS, 103, 1147 (2006)) was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 ?L reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously-untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (~25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease.
Proceedings of the National Academy of Sciences	Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease Zheng W, Padia J, Urban D, Jadhav A, Simeonov A, Goldin E, Auld DS, LaMarca ME, Inglese J, Austin CP, Sidransky E. Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure-activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40-90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders.
Analytical Biochemistry	Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction. Simeonov A, Yasgar A, Jadhav A, Lokesh GL, Klumpp C, Michael S, Inglese J, Austin CP, Natarajan A. Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3ul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.
Journal of Medicinal Chemistry	Characterization of Chemical Libraries for Luciferase Inhibitory Activity. Auld DS, Southall N, Jadhav A, Johnson RL, Diller D, Simeonov S, Austin CP, Inglese J. To aid in the interpretation of HTS results derived from luciferase-based assays we used quantitative HTS (qHTS), an approach that defines the concentration-response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against >70,000 samples. We found approximately 3% of the library was active, containing only compounds with inhibitory concentration-responses of which 681 (0.9%) exhibited IC50s < 10 uM. Representative compounds were shown to inhibit purified P. pyralis as well as several commercial luciferase-based detection reagents but were found to be largely inactive against Renilla reniformis luciferase. Light attenuation by the samples was also examined and found to be more prominent in the blue-shifted bioluminescence produced by R. reniformas luciferase than with bioluminescence produced by P. pyralis luciferase. We describe the SAR of the luciferase inhibitors and discuss the use of this data in the interpretation of HTS results, and configuration of luciferase-based assays.
Journal of Medicinal Chemistry	Fluorescence Spectroscopic Profiling of Compound Libraries. Simeonov S, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall N, Shinn P, Smith J, Austin CP, Inglese J. Chromo/fluorophoric properties often accompany the conjugated, aromatic and heterocyclic features of many of the scaffolds and impurities that make up library samples used for high throughput screening (HTS). These properties impart highly variable effects on assay outputs employing optical detection, thus complicating the interpretation of data and leading to false positives and negatives. Here, we report the comprehensive fluorescence profile of >70,000 samples across multiple spectral regions commonly utilized in HTS assays. The quantitative HTS (qHTS) paradigm was utilized to test each sample at seven or more concentration points over a 4-log concentration range in 1536-well format, with raw fluorescence response collected using a CCD-based imager. The resulting output was compared with fluorophore standards to compute a normalized fluorescence response (termed fluorophore-equivalent concentration, FEC) for each sample, concentration, and relevant spectral region. The greatest fraction of fluorescent compounds appeared in the UV-end of the light spectrum, where over 5% of library members matched or exceeded 10 nM FEC of 4-methylumbelliferone and AlexaFluor 350, while approximately 1.8% of the library matched or exceeded 100 nM FEC of these standards. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, scaffold overlap with known fluorophores, fluorescent impurities, novel fluorescent compounds, and the ability to discriminate generalities of fluorescent interferences and devise strategies to identify them are discussed.
Combinatorial Chemistry & High Throughput Screening	A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction. Kimple AJ, Yasgar A, Hughes M, Jadhav A, Willard FS, Muller RE, Austin CP, Inglese J, Ibeanu GC, Siderovski DP, Simeonov A. The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
Analytical Biochemistry	A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression. Johnson RL, Huang W, Jadhav A, Austin CP, Inglese J, Martinez ED. Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced GFP reporter to identify novel classes of compounds involved in epigenetic regulation. This Locus Derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, six distinct chemical series were chosen for further study. Forty-eight qHTS actives and analogs were counter screened using the parental line of the LDR cells, which lack the GFP reporter. Three series, 8-hydroxy quinoline, quinoline-8-thiol and 1,3,5- thiadiazinane-2-thione, were not fluorescent and re-confirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.
Journal of Medicinal Chemistry	A Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens Against Beta-Lactamase. Babaoglu K, Simeonov A, Irwin, J, Nelson M, Feng BY, Thomas C, Cancian L, Costi MP, Maltby D, Jadhav A, Inglese J, Austin CP, Shoichet BK. High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against beta-lactamase using quantitative HTS (qHTS). Of the 1,274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting beta-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 microM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.
Joural of the Association for Laboratory Automation	Compound Management for Quantitative High-Throughput Screening. Yasgar A, Shinn P, Jadhav A, Auld DS, Michael S, Zheng W, Austin CP, Inglese J, Simeonov A. An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes which enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently-established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing >200,000 members, at a series of dilutions to construct a full concentration-response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a vertically-developed plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1,536- well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable and parallel compound manipulation in 384- and 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues.
Combinatorial Chemistry & High Throughput Screening	A Miniaturized Glucocorticoid Receptor Translocation Assay using Enzymatic Fragment Complementation Evaluated with qHTS. Zhu PJ, Zheng W, Auld DS, Jadhav A, MacArthur R, Olson KR, Peng K, Dotimas H, Austin CP, Inglese J. Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells [Fung, P., et al. Assay Drug Devel. Technol. 2006, 4(3): 263-272]. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC50 values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 uM screening concentration the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3? or 6? threshold, respectively. This corresponds to a confirmation rate of ~30% or ~50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.
European Pharmaceutical Review	HTS technologies to facilitate chemical genomics Auld DS, Inglese J, Jadhav A, Austin CP, Sittampalam GS, Montrose-Rafizadeh C, Mcgee JE Iversen PW. Industrial scale technologies developed and applied within the pharmaceutical industry for the purpose of drug discovery have recently been adopted by many research laboratories for the purpose of facilitating chemical genomics. Taking full advantage of these technologies will require education in highthroughput screening assay systems as well as new methods that exploit the capabilities of existing technologies.
Journal of Medicinal Chemistry	A high-throughput screen for aggregation-based inhibition in a large compound library. Feng BY, Simeonov A, Jadhav A, Babaoglu K, Inglese J, Shoichet BK, Austin CP. High-throughput screening (HTS) is the primary technique for new lead identification in drug discovery and chemical biology. Unfortunately, it is susceptible to false-positive hits. One common mechanism for such false-positives is the congregation of organic molecules into colloidal aggregates, which nonspecifically inhibit enzymes. To both evaluate the feasibility of large-scale identification of aggregate-based inhibition and quantify its prevalence among screening hits, we tested 70,563 molecules from the National Institutes of Health Chemical Genomics Center (NCGC) library for detergent-sensitive inhibition. Each molecule was screened in at least seven concentrations, such that dose-response curves were obtained for all molecules in the library. There were 1274 inhibitors identified in total, of which 1204 were unambiguously detergent-sensitive. We identified these as aggregate-based inhibitors. Thirty-one library molecules were independently purchased and retested in secondary low-throughput experiments; 29 of these were confirmed as either aggregators or nonaggregators, as appropriate. Finally, with the dose-response information collected for every compound, we could examine the correlation between aggregate-based inhibition and steep dose-response curves. Three key results emerge from this study: first, detergent-dependent identification of aggregate-based inhibition is feasible on the large scale. Second, 95% of the actives obtained in this screen are aggregate-based inhibitors. Third, aggregate-based inhibition is correlated with steep dose-response curves, although not absolutely. The results of this screen are being released publicly via the PubChem database.
Methods in Enzymology	Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate format. Auld DS, Johnson RL, Zhang YQ, Veith H, Jadhav A, Yasgar A, Simeonov A, Zheng W, Martinez ED, Westwick JK, Austin CP, Inglese J. Microtiter plate readers have evolved from photomultiplier and charged-coupled device-based readers, where a population-averaged signal is detected from each well, to microscope-based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536-well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high-throughput screening applications using laser-scanning microplate cytometry, we used green fluorescent protein- and yellow fluorescent protein-expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein-coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput.
Proceedings of the National Academy of Sciences	Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.
Analytical Chemistry	Streamlined System for Purifying and Quantifying a Diverse Library of Compounds and the Effect of Compound Concentration Measurements on the Accurate Interpretation of Biological Assay Results Popa-Burke IG, Issakova O, Arroway JD, Bernasconi P, Chen M, Coudurier L, Galasinski S, Jadhav AP, Janzen WP, Lagasca D, Liu D, Lewis RS, Mohney RP, Sepetov N, Sparkman DA, Hodge CN. As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.