High-Throughput Screening and Triage Assays Identify Small Molecules Targeting c-MYC in Cancer Cells.

While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Förster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described.

Cell-Based Drug Discovery: Identification and Optimization of Small Molecules that Reduce c-MYC Protein Levels in Cells.

Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

Benzofuran-substituted urea derivatives as novel P2Y(1) receptor antagonists.

Benzofuran-substituted urea analogs have been identified as novel P2Y(1) receptor antagonists. Structure-activity relationship studies around the urea and the benzofuran moieties resulted in compounds having improved potency. Several analogs were shown to inhibit ADP-mediated platelet activation.

High-Throughput Cellular Thermal Shift Assays in Research and Drug Discovery.

Thermal shift assays (TSAs) can reveal changes in protein structure, due to a resultant change in protein thermal stability. Since proteins are often stabilized upon binding of ligand molecules, these assays can provide a readout for protein target engagement. TSA has traditionally been applied using purified proteins and more recently has been extended to study target engagement in cellular environments with the emergence of cellular thermal shift assays (CETSAs). The utility of CETSA in confirming molecular interaction with targets in a more native context, and the desire to apply this technique more broadly, has fueled the emergence of higher-throughput techniques for CETSA (HT-CETSA). Recent studies have demonstrated that HT-CETSA can be performed in standard 96-, 384-, and 1536-well microtiter plate formats using methods such as beta-galactosidase and NanoLuciferase reporters and AlphaLISA assays. HT-CETSA methods can be used to select and characterize compounds from high-throughput screens and to prioritize compounds in lead optimization by facilitating dose-response experiments. In conjunction with cellular and biochemical activity assays for targets, HT-CETSA can be a valuable addition to the suite of assays available to characterize molecules of interest. Despite the successes in implementing HT-CETSA for a diverse set of targets, caveats and challenges must also be recognized to avoid overinterpretation of results. Here, we review the current landscape of HT-CETSA and discuss the methodologies, practical considerations, challenges, and applications of this approach in research and drug discovery. Additionally, a perspective on potential future directions for the technology is presented.

Evidence for allosteric interactions of antagonist binding to the smoothened receptor.

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

M3 muscarinic acetylcholine receptor antagonists: SAR and optimization of bi-aryl amines.

Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M(3) muscarinic acetylcholine receptor antagonists such as 14 (pA(2)=11.0) possessing good sub-type selectivity for M(3) over M(2). The structure-activity relationships (SAR) and optimization of the bi-aryl amine series are described.

Identification via a Parallel Hit Progression Strategy of Improved Small Molecule Inhibitors of the Malaria Purine Uptake Transporter that Inhibit Plasmodium falciparum Parasite Proliferation.

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and ∼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.

Tetrahydro-4-quinolinamines identified as novel P2Y(1) receptor antagonists.

High-throughput screening of the GSK compound collection against the P2Y(1) receptor identified a novel series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet aggregation.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

A High-Content Imaging Screen for Cellular Regulators of ß-Catenin Protein Abundance.

Abnormal accumulation of ß-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, ß-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the ß-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to ß-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/ß-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect ß-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated ß-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce ß-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of ß-catenin degradation. This study demonstrates that detecting cell-based ß-catenin protein stability is a viable approach to identifying novel mechanisms of ß-catenin regulation as well as small molecules of therapeutic potential.

A novel approach applying a chemical biology strategy in phenotypic screening reveals pathway-selective regulators of histone 3 K27 tri-methylation.

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.

Identification of selective small molecule inhibitors of the nucleotide-binding oligomerization domain 1 (NOD1) signaling pathway.

NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.

Identification of benzimidazole diamides as selective inhibitors of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway.

NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.

The development of a high-content screening binding assay for the smoothened receptor.

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.

Development of a high-throughput cell-based assay for superoxide production in HL-60 cells.

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC(50) reproducibility and met a desired Z' value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 microM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

Discovery of novel and long acting muscarinic acetylcholine receptor antagonists.

High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.

Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation.

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.