Phenotypic Cell-based Screening Research Articles

Predicting drug-induced liver injury by normalising liver microtissue high content screening data to in vivo tissue concentrations

Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1α and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

High-Throughput Screen for Inhibitors of Androgen Receptor-RUNX2 Transcriptional Regulation in Prostate Cancer.

Runt-related transcription factor 2 (RUNX2) plays a critical role in prostate cancer progression. RUNX2 interacts with the androgen receptor (AR) and modulates its transcriptional activity in a locus-specific manner. RUNX2 and AR synergistically stimulate a subset of genes, including the pro-oncogene snail family zinc finger 2 (SNAI2). AR-RUNX2 signaling cooperatively induces invasiveness of prostate cancer cells via SNAI2; and coexpression of AR, RUNX2, and SNAI2 in prostate cancer biopsy samples predicts disease recurrence. Competitive inhibition of AR alone could not disrupt the synergistic activation of SNAI2. We therefore established a phenotypic cell-based screening assay for compounds that could inhibit AR-RUNX2 synergistic activity either directly or indirectly. This assay was used to screen 880 compounds as a proof of concept, resulting in identification of several compounds that disrupted the synergistic stimulation of genes. Further investigation suggested the involvement of epidermal growth factor receptor (EGFR) signaling in AR/RUNX2 synergistic activity. Our assay is amenable to high-throughput screening and can be used to identify inhibitors of the AR-RUNX2 interaction in prostate cancer cells.

Abstract 3059: Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry

Abstract As part of a multi-year technology integration strategy to identify unprecedented targets, AbbVie has committed to building broad-endpoint profiling assays to enable phenotypic screening campaigns and compound prioritization. Early on, phenotypic cell-based screening employing a panel of protein-fragment complementation assays (PCAs) identified A-1107604 as a hit with potent activity against a subset of endpoints. While its activity profile had some commonalities with known anti-cancer agents, the overall profile of PCAs that were significantly and concomitantly modulated represented a unique signature. Further analysis revealed A-1107604 to have potent and selective activity in a panel of human tumor cell lines. Inhibitor affinity capture from cellular lysates coupled with mass spectrometry identified the BET family of proteins as the putative cellular targets of A-1107604. Binding was localized to the bromodomain of the target proteins using affinity capture-protease digestion and was confirmed by thermal shift assay, solution binding and X-ray crystallography. This binding was found to be highly selective when A-1107604 was counter-screened against a 150-member kinase panel and an 80-member receptor panel. To correlate target affinity with cellular efficacy, a series of analogs were prepared with affinities spanning 3 orders magnitude. Affinity for BRD4, a BET family member, strongly correlated with efficacy in human tumor cell lines. Finally, A-1107604 was evaluated in human tumor xenograft models where it demonstrated significant tumor growth inhibition. This discovery effort laid the foundation for our BET inhibitor program. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Scott E. Warder, Shaun M. McLoughlin, T. Matthew Hansen, Paul L. Richardson, Denise M. Wilcox, Sadiya N. Addo, Hua Tang, Chaohong Sun, Andrew M. Petros, Sanjay C. Panchal, Chang H. Park, M. Shannon Duggan, Melanie J. Patterson, F. Greg Buchanan, Dong Cheng, Heather M. Davis, David J. Calderwood, Steven W. Elmore, Yu Shen. Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3059.

Abstract 4701: A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds

Abstract Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. In an effort to identify novel targets that can reactivate hypermethylated silenced genes, our lab developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. By screening an NDL-3040 library and grouping the molecules based on chemical structures, we were able to identify three main drug classes. The Moulder Center then synthesized 60 new analogs based on class #1's lead's structure and 15 are positive in the YB5 system. The most potent analog can induce 10% GFP+ cells upon 500nM treatment. All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes can be reactivated upon drug treatment. Also, GFP positive cells show higher endogenous gene reactivation than unsorted and GFP negative cells. By performing RNA-seq analysis upon class #1 top lead treatment followed by connectivity mapping, we identified X as the class #1 drug target. The on-target effect can be validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Proliferation assays showed differential sensitivities of a panel of colon cancer cell lines compared to normal cells. These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Besides class #1 drugs, a novel class of LSD1 inhibitors was identified and the most active drug can induce 15% GFP at 5uM. Consistent with LSD1 inhibition, many known LSD1 target genes can also be upregulated. Like known LSD1 inhibitors, these compounds significantly inhibited proliferation of AML cells. We also identified some known natural compounds that have epigenetic activities, including arsenic trioxide, cardiac glycosides, and toyocamycin. Thus, many novel epigenetic drug classes derived from natural compounds were identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judit Garriga, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4701.

NMR Binding and Functional Assays for Detecting Inhibitors of S. aureus MnaA

Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated β-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore β-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P.

A BET inhibitor workaround

A phenotypic cell-based screen identifies an inhibitor of BET bromodomain transcriptional activity via inhibition of the alternative bromodomain-containing protein TAF1.

Discovery, Optimization, and Biological Evaluation of Sulfonamidoacetamides as an Inducer of Axon Regeneration.

Axon regeneration after injury in the central nervous system is hampered in part because if an age-dependent decline in the intrinsic axon growth potential, and one of the strategies to stimulate axon growth in injured neurons involves pharmacological manipulation of implicated signaling pathways. Here we report phenotypic cell-based screen of chemical libraries and structure-activity-guided optimization that resulted in the identification of compound 7p which promotes neurite outgrowth of cultured primary neurons derived from the hippocampus, cerebral cortex, and retina. In an animal model of optic nerve injury, compound 7p was shown to induce growth of GAP-43 positive axons, indicating that the in vitro neurite outgrowth activity of compound 7p translates into stimulation of axon regeneration in vivo. Further optimization of compound 7p and elucidation of the mechanisms by which it elicits axon regeneration in vivo will provide a rational basis for future efforts to enhance treatment strategies.

Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling.

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.

Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response.

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.

Identification and biological activities of a new antiangiogenic small molecule that suppresses mitochondrial reactive oxygen species

Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1α and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

Inhibition of Chk1 by the G2 DNA damage checkpoint inhibitor isogranulatimide

Inhibitors of the G(2) DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G(2) checkpoint inhibitor containing a unique indole/maleimide/imidazole skeleton identified in a phenotypic cell-based screen; however, the mechanism of action of isogranulatimide is unknown. Using natural and synthetic isogranulatimide analogues, we show that the imide nitrogen and a basic nitrogen at position 14 or 15 in the imidazole ring are important for checkpoint inhibition. Isogranulatimide shows structural resemblance to the aglycon of UCN-01, a potent bisindolemaleimide inhibitor of protein kinase C beta (IC(50), 0.001 micromol/L) and of the checkpoint kinase Chk1 (IC(50), 0.007 micromol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC(50), 0.1 micromol/L) but not protein kinase C beta. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3beta (IC(50), 0.5 micromol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimide. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu(85) and the amide nitrogen of Cys(87). Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu(17), causing a conformation change in the kinase glycine-rich loop that may contribute importantly to inhibition. The mechanism by which isogranulatimide inhibits Chk1 and its favorable kinase selectivity profile make it a promising candidate for modulating checkpoint responses in tumors for therapeutic benefit.

Small molecules that induce cardiomyogenesis in embryonic stem cells.

A phenotypic cell-based screen of a large combinatorial chemical library led to the identification of a class of diaminopyrimidine compounds (cardiogenol A-D) which can selectively and efficiently induce mouse embryonic stem cells (ESCs) to differentiate into cardiomyocytes. ESC-derived cardiomyocytes were shown to express multiple cardiac muscle markers, including myosin heavy chain, GATA-4, MEF2, and Nkx2.5, and spontaneously form beating regions. Such small molecules will serve as useful chemical probes to study cardiac muscle differentiation and may ultimately facilitate the therapeutic application of ESCs for cardiac repair.

Synthetic small molecules that control stem cell fate.

In an attempt to better understand and control the processes that regulate stem cell fate, we have set out to identify small molecules that induce neuronal differentiation in embryonic stem cells (ESCs). A high-throughput phenotypic cell-based screen of kinase-directed combinatorial libraries led to the discovery of TWS119, a 4,6-disubstituted pyrrolopyrimidine that can induce neurogenesis in murine ESCs. The target of TWS119 was shown to be glycogen synthase kinase-3beta (GSK-3beta) by both affinity-based and biochemical methods. This study provides evidence that GSK-3beta is involved in the induction of mammalian neurogenesis in ESCs. This and such other molecules are likely to provide insights into the molecular mechanisms that control stem cell fate, and may ultimately be useful to in vivo stem cell biology and therapy.