Droplet Microfluidics-Based Fabrication of Monodisperse Poly(ethylene glycol)-Fibrinogen Breast Cancer Microspheres for Automated Drug Screening Applications.

Spheroidal cancer microtissues are highly advantageous for a wide range of biomedical applications, including high-throughput drug screening, multiplexed target validation, mechanistic investigation of tumor-extracellular matrix (ECM) interactions, among others. Current techniques for spheroidal tissue formation rely heavily on self-aggregation of single cancer cells and have substantial limitations in terms of cell-type-specific heterogeneities, uniformity, ease of production and handling, and most importantly, mimicking the complex native tumor microenvironmental conditions in simplistic models. These constraints can be overcome by using engineered tunable hydrogels that closely mimic the tumor ECM and elucidate pathologically relevant cell behavior, coupled with microfluidics-based high-throughput fabrication technologies to encapsulate cells and create cancer microtissues. In this study, we employ biosynthetic hybrid hydrogels composed of poly(ethylene glycol diacrylate) (PEGDA) covalently conjugated to natural protein (fibrinogen) (PEG-fibrinogen, PF) to create monodisperse microspheres encapsulating breast cancer cells for 3D culture and tumorigenic characterization. A previously developed droplet-based microfluidic system is used for rapid, facile, and reproducible fabrication of uniform cancer microspheres with either MCF7 or MDA-MB-231 (metastatic) breast cancer cells. Cancer cell-type-dependent variations in cell viability, metabolic activity, and 3D morphology, as well as microsphere stiffness, are quantified over time. Particularly, MCF7 cells grew as tight cellular clusters in the PF microspheres, characteristic of their epithelial morphology, while MDA-MB-231 cells displayed elongated and invasive morphology, characteristic of their mesenchymal and metastatic nature. Finally, the translational potential of the cancer microsphere platform toward high-throughput drug screening is also demonstrated. With high uniformity, scalability, and control over engineered microenvironments, the established cancer microsphere model can be potentially used for mechanistic studies, fabrication of modular cancer microtissues, and future drug-testing applications.

In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators.

Understanding the mechanisms of modulators' action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17's broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New 'exosite' (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity.

A Novel Probe for Spliceosomal Proteins that Induces Autophagy and Death of Melanoma Cells Reveals New Targets for Melanoma Drug Discovery.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.

AlphaLISA-based high-throughput screening assay to measure levels of soluble amyloid precursor protein α.

Activation of nonamyloidogenic processing of amyloid precursor protein (APP) has been hypothesized to be a viable approach for Alzheimer's disease drug discovery. However, until recently, the lack of HTS-compatible assay technologies precluded large scale screening efforts to discover molecules that potentiate nonamyloidogenic pathways. We have developed an HTS-compatible assay based on AlphaLISA technology that quantitatively detects soluble APPα (sAPPα), a marker of nonamyloidogenic processing of APP, released from live cells in low volume, 384-well plates. The assay exhibited good QC parameters (Z'>0.5, S/B>2). A pilot screen of 801 compounds yielded a novel chemotype that increased the release of sAPPα 2-fold at 5µM. These results suggest that the AlphaLISA-based HTS assay is robust and sensitive and can be used to screen large compound collections to discover molecules that potentiate the release of sAPPα. Additionally, we demonstrated that increase of APP processing by nonamyloidogenic pathways will result in decrease of release of amyloidogenic Aß40 fragments.

Discovery of novel inhibitors of a disintegrin and metalloprotease 17 (ADAM17) using glycosylated and non-glycosylated substrates.

A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3-19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118-122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.

High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate.

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n approximately 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.

Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II).

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.