Development of a High-Throughput Screening–Compatible Assay for the Discovery of Inhibitors of the AF4-AF9 Interaction Using AlphaScreen Technology

Watson, Venita Gresham; Drake, Katherine M.; Peng, Yu; Napper, Andrew D.

doi:10.1089/adt.2012.495

Cited by 12 publications

(11 citation statements)

References 24 publications

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“…Alpha‐based assays provide a versatile and sensitive technique for characterizing PPIs as well as their modulators in an HTS format . They have the following technical advantages: (1) broad energy transfer distance (200 nm) compared with FRET (∼10 nm), which allows a greater range of analyte that can be studied, such as full‐length proteins and immuno‐complexes; (2) detection of interactions with a wide range of affinities ( K d values ranging from picomolar to millimolar); (3) ease of use.…”

Section: Biochemical Methodsmentioning

confidence: 99%

Current Experimental Methods for Characterizing Protein–Protein Interactions

2016

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Protein molecules often interact with other partner protein molecules in order to execute their vital functions in living organisms. Characterization of protein-protein interactions thus plays a central role in understanding the molecular mechanism of relevant protein molecules, elucidating the cellular processes and pathways relevant to health or disease for drug discovery, and charting large-scale interaction networks in systems biology research. A whole spectrum of methods, based on biophysical, biochemical, or genetic principles, have been developed to detect the time, space, and functional relevance of protein-protein interactions at various degrees of affinity and specificity. This article presents an overview of these experimental methods, outlining the principles, strengths and limitations, and recent developments of each type of method.

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Section: Biochemical Methodsmentioning

confidence: 99%

Current Experimental Methods for Characterizing Protein–Protein Interactions

2016

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“…The AF4-derived peptide of amino acid sequence PFWT was shown to inhibit AF9 when used in combination with established chemotherapeutic agents [261,262], while some non-peptidic inhibitor candidates have been identified by high-throughput screening [263]. The protein-tyrosine phosphatase 1B (PTP1B), a reticular non-transmembrane enzyme, has been validated as therapeutic target for diabetes, obesity, and breast cancer, due to its role as negative regulator of insulin and leptin signaling [264]. Protein-tyrosine phosphatase 1B has an elongated disordered carboxy-terminus, to which trodusquemine (Table 1) (MSI-1436, a natural product) binds [265].…”

Section: Known Drugs Acting On Idpsmentioning

confidence: 99%

Modulation of Disordered Proteins with a Focus on Neurodegenerative Diseases and Other Pathologies

Martinelli

Lopes

John

et al. 2019

IJMS

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Intrinsically disordered proteins (IDPs) do not have rigid 3D structures, showing changes in their folding depending on the environment or ligands. Intrinsically disordered proteins are widely spread in eukaryotic genomes, and these proteins participate in many cell regulatory metabolism processes. Some IDPs, when aberrantly folded, can be the cause of some diseases such as Alzheimer′s, Parkinson′s, and prionic, among others. In these diseases, there are modifications in parts of the protein or in its entirety. A common conformational variation of these IDPs is misfolding and aggregation, forming, for instance, neurotoxic amyloid plaques. In this review, we discuss some IDPs that are involved in neurodegenerative diseases (such as beta amyloid, alpha synuclein, tau, and the “IDP-like” PrP), cancer (p53, c-Myc), and diabetes (amylin), focusing on the structural changes of these IDPs that are linked to such pathologies. We also present the IDP modulation mechanisms that can be explored in new strategies for drug design. Lastly, we show some candidate drugs that can be used in the future for the treatment of diseases caused by misfolded IDPs, considering that cancer therapy has more advanced research in comparison to other diseases, while also discussing recent and future developments in this area of research. Therefore, we aim to provide support to the study of IDPs and their modulation mechanisms as promising approaches to combat such severe diseases.

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“…The pilot screen library consisted of the Spectrum Collection compound library that contains 2000 U.S. Food and Drug Administration (FDA)-approved drugs and bioactive natural products in DMSO (MicroSource, Inc., Gaylordsville, CT) and was reformatted into a 384-well format as described previously, 29 plus additional investigational compounds available in our compound collections.…”

Section: Compound Library and Pilot Screenmentioning

confidence: 99%

Implementation of a High-Throughput Pilot Screen in Peptide Hydrogel-Based Three-Dimensional Cell Cultures

Worthington

Drake

et al. 2019

SLAS Discovery

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Cell-based high-throughput drug screening (HTS) is a common starting point for the drug discovery and development process. Currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. However, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration challenging. Here, we used the peptide-based shear-thinning hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in three dimensions and showed a preliminary implementation of the scaffold within an automated HTS setup using a pilot drug screen targeting medulloblastoma, a pediatric brain cancer. A total of 2202 compounds were screened in the 384-well format against cells encapsulated in the hydrogel as well as cells growing on traditional two-dimensional (2D) plastic. Eighty-two compounds passed the first round of screening at a single point of concentration. Sixteen-point dose–response was done on those 82 compounds, of which 17 compounds were validated. Three-dimensional (3D) cell-based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment, getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide-based hydrogel into the high-throughput format.

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Development of a High-Throughput Screening–Compatible Assay for the Discovery of Inhibitors of the AF4-AF9 Interaction Using AlphaScreen Technology

Cited by 12 publications

References 24 publications

Current Experimental Methods for Characterizing Protein–Protein Interactions

Current Experimental Methods for Characterizing Protein–Protein Interactions

Modulation of Disordered Proteins with a Focus on Neurodegenerative Diseases and Other Pathologies

Implementation of a High-Throughput Pilot Screen in Peptide Hydrogel-Based Three-Dimensional Cell Cultures

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