Bill Janzen is Professor and Director of Assay Development and Compound Profiling in the Center for Integrative Chemical Biology and Drug Discovery (CICBDD) within the Eshelman School of Pharmacy at The University of North Carolina (UNC) at Chapel Hill. Bill has 20 years of experience in innovative drug discovery and has been a leader in high throughput screening for lead generation in startup and large pharmaceutical ventures. Prior to joining the faculty of UNC at Chapel Hill, he was President and Chief Operating Officer of Amphora Discovery. In this position, Bill established and managed a team that developed a unique drug discovery process based on micro fluidic technologies, and ran over 100 high throughput screens to create an SAR quality database for lead discovery.
The CICBDD provides an important link between academic research and the drug development pipeline. The research culture of the University is based upon fostering innovation and nurturing new ideas – qualities that are essential for improving the success of early discovery efforts. The CICBDD provides an experienced and dedicated chemistry/biology team to drive projects from target identification through to lead or candidate discovery. As a result, this center enables translation of basic scientific discoveries into potential human therapeutics.
Epigenetics is an active area of research at the CICBDD. Scientists are studying the methylation of lysine and arginine residues present mainly in the flexible N-terminal tails that protrude from the core of histone proteins. The methylation patterns result in the recruitment of proteins containing domains that dynamically recognize and bind to these markers. This controls whether chromatin is in an open or closed state and ultimately turns the expression of certain genes on or off. Small molecule probes that disrupt the interaction between histone-binding proteins and chromatin would enable a systematic chemical/biological study of proteins that read the histone code, and could potentially reveal new targets for drug discovery. However, no such small molecules are currently known.
Researchers have recently used AlphaScreen assay technology to study histone-binding proteins in an effort to identify small molecules that can regulate chromatin structure and, ultimately, modulate gene expression.
Tim Wigle, a post-doctoral fellow working at CICBDD, says, “Consider this: Every cell in the human body has 1.8 meters of DNA in it, yet is somehow condensed into an area not visible to the eye. On top of this, every cell has the same copy of DNA, yet a glimpse inside the human reveals a complex and diverse array of cell types.” At the heart of this conundrum is chromatin, a sophisticated and dynamic packaging system comprising DNA wrapped around repeating units of histone protein octamers, collectively known as a nucleosome. Post translational modifications to these histone proteins control the way DNA associates with them, influencing the positioning of nucleosomes. This has a profound effect on the way the genome is accessed and ultimately dictates cellular fate. These modifications form a code that is not hardwired into the genome, yet is heritable and retained through rounds of cell division. Deciphering this code may unlock the potential to treat human diseases in an unprecedented and truly novel way.
Tim and his colleagues at CICBDD have developed an original high-throughput screening assay, based on AlphaScreen assay technology, which has been used to initiate a full-scale discovery effort of small molecule modulators of methyl-lysine recognition. Researchers have synthesized histone peptides containing methylated lysine residues and a terminal biotin group that allows the capture of streptavidin-coated donor beads. Histone-binding proteins were purified using affinity tags that were left intact following purification, providing a site for the binding of AlphaScreen Acceptor beads (i.e. a hexahistidine tag and nickel chelate acceptor beads). When these proteins are bound to their cognate peptides, they bring the donor and acceptor beads into proximity, resulting in the generation of an AlphaScreen signal (Figure 1).
Figure 1: Schematic diagram of the AlphaScreen assay used to monitor the interaction of histone binding proteins with their cognate peptides. His-tagged histone proteins were detected using PerkinElmer AlphaScreen nickel chelate Acceptor beads and streptavidin-conjugated Donor beads. Assays were read on a PerkinElmer EnVision® Multi-Label Reader.
CICBDD scientists have implemented this assay as a modular “plug and play” concept, where proteins and peptides are matched up by virtue of being able to generate an AlphaScreen signal and form screening pairs (Figure 2). As a result, they have generated a screening panel of histone binding proteins for the broad profiling of small molecules that antagonize methyl-lysine recognition.
Figure 2: Development of a histone-binding protein assay panel using the AlphaScreen platform. Grid titrations of various biotinylated histone peptides and His6-tagged methyl-lysine recognition domains were set up and evaluated for the generation of an AlphaScreen signal. Those that do form a peptide-protein pair are systematically screened against small molecule inhibitors in a dose response format and the selectivity and potency of these compounds is rapidly evaluated.
To find out more about the research of the Janzen lab, please visit
http://pharmacy.unc.edu/faculty-research/faculty-directory/billjanzen
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References
1. Wigle TJ, Herold JM, Senisterra GA, Vedadi M, Kireev DB, Arrowsmith CH, Frye SV and Janzen WP (2010). Screening for Inhibitors of Low-Affinity Epigenetic Peptide-Protein Interactions: An AlphaScreen™-Based Assay for Antagonists of Methyl-Lysine Binding Proteins. Journal of Biomolecular Screening, 15 (1): 62-71
The Janzen laboratory gratefully acknowledges the financial support of NIH, NCI, TraCS Institute, The UNC Carolina Partnership and the University Cancer Research Fund.