Improving scientific methods and tools is a key part of the NCATS approach to solving translational problems on a system-wide level, with the goal of delivering more treatments to more patients more quickly. A recent advance in this area — made possible through a project collaboration involving NCATS experts, academic researchers, a patient advocacy group and a pharmaceutical company — already has enabled a research team to identify a potential drug target for an inherited neurological disorder called Charcot-Marie-Tooth disease (CMT). The disease affects the peripheral nerves (i.e., the nerves outside the brain and spinal cord), and individuals with the CMT1A subtype, which affects approximately 1 in 2,500 people in the United States, experience progressive muscle weakness, movement problems, chronic pain and fatigue.
The CMT project involved the first combined use of a new type of assay (test) along with a version of high-throughput screening (HTS) developed at NCATS, called quantitative HTS (qHTS), to identify CMT1A therapeutic candidates. In qHTS, researchers use robotics to quickly conduct millions of chemical tests at multiple concentrations to identify compounds with therapeutic potential. Screening at multiple concentrations increases the probability of finding compounds with active pharmacology.
James Inglese, Ph.D., director of the NCATS Assay Development and Screening Technology Laboratory, and John Svaren, Ph.D., professor of comparative biosciences at the University of Wisconsin–Madison Waisman Center, led the work with funding from NCATS, the CMT Association (CMTA), the National Institute of Neurological Disorders and Stroke, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The group’s work was published in the September 2014 issue of ACS Chemical Biology.
The scientists were looking for agents that could reduce the activity of the gene PMP22, which is over-expressed in patients with CMT1A. To create an assay for this screen, the team, including CMTA-supported postdoctoral fellow Sung-Wook Jang, Ph.D., used a technique called genome editing to insert biological tools known as “reporter” genes directly into the DNA sequence of PMP22 in cells grown in culture. The reporter genes then produced detectable signals — in this case, light — when PMP22 was expressed.
This newly developed assay differs from previous versions in an important way. In earlier assays, scientists attached a reporter to a piece of the gene of interest that helps control its expression and then inserted the pair randomly into a cell’s DNA. In contrast, the new assay used targeted integration, meaning the group placed the reporter directly within the sequence of the gene of interest, making the reporter assay more sensitive to a wider range of mechanisms regulating gene expression and, thus, more biologically relevant. Targeted integration of reporter genes provides more assurance that a screen can identify more potential compounds that affect the expression of the gene under study.
Inglese, Svaren and colleagues showed that compared with the previous version of the assay, a screen using the new assay produced an expanded list of compounds that decreased PMP22 expression. One of those compounds reduced the gene’s activity through a previously unrecognized mechanism, known as the protein kinase C (PKC) pathway. This finding could spur further exploration of the PKC pathway as a target for potential therapies.
Notably, these results also validate the combined use of the targeted integration reporter assay with qHTS, which now can be used in future drug discovery efforts. “In close collaboration with Dr. Inglese’s group at NCATS, we’ve developed an assay that is suitable for screening even larger chemical libraries in pursuit of novel CMTA treatment targets,” Svaren said.
CMT patients will not be the only ones to benefit. “This work is a key proof-of-principle and provides a template for drug screens for other types of disorders,” Inglese said. According to the NCATS “3-Ds” philosophy, the team has developed a new approach and demonstrated its potential for improving the search for drug targets, with CMTA as the test case. Now, the group is disseminating this innovative tool to the broader scientific community. The scientists and CMTA have partnered with pharmaceutical company Sanofi-Genzyme to provide the new assay for screens of compound libraries that include more than 2 million small molecules. CMTA will pursue further testing of promising candidates that emerge from these screens and those conducted at NCATS, validating them in animal models and refining them for eventual studies in humans.
The team’s collaborative effort to create a state-of-the-art tool for drug discovery embodies the NCATS mission to develop innovative approaches that make translational science more efficient and accelerate the drug development process.
Posted November 2014