8 January 2007. High-throughput screening for small molecule compounds that prevent neurodegeneration is a relatively new undertaking but growing rapidly. A paper published online in Nature Chemical Biology on the last day of 2006 describes a cell-based screening scheme for the Huntington disease protein. Brent Stockwell of Columbia University in New York and various collaborators powered through a 43,685-compound library to come up with four new hits that may be useful as both mechanistic probes and potential drug leads. Might this example encourage some Alzheimer researchers to make a New Year’s resolution to devise some new and clever assays to find the needle in their own haystacks?
Lead author Hemant Varma, a postdoc in the Stockwell lab, set up the screen to find inhibitors of the cytotoxic actions of polyglutamine-expanded huntingtin protein, the cause of Huntington disease. Varma started with the immortalized striatal neuron line ST14A developed by coauthor Elena Cattaneo at the University of Milan in Italy (Cattaneo and Conti, 1998), and a matching line stably transfected with an N-terminal 548-amino-acid fragment of a mutant huntingtin sporting 120 glutamine repeats. By measuring viability in a high-throughput format, they were able to identify 29 compounds that blocked cell death in the mutant-bearing cells, but did not affect the parental line.
To further characterize the hits, they used additional HD models: coauthor Anne Hart from Harvard Medical School in Boston helped design a C. elegans-based screen, while Donald Lo from Duke University contributed a brain slice HD model. They also tested the compounds in huntingtin-expressing PC12 cells and yeast, narrowing the field to four candidates that were active in at least one of the models.
Since the compounds selectively blocked huntingtin toxicity, the investigators used them to probe the mechanism of cell death. Their results suggest that caspase 3 activation was to blame, a result that agrees with other studies suggesting that abnormal caspase 3 activity may be important in HD (Rigamonti et al., 2000; Sanchez Meija and Friedlander, 2001).
Three of the four most active compounds passed medicinal chemistry muster, their structures suggesting they would be suitable for taking by mouth. The investigators tested 57 analogs of these three, identifying relatives that maintained activity, and defining a jumping-off point for making new versions with higher potency.
Other high-throughput screens for Huntingtin toxicity have focused on protein aggregation rather than cell death (see ARF related news story), and it’s unclear which endpoints will yield therapeutically useful hits. Fortunately, the number of endpoints amenable to high-capacity assays in neurons is increasing all the time—besides protein aggregation or cell death, screens can track protein levels, cellular localization, or protein or cell function (for a recent review, see Stockwell, 2002). That flexibility will open up assay opportunities in other diseases—the Stockwell lab is currently working on spinal muscular atrophy and ALS target proteins, in addition to huntingtin.—Pat McCaffrey.
Varma H, Voisine C, Demarco CT, Cattaneo E, Lo DC, Hart AC, Stockwell BR. Selective inhibitors of death in mutant huntingtin cells. Nat Chem Biol. 2006 Dec 31; [Epub ahead of print] Abstract