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Chemical screens generally search for either agonists or antagonists—looking for the up-or down-regulation of a particular cellular phenotype, say the activation of an ion channel or a membrane receptor protein. And while this approach has worked for many drug screens in the past, when it comes to finding a new pharmacoperone, such screens might not prove the most effective approach. “When screening for agonists and antagonists, it is likely that many molecules that regulate trafficking would have been missed,” notes Conn.

When hunting for a pharmacoperone or even a chemical chaperone with therapeutic potential, it's all about finding molecules that specifically stabilize a misfolded protein of interest and avoiding antagonists. This creates a challenge for developing endpoint assays since several compounds previously identified as potential pharmacoperones also act as antagonists, the likely reason few such screens have been described to date.

Screens for small molecules that affect protein folding need to start with proteins that are intentionally misfolded. In Conn's case, his team has generated stable cell lines expressing mutant versions of two different GPCRs for testing small molecules. For an endpoint, the researchers decided to assay effector coupling (such as IP or cAMP production) since this provides the most effective means of measuring receptor activation and signaling following proper refolding and membrane localization.

“It is becoming clear that many diseases are caused by misrouting of mutants of ion channels, receptors, and enzymes. This is likely a broadly applicable approach to a new class of therapeutic agents,” notes Conn, who is currently applying those mutant GPCR cell lines in a project to screen a 660,000 compound library at the new Scripps Research Institute campus in Jupiter, Florida.

But pharmacoperone screens are not the only misfolded protein screens that have been initiated in recent years; a number of other screens searching for small molecules that modify general protein folding activity have also been reported in the literature.

The chemistry-biology connection

In his Hsp70 work, Jason Gestwicki also takes advantage of small molecules. “Our focus is on finding new compounds that block, or promote, protein-protein interactions between Hsp70 and its co-chaperones,” explains Gestwicki. In 2008, Gestwicki, along with Susanne Wisen, described an early small molecule assay wherein firefly luciferase activity was used to monitor folding reaction progress. That study uncovered five new inhibitors of the protein DnaK, a bacterial ortholog of the human heat shock protein 70 (Hsp70), and one other molecule that seemed to promote folding (4).

According to Gestwicki, despite the challenges of identifying small molecules, they do present a unique opportunity to uncover fundamental biological insights. “An advantage of this approach is that the molecules might reveal how the Hsp70 system behaves after acute disruption of its equilibrium, before the system has time to compensate.” And when coupled with other molecular biology or genetic techniques, the information opportunity is even greater.

“It is very important to keep in mind that any small molecule-based project can, and should, be supported by results obtained using orthogonal methods, such as genetics,” notes Gestwicki. As an example, Gestwicki points to a case where an inhibitor of a protein and a catalytically dead mutant produce the same phenotype, providing one with tremendous confidence that the activity of that protein is important in the biology being uncovered.

Brent Stockwell is an associate professor in the Biological Sciences and Chemistry departments at Columbia University in New York City, New York, and an Early Career Scientist of the Howard Hughes Medical Institute. For years, Stockwell's lab has been using small molecule approaches to study cell death and neurodegeneration. In 2010, Stockwell and his colleagues reported in Nature Chemical Biology on a small molecule screenthey performed that identified five distinct compounds capable of preventing the cell death induced by a mutant huntingtin protein (5). Using this information and results from a series of complementary genetic techniques, Stockwell was able to confirm that the small molecules his team had identified were in fact acting on protein disulfide isomerase (PDI), a known protein chaperone. “PDI accumulation was leading to cell toxicity,” explains Stockwell. His work provided a new connection between apoptosis and protein chaperones.

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