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Still, alterations in a cell's chemical environment alone might not necessarily determine the folding state of a particular protein. In some cases, other proteins, know as molecular chaperones, or even small synthetic molecules can act as molecular scaffolds to help drive a protein towards a final, properly folded structure. But the question for researchers interested in altering the amount of misfolded protein in a cell is whether or not they can “hijack” these chaperones for their own purposes.

Pharma meets folding

Michael Conn, Professor of Internal Medicine and Cell Biology-Biochemistry and Senior VP at Texas Tech University Health Science Center in Lubbock, Texas, is taking a more specific approach in his efforts to correct protein misfolding in cells. Conn works on a subset of small molecules that he has collectively termed “pharmacoperones,” or “pharmacological chaperones.” According to Conn, “these exceedingly specific molecules” can enter cells, target a misfolded protein for proper folding, and then traffic that protein to the correct location. Specificity for a target protein is what separates pharmacoperones from chemical chaperones, and that makes the therapeutic possibilities of pharmcoperones extremely interesting to scientists.

A dramatic example of this therapeutic promise came in 2013 when Conn and his colleagues reported the rescue and expression of a misfolded G-protein coupled receptor (GPCR), the gonadotropin releasing hormone (GnRHR) receptor, in a GnRHR mutant mouse using a pharmacological small molecule called IN3 (3).

“We selected peptidomimetics that we knew interacted with the GnRHR because they were small and hydrophobic and, so, could enter cells,” explains Conn. At the time, they decided to focus their efforts on antagonists to avoid activating the receptor. But that decision also meant that the pharmacology would be complex to interpret, and care had to taken to remove IN3 before testing receptor recovery. “The drugs [pharmacoperones] act to rescue the receptor but then have to be removed to allow endogenous GnRH to bind.”

The specific GnRHR mutation that Conn and his colleagues were studying was a single amino acid change, a negatively charged glutamic acid substituted with a positively charged lysine. The result of this single amino acid modification is a condition known as hypoganadotrophic hypogondism, which causes a loss of gonadal function and decreases in the levels of many gonadal hormones. Through a series of early experiments (see sidebar “Understanding cell biology with small molecules” on page 215), Conn's research team learned that the mutant GnRHR protein was not properly folding and was being misrouted in the cell, not ending up in the plasma membrane as needed for proper function. This was not to say the mutant protein could not function if properly folded, merely that it was misfolded and mislocalized in this experimental model. Using IN3, the team was able to rescue the GnRHR localization defect, driving the receptor to the plasma membrane (an indication of proper folding of the mutant GnRHR) and restoring proper testis function in the mutant mice.

IN3 is not the first example of a pharmacoperone-like or even a chemical chaperone-like molecule being applied for the treatment of diseases associated with misfolded proteins. But the results from Conn's group do add weight to the argument that small molecules might have broader utility when it comes to treating certain diseases, especially given the number of diseases believed to be the direct result of protein misfolding. The challenge for interested researchers, however, is finding other effective small molecules like IN3.

Hunting for small molecules

Conn obtained the IN3 small molecule for his GnRHR studies from the pharmaceutical company Merck, which was using it at the time for another purpose. It was known that IN3specifically targeted the GnRHR receptor, so it was a good starting point for Conn's studies. But if the general use and development of pharmacoperones and other small molecule chaperones is going to become widespread, there needs to be a more effective approach to their identification. Enter the high-throughput screen.

Once the realm of pharmaceutical companies with deep pockets, high-throughput, small molecule screens have become increasingly commonplace in academic research in recent years. “[There has been] a rise in academic high-throughput screening centers, which create more ready access to the infrastructure required to identify new molecules. While these capabilities have been present in the pharmaceutical and biotechnology realism for a long time, they can now be found at nearly all major research universities,” explains Jason Gestwicki, an associate professor in the Department of Pharmaceutical Chemistry at the University of California, San Francisco who works on the protein chaperone Hsp70. According to Gestwicki, once the infrastructure is in place, anyone with a good idea—whether fundamental or translational—can pursue molecule discovery.

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