Some targets are considered “undruggable” because what’s known about their structure says that they lack traditional binding pockets for small molecules or the native ligand binds too tightly to be out-competed. X-ray crystallography, the preferred way of discerning the structure of a protein, freezes a protein into one particular conformation.
But proteins are plastic, points out Joshua Salafsky, Ph.D., CSO of Biodesy: “You’re not able to see all the conformations it’s adopting under physiological conditions, and therefore, just using crystallography, you’re unlikely to find drugs that perturb the conformation in specific ways.
“There are likely transient pockets that open up that aren’t visible in the crystal structure that you would like to develop a molecule to bind to and stabilize a particular conformation of the protein, to render it inactive, for example. Or you’d like to develop an allosteric drug, again that binds to a pocket that may or may not be visible in the crystal structure.”
While a post-doc at Columbia University, Dr. Salafsky developed a novel way to detect biomolecules, based on a technique used in physics and physical chemistry research called second harmonic generation (SHG), by labeling them with SHG-active dyes.
Subsequently, at Biodesy, the company he founded, he developed this advance into a tool to monitor a protein as its conformation changes in real time. Upon excitation, immobilized biomolecules labeled with SHG-active dyes will re-radiate two photons of red light as a single photon of blue light (the “second harmonic”).
The key, he said, is that the amount of blue light produced is very sensitive to the orientation of the dye, and so “we can detect a very small shift in the average orientation of the probe due to protein conformational change.”
By labeling a protein at a specific amine or cysteine, different parts of the protein can be monitored.
Biodesy recently used this technique to identify activators and inhibitors of Ras activity. “We not only were able to show that these compounds in fact did change the conformation of Ras directly, but that the label site also told you whether conformation changed at that particular site,” Dr. Salafsky said.
"It was really exciting that the two active compounds in our hands that changed conformation of Ras were also the two inhibitors that other people had found in cell-based assays, and that the third compound that had no effect in the cell-based assays had no effect on the conformation, in our case, either when Ras was labeled at the cysteine or at the amines.”