One major challenge in drug discovery is determining whether a drug is behaving as intended, binding the “right” protein, inhibiting the correct target — when such events happen on a scale that is much too small to be seen. To overcome this problem, scientists have come up with various methods to indirectly observe biochemical changes, and they are always looking to refine and improve on these methods.
Protein phosphorylation is one particularly important and common biological event and Assay Depot lists many vendors offering techniques to measure protein phosphorylation. When a protein is phosphorylated, it picks up a phosphate “tag”, which is enough to help set up a whole cascade of changes in the body. Phosphorylation is key to letting a cell know when to grow, when to divide, and when to die. As can be imagined, abnormal phosphorylation is associated with a host of problems, including cancer, diabetes, and Alzheimer’s disease.
Consequently, in drug discovery considerable effort has been spent on compounds that affect kinases, the enzymes responsible for protein phosphorylation. But how to test them? As Bhalla et al. have pointed out, many different assays have been developed, but the authors argue that they either have drawbacks, such as toxicity or expense, or that their application is often limited .
In response, Bhalla et al. took advantage of one little fact about protein phosphorylation: Protons are released as a result of the reaction. The researchers devised two means to detect these protons. One way is to use electrolyte-insulator-semiconductor capacitor structures that detect protons via gate capacitance at the oxide-semiconductor interface. This is followed by a modulation in the gate bias voltage of the capacitor.
The second method is to use a commercially available micro-pH electrode. Even though the change in pH was small, because a buffered solution is needed to keep the protein from falling apart, the electrode is sensitive enough to pick up the minute change. Both of these approaches were successful in detecting phosphorylation of myelin basic protein by its kinase.
The authors imagine that these approaches will be useful in high throughput screening (when numerous different conditions in drug discovery are tested all at once), in order to improve the chances of finding something of interest. They even imagine a day when a subset of proteins are immobilised on the same microchip (a protein microarray), and the researchers’ detection systems could tell you which ones are phosphorylated by a kinase. If all goes well, this system may be an important tool in making drug discovery faster and more efficient.
1. Bhalla et al. 2014. Protein phosphorylation analysis based on proton release detection: Potential tools for drug discovery. Biosens. Bioelectron. 54:109-114.