Sensitivity and safety

UNIVERSITY PARK, Pa.—Drug safety (and toxicity) remains one of the biggest concerns and stumbling blocks in development, with more than half of drugs failing in late-stage trials due to safety concerns not seen in in-vitro work. Some advancements for testing drug safety have focused on in-vivo models using lab-grown organoids, but the technology is still fairly young. A new method out of Penn State this quarter offers a different approach for assessing drug safety that could be safer and more sensitive. The work was covered in a paper titled “AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress,” which appeared in Angewandte Chemie International Edition.

“Drug-induced protein stress in cells is a key factor in determining drug safety,” said Xin Zhang, assistant professor of chemistry and of biochemistry and molecular biology at Penn State and senior author of the paper. “Drugs can cause proteins—which are long strings of amino acids that need to be precisely folded to function properly—to misfold and clump together into aggregates that can eventually kill the cell. We set out to develop a system that can detect these aggregates at very early stages and that also uses technology that is affordable and accessible to many laboratories.”

This approach is based on detecting stress on cells. While current methods also take this approach, they generally detect cell death, while the new technique is capable of detecting stress earlier in the process. The key is a fluorescent sensor that turns on in a cell when misfolded proteins start to aggregate, which is an early sign of stress. It features AgHalo, an unstable protein tagged with a fluorescent dye that becomes active in a hydrophobic environment. In normally folded proteins, any hydrophobic segments are generally hidden within the protein's structure. When the AgHalo protein starts to misfold and aggregate, with normally hidden sections exposed, the fluorescent dye interacts with the hydrophobic segments and lights up. While previous methods have also used sensors, they were always on, which meant protein stress could only be detected when the proteins aggregated.

The team tested the sensor by tracking the level of protein stress resulting from five common anti-cancer drugs. None of the drugs were noted to cause significant cell death in previous safety tests, the AgHalo sensor was able to detect some level of protein stress in the wake of all five drugs.

“Because we tested the anti-cancer drugs at much higher doses than typically used for treatment, our results do not necessarily call into question the continued use of these drugs,” explained Yu Liu, a postdoctoral researcher at Penn State and the first author of the paper. “However, because protein stress from long-term treatments could have lasting effects, evaluating drugs with our new sensor will help in the development of safer drugs.”

“An additional advantage of our system is that the level of fluorescence is correlated to the amount of protein aggregation in the cell, so we can quantify the level of stress,” he added. “Also, because our method measures the level of fluorescence, rather than having to identify the fluorescence under a microscope, it can be done using more accessible technology, like plate readers, and it is much more high-throughput.”

It's thought that there's also potential to use this technique in diseases such as Alzheimer's disease, which is hallmarked by the aggregation of misfolded proteins, or Parkinson's disease.

Other members of the research team include Matthew Fares, Noah P. Dunham, Zi Gao, Kun Miao, Xuenyuan Jiang, Samuel S. Bollinger and Amie K Boal at Penn State. The research was funded by a Burroughs Wellcome Fund Career Award at the Scientific Interface; the Paul Berg Early Career Professorship; the Lloyd and Dottie Huck Early Career Award; the U.S. National Institutes of Health; and the Searle Scholars Program. Additional support came from the Huck Institutes of the Life Sciences at Penn State.

Source: Penn State press release by Sam Sholtis