“Identification and validation of drug targets within living organisms is the bottle neck in developing new drugs that are both effective and safe. Even for many medicines that have been previously developed and are currently being used, we do not know why they work and what might be responsible for their side effects,” said Dr. Xuewen Pan, assistant professor of biochemistry and molecular biology at BCM, and senior author of the report. “Another significant problem faced in medicine is drug resistance, which is responsible for most treatment failures. The causes for drug resistance are often difficult to pinpoint. This new technique is developed to effectively address both of these problems.”
Cause for drug resistance
The theory behind this technique is the fact that genetic variations in drug targets are often among the major causes for drug resistance.
“You have a mutation in a drug target that does not allow the drug to be effective, that is, to bind to the cell,” he said. “The cell becomes resistant. If we can find the gene(s) that cause resistance to the drug, we could well find its target(s). So the difficulty has been discovering drug resistance genes.”
The approach he and his colleagues developed allows them to find almost all genes that, when mutated or modestly overexpressed (causing high levels of protein), lead to drug resistance.
Mega-library of mutants
“We have made a mega-library of numerous mutants of the model organism baker’s yeast that affect almost all nucleotides of its genome. By mixing all the mutants together and throwing them against a drug, we can find almost all mutations that cause resistance,” he said. “It’s a very high throughput, systematic and unbiased approach.”
Using this tool, he and his colleagues discovered almost all the genes that, as a consequence of either mutation or modest overexpression, confer resistance to three drugs–rapamycin (an immune system suppressant), cycloheximide (an anti-bacterial drug) and amphotericin B (an antifungal agent).
“The next step is to develop a similar tool in human cells in the laboratory,” he said.
“This proof of principle study in yeast was remarkably successful. A similar tool in human cells will be more exciting but also more challenging to develop.”
Others who took part in this work include Zhiwei Huang, Kaifu Chen, Jianhuai Zhang, Yongxiang Li, Hui Wang, Dandan Cui, Xiaomin Shi, Wei Li, Dan Liu, Rui Chen and Richard S. Sucgang, all of BCM, and Jiangwu Tang and Yong Liu of Zhejiang Academy of Agricultural Sciences in Hangzhou, China. Huang is also with Donghua University in Shanghai, China.
Funding for this work came from the National Institutes of Health (NIH grants HG004840 to X.P. and 1S10RR026550 to R.C), the Dan L. Duncan Cancer Center (P30CA125123) and the Administrative and Genome-wide RNAi Screens Cores (IDDRC P30HD024064).