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5’ stem-loop is unique regulatory element of type I collagen mRNAs which regulates their translation by binding protein LARP6. Drugs that can directly interact with the 5’ stem-loop RNA sequence and interfere with LARP6 recognition can be regarded as truly specific antifibrotic drugs. This invention is to enable screening for such drugs by designing two sensor 5’ stem-loop RNAs which can report drug or protein binding. The sensors are modified by incorporation of a fluorescent nucleotide analog at the critical position in the active site to report the change in fluorescence intensity upon protein or drug binding. Each sensor RNA has only one nucleotide replaced by a fluorescent analog, but they are at different positions in the two sensors so that the sensor RNAs can detect a change in molecular environment at the two positions within the LARP6 recognition site. By measuring the fluorescence intensity of sensors, protein or drug binding to the active site can be detected and quantified. The main application of the sensors is for high throughput screening of compounds that directly interact with 5’ stem-loop RNA, as hits for developing truly specific antifibrotic drugs.
Fibrosis affects 45% of the population in the USA. It is characterized by excessive synthesis of type 1 collagen and scarring of various organs. This leads to organ insufficiency and death. The process is chronic and progressive and there are no approved drugs that can inhibit collagen synthesis. Aspects of the regulation of type 1 collagen production have been delineated and a drug screening procedure based on disruption of the regulatory pathway has been devised. Using this screening procedure a library of chemicals compounds has been screened and nine compounds that can inhibit collagen synthesis in cultured cells between 50-90% have been found.
This is a completely novel approach to finding antifibrotic drugs. If these compounds prove to be effective in humans, they will be the first chemicals that can directly inhibit collagen production. Since there is no cure for fibrosis, they may represent the first specific antifibrotic drugs.
This technology consists of two novel assay systems and three potential antifibrosis drugs.