Project 1: Screening for New Drug Targets
The increase of multidrug resistance among human bacterial pathogens is becoming alarming. Many antibiotics are loosing their potency even at very high doses and the need for new antibiotics is becoming urgent.
There are several ways to search for new antibiotics.
Use chemistry to modify existing drugs to make them more potent or counter resistance in bacteria.
- Screen for new compounds that target the same molecules.
- Identify new cellular molecules that could be good candidates for drug targets.
You have decided to use the third approach.
Most of the current antibiotics target protein synthesis and the ribosome in particular. You want to target a different part of the protein synthesis apparatus and decide to focus on aminoacyl-tRNA synthetases (AARSs).
At the end of the day you must convince a big investor that your selected target is promising and has all of the characteristics required of a good cellular target.
Try to Address the Following Questions
- What are the basic principles that would make AARSs (or any other enzyme) a good drug target for antimicrobials? Why target protein synthesis?
- How would you determine if the isoleucyl-tRNA synthetase is a good target for antimicrobials? List 5 characteristics that would make it a good target.
- Develop an assay that will allow you to select for small molecule inhibitors of isoleucyltRNA synthetase. This assay should be amenable to high throughput. Discuss the strength and weaknesses of an in vitro vs. in vivo assay.
- What are the essential controls that you need to have an effective screening assay? Bear in mind that a good drug must have low toxicity.
- What information and materials do you need to obtain for your preliminary experiments?
Project 2: Site-Specific Incorporation of L-Acetyl-Phenylalanine and L-Benzoyl-Phenylalanine into a Target Protein (X) in Mammalian Cells
Protein X has been identified as a potential target for several clinical drugs. It is part of a signal transduction pathway and, upon binding of a specific ligand, induces a definite cascade of downstream events. Protein X is ~200 amino acids long; its crystal structure has been solved, but fairly little is known about its localization within the cell and its interaction with other proteins. Therefore, it has been decided to address these questions using specific amino acid analogues incorporated at position 50 (a surface exposed residue) of protein X.
L-acetyl-phenylalanine (ap) and L-benzoyl-phenylalanine (bp) are tyrosine derivatives:
- ap carries a reactive keto group, which can be derivatized further to introduce a fluorescent tag to monitor the intracellular localization of the target protein during maturation
- bp allows both in vivo and in vitro crosslinking to study inter- and intramolecular interactions
Think about this project as a proposal to be submitted to a national funding agency, such as NIH. In addition to extensive background information, such a grant proposal requires a detailed description of the experiments that you are planning and a discussion of their possible outcome.
Try to Address the Following Questions
- Which basic principle are you using to introduce ap or bp site-specifically into a protein in mammalian cells? What are the basic requirements of the system?
- Can you use previous research as a useful starting point for your own project? Which paper(s) would you use as reference?
- What materials are needed for your project?
- How do you monitor the efficiency and specificity of incorporating these analogues into a protein in mammalian cells? Which control experiments are critical to clearly demonstrate the efficiency and specificity of the system?
- How do you prove the presence of ap or bp in position 50 of target protein X?
- What are the possible limitations of the system and can you think of strategies to overcome them?