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Chemical Biology for Tuberculosis Research

Final Report Summary - CHEMBIO4TB (Chemical Biology for Tuberculosis Research)

Small molecule drugs against pathogenic organisms generally act by binding one or more protein targets in these organisms which are essential for their survival. The identification of these protein targets is crucial for the understanding of the mechanism of action of a certain drug and for the development of new drugs against a pathogen.

This project aimed at identifying new potential drugs and drug targets against Mycobacterium tuberculosis, the causative bacterium of tuberculosis (TB). Tuberculosis is a worldwide menace, and the drugs that are applied for the treatment of the disease have already been in use for decades. An urge exists for the identification of new drug targets against Mycobacterium tuberculosis, as multidrug resistant strains readily emerge during the conventional medication. To achieve this goal we proposed to establish a system based on yeast (the so-called yeast-three-hybrid, Y3H) that allows the identification of mycobacterial proteins that bind to selected small molecules. The main advantages of this system are the following:

(i) it enables the detection of drug-protein interactions in living cells;
(ii) the use of a non-pathogenic organism for the screening of small molecule-protein interactions of proteins of a pathogenic bacterium; and
(iii) the system is 'low-tech' and does not require expensive machines as for example mass spectrometers.

In this simple approach, the interaction of a protein of interest, which is a member of a protein library, and a certain drug leads to growth of yeast colonies. Every yeast colony carries a different DNA insert encoding for a different protein of the library. The library has been constructed by cutting the genomic DNA of the mycobacterium into random fragments. These fragments cover statistically every possible gene on the genome of Mycobacterium tuberculosis. The cut DNA is then inserted in a shuttle, enabling them to enter the yeast cells which have been engineered for the Y3H.

Having this tool in hand, we employed our Y3H system to a diverse set of 'candidate' molecules, which have been modified by chemical synthesis to be suitable for the Y3H screening. This set comprises known TB drugs, drug leads that we received from companies working on TB, and natural products or clinically approved drugs used to treat different diseases, all of them having a reported activity against Mycobacterium tuberculosis.

The performed screenings yielded several hits, which were further characterised. Two of them could be confirmed by affinity enrichment, a different strategy for target identification proving the reliability of our system. Furthermore, a possible new drug target against TB could be identified. This putative new target will be subject of further studies and focus of collaborations within the European Commission (EC) initiative 'More molecules for tuberculosis' (MM4TB), a network of researchers working to combat the disease.

The successful establishment of a Y3H system as a complementary approach for target identification of mycobacteria adds a new method to the 'toolbox' for drug discovery against tuberculosis. Due to the MM4TB network, we expect to receive further drug candidates for our Y3H system, which will establish the Y3H screenings as valuable tool for drug discovery against tuberculosis.

The establishment of a system for drug discovery that uses genomic random fragment libraries constructed from bacteria for target identification is a general method and not restricted to mycobacteria. We could envision constructing libraries from other pathogens or interesting organisms for use in our Y3H screenings.