Overlooked Potential of EPSP Synthase as an Antimicrobial Target
The rise of antibiotic resistance has necessitated a need to discover novel drug candidates. We targeted an enzymatic protein found in Escherichia coli (E. coli) bacteria called 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is an enzyme found in the shikimate pathway that forms EPSP and is essential for the downstream synthesis of vital aromatic amino acids: tryptophan, tyrosine, and phenylalanine. Using high-throughput (HTS) screening, we targeted a library of 1000+ compounds against the target of interest to discover inhibitors that significantly limited cellular growth. M9 minimal media was used in HTS to render EPSPS essential for bacteria. This media does not contain the aromatic amino acids, which would otherwise make EPSPS unnecessary for survival. Thus, we rationalized that minimal media had to be used to avoid cell survival from external sources of amino acids. After conducting HTS and normalizing the data, 8 successful inhibitors of the enzyme were identified. However, these findings cannot tell us whether or not the compounds were targeting EPSPS or inhibiting cellular growth via another mechanism. Therefore, a series of secondary screens have been proposed to hone in the target specificity of these 8 hits towards the shikimate pathway, EPSPS, and shikimate-3-phosphate binding site, in the given order.
(2) Breithaupt, H. (1999). The new antibiotics. Nat Biotech, 17(12), 1165–1169.
http://doi.org/10.1038/70705 (accessed April 5, 2016).
(3) Maeda, H.; Dudareva, N. The Shikimate Pathway and Aromatic Amino Acid Biosynthesis in Plants. Annu. Rev. Plant Biol. 2012, 63, 73-105 (accessed April 5, 2016).
(4) Herrmann, K. M.; Weaver, L. M. The Shikimate Pathway. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999, 50, 473–503 (accessed April 5, 2016).
(5) George, J., Prasad, S., Mahmood, Z., & Shukla, Y. (2010). Studies on glyphosate-induced carcinogenicity in mouse skin: A proteomic approach. Journal of Proteomics, 73(5), 951 –964. http://doi.org/10.1016/j.jprot.2009.12.008 (accessed April 5, 2016).
(6) Ramachandran, V., Singh, R., Yang, X., Tunduguru, R., Mohapatra, S., Khandelwal, S., … Datta, S. (2013). Genetic and chemical knockdown: a complementary strategy for evaluating an anti infective target. Advances and Applications in Bioinformatics and Chemistry : AABC, 6, 1 –13. http://doi.org/10.2147/AABC.S39198 (accessed April 5, 2016).
(7) Eschenburg, S., Healy, M. L., Priestman, M. A., Lushington, G. H., & Schönbrunn, E. (n.d.). How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3- phosphate synthase from Escherichia coli. Planta, 216(1), 129–135. http://doi.org/10.1 007/s00425-002-0908-0 (accessed April 5, 2016).
(8) Zlitni, S.; Blanchard, J. E.; Brown, E. D. High-Throughput Screening of Model Bacteria. Methods Mol. Biol. 2009, 486, 13–27 (accessed April 5, 2016).
(9) Campbell, T. (2016). Lead Discovery: Planning and Implementing an HTS Campaign (accessed April 5, 2016).
(10) French, S. (2016). Data Normalization in High Throughput Screening (accessed April 5, 2016).
(11) Irvine, G. B. (2001). Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration). In Current Protocols in Cell Biology. John Wiley & Sons, Inc. Retrieved from http://dx.doi.org/10.1002/0471143030.cb0505s06 (accessed April 5, 2016).
(12) Herrmann, K. M. The Shikimate Pathway: Early Steps In The Biosynthesis Of Aromatic Compounds. Plant Cell. 1995, 7, 907-919 (accessed April 5, 2016).
(13) Feng, J., Chen, Y., Pu, J., Yang, X., Zhang, C., Zhu, S., Liao, F. An improved malachite green assay of phosphate: Mechanism and application. Anal. Biochem. 2011. 409, 144–149.
(14) Boocock, M. R., & Coggins, J. R. (n.d.). Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Letters, 154(1), 127–133. http://doi.org/10.1016/0014-5793(83)80888-6 (accessed April 5, 2016)