Development of a NanoBRET screening assay for buruli ulcer drug leads with a novel mode of action Isobel Beard, Eftychia Mavrogiannaki, Daniel Whelligan, Rachel Simmonds University of Surrey, UK Mycolactone A/B (MycA/B) is the compound which confers virulence to Mycobacerium ulcerans which causes the neglected tropical disease Buruli ulcer. 1 Simmonds discovered that MycA/B’s mechanism of action is blockade of the protein channel Sec61, 2 which is responsible for the movement of proteins (translocation) into the endoplasmic reticulum (ER) for incorporation into the cell membrane or secretion from the cell. This blockade leads not only to cell death, which causes the characteristic skin necrosis, but also inhibition of inflammation and the immune response.
Figure 1: Structure of MycA/B We intend to semi-synthesise MycA/B (Figure 1) and derivatives with conjugable ‘handles’ for the attachment of fluorophores. The resulting probe molecules would permit intracellular (or microsomal) measurement of binding to Sec61 in a bioluminescence resonance energy transfer (BRET) assay, specifically using Promega’s NanoBRET biotechnology . The shortest literature syntheses of MycA/B require 13 steps for the core (in black), 3 17 for the fatty acid tail (in red) 4 and 2-3 more for coupling and deprotection. Furthermore, isolation of natural MycA/B from M. ulcerans is slow and hazardous. Alternatively, M. marium is a faster-growing, less hazardous bacterium which produces mycolactone F, 5 which has the same core structure but a different fatty acid southern chain. This project focuses on isolating MycF from bacterial cultures via an optimised extraction method. This is achieved by quantification of MycF through the process by LCMS. The extracted MycF will be hydrolysed to give the common core which will then be combined with the fatty acid southern chain of MycA/B synthesised by a novel route. The fatty acid tail will be broken into two main parts for synthesis, a penta-ene and a triol precursor. The penta- ene is synthesised by employing Burke’s iterative MIDA-boronate methodology involving bifunctional halide alkyene MIDA boronates which can be cross-coupled before deprotection of the boronate ready for another coupling reaction. The triol precursor is synthesised via a Horner-Wadsworth-Emmons reaction followed by a Sharpless asymmetric dihydroxylation. Current work is examining the diastereoselective coupling between the triol precursor (an aldehyde) and the penta-ene boronate to complete the fatty acid southern chain. References 1. Chany, A. C.; Tresse, C.; Casarotto, V.; Blanchard, N., Nat. Prod. Rep. 2013 , 30, 1527. 2. Hall, B. S.; Hill, K.; McKenna, M.; Ogbechi, J.; High, S.; Willis, A. E.; Simmonds, R. E., PLoS Pathogens 2014 , 10, e1004061. Brown, C. A.; Aggarwal, V. K., Chem. Eur. J. 2015 , 21, 13900. 3. Wang, G.; Yin, N.; Negishi, E., Chem. Eur. J. 2011 , 17, 4118. Ranger, B. S.; Mahrous, E. A.; Mosi, L.; Adusumilli, S.; Lee, R. E.; Colorni, A.; Rhodes, M.; Small, P. L., Infect. Immun. 2006, 74, 6037
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