In recent years, the problem of antibiotic resistance has been growing rapidly 1. Multi-drug resistant pathogens cause a heavy financial burden and reduce the efficiency of hospitals around the world. Part of the issue has been thought to be the limited number of bacterial processes that current antibiotics target. There is a need for novel antibiotics with new chemical scaffolds and mechanisms of action in order to alleviate this problem. Isoprenoids are a diverse group of molecules that are essential in all living organisms. All isoprenoids are synthesized from two universal precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) 2. There are two pathways in nature that synthesize these precursors: the mevalonate (MEV) pathway utilised, by most eukaryotes and archaebacteria, and the unrelated 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway used by many eubacteria (a notable exception being Staphylococcus aureus), by the plastids of plants, and the apicoplast of organisms such as Plasmodium falciparum (malaria). The essentiality of the DOXP pathway in a broad spectrum of pathogens, and the absence of it in humans makes it an ideal target for antibiotics since drugs targeting pathway.
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Only one inhibitor of the DOXP pathway, fosmidomycin, has been advanced into clinical trials to date. Fosmidomycin is also natural product isolated from in 1979 by the Fujisawa Pharmaceutical Co. 3. It has been shown to be effective against most Gram-negative and some Gram-positive bacteria in vitro and in mouse models. 4, 5 However, the clinical use of fosmidomycin has been limited due to its poor pharmacokinetics.6 With the great potential to be a novel and potent class of antibiotics, there has been an increasing interest in clinically-relevant inhibitors of the DOXP pathway. In recent years, groups have attempted many approaches to accomplish this, including designing substrate-based inhibitors7, synthesizing derivatives of fosmidomycin with better pharmacokinetics8, in silico screening9, 10, and high-throughput screening against individual enzymes of the pathway.11
Despite this concerted effort to find new inhibitors of this pathway, no new drugs have advanced into clinical trial. There is a need for new approaches in this field. Natural products, particularly from soil bacteria have historically been the best source chemical scaffolds for antibiotics, including penicillin.1 Since natural products have been optimized over years of evolution, they are often extremely potent and target the best antibacterial targets. In this project, a library, generated by our collaborator Dr. Gerry Wright, of ~5000 natural product extracts from soil bacteria grown in different conditions, will be screened on wild-type Bacillus subtilis for bioactive extracts. A counter-screen will be performed in parallel to determine if any contain DOXP pathway specific inhibitors (Fig. 2). This will involve a B. subtilis strain (EB2337) that a former lab member, Alexandra Perri, had engineered to express the enzymes of the S. aureus MEV pathway that convert mevalonate, an intermediate in the pathway, to IPP and DMAPP. These enzymes are under xylose control using the Psweet plasmid 12. The bioactives will be lethal to wild-type B. subtilis. However, when EB2337 is grown in media supplemented with xylose and mevalonate, which allows for an alternative route for isoprenoid biosynthesis, and rescues the B. subtilis from DOXP pathway specific inhibitors.
There are three advantages of this approach: 1) It will identify only DOXP pathway specific inhibitors, 2) Inhibitors of any of the DOXP pathway enzymes are screened simultaneously, and 3) Since the screen is performed on whole cells, only molecules able to enter the cell will be found.13 This approach overcomes traditional problems in whole cell screening where the target is not known, and enzyme-based screens where the inhibitors may fail to kill cells. After identifying the extracts with DOXP pathway specific inhibitors in the library, the hits will be validated and additional extract will be generated using the same growth conditions and extraction methods used to generate the extract in the library. To identify the active compound, bioactivity-guided purification will then be used.14, 15 This will involve the use of HPLC and different solvent gradients to generate fractions of the extract and testing each fraction for DOXP pathway specific inhibition. This process will be repeated with the active fractions until a pure active compound is obtained. NMR and mass spectrometry will be used to elucidate the structure of the natural product. The identification of a DOXP-specific inhibitor will greatly aid in efforts in combating multi-drug resistant pathogens. It will represent a novel antibiotic lead with a largely unutilised mechanism of action and a broad spectrum of activity.
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Figure 1 - The DOXP Pathway. The pathway begins with the transfer of acetaldehyde from pyruvate to glyceraldehyde-3-phosphate (1), catalyzed by DXS, to form 1-deoxy-D-xylulose 5-phosphate (2)2. IspC then catalyzes a rearrangement and subsequent reduction of (2) into 2-C-methyl D-erythritol 4-phophate (3). IspD, IspE, and IspF then catalyze the formation the cyclic, 2-C-methyl-D-erythritol-2-4-cyclodiphosphate (4). IspG and IspH subsequently convert (4) into IPP and DMAPP (5) through an unknown mechanism involving flavodoxin as a cofactor and likely free radical intermediates. Fosmidomycin is a known inhibitor of IspC, the first committed step in the DOXP pathway.