Final Report Summary - DPRETB (Exploring decaprenyl-phosphoryl ribose epimerase (DprE1) as a validated target for TB drug discovery: assay development, high-throughput screening )
Tuberculosis (TB) is the leading cause of infectious disease mortality by a bacterial pathogen, Mycobacterium TB (Mtb), and was responsible for approximately 1.8 million deaths worldwide in 2007. Current therapeutic regimens for TB involve the use of at least four different drugs for at least 6 months. Mtb strains resistant to currently used drugs have emerged in recent years. Additionally, co-infection with Human immunodeficiency virus / Acquired immunodeficiency syndrome (HIV / AIDS) has brought further difficulties into TB therapeutics. No new drugs have been approved for TB in the last 40 years, and thus new drugs with novel mechanisms of action are urgently needed.
Mtb has a thick, impermeable cell wall which shields the bacterium from environmental stress, protects them from many drugs and plays key roles in the pathogenesis of TB infections. Arabinans, lipoarabinomannan and arabinogalactan are crucial structural components of the Mtb cell wall, containing arabinose sugars. Decaprenyl-phosphoryl arabinose (DPA) is produced by Mtb and serves as the sole arabinose donor for cell wall biosynthesis. Without DPA, Mtb bacteria are not able to survive. DprE1 is one of the enzymes responsible for the biosynthesis of DPA in Mtb. This enzyme is the target of BTZ043, a potent TB drug currently in the late stages of preclinical development. The main objectives of this project were to further understand the mode of action of BTZ043 and improve the knowledge on the structure and function of DprE1. Furthermore, by designing and synthesising fluorescent derivatives of BTZ043, we aimed to develop a new assay to test potential inhibitors of DprE1 and therefore provide tools for the development of new DprE1 inhibitors.
The research work of this project took place at the laboratories of Microbial Pathogenesis (Global Health Institute, Prof. Stewart Cole) and the Laboratory of Organometallic and Medicinal Chemistry (Institute of Chemical Sciences and Engineering, Prof. Paul Dyson), at EPFL. The main results are summarised in the following sections.
Crystal structure of DprE1
In order to have a detailed view and understanding of the way through which BTZ043 binds to and inhibits DprE1, we initiated crystallisation trials for recombinant DprE1. The protein was produced in E. coli. cells transformed with a plasmid vector containing the dprE1 gene. The protein was purified and subjected to crystallisation trials, initially in 96-well plate format, where about 1 200 crystallisation solutions with different compositions.
We attempted crystallisation of DprE1 alone and in presence of various inhibitors and substrates. A crystallisation condition was found that provided protein crystals of satisfactory quality. This condition was optimised by varying the composition of its components, and screening various additives. Finally, good quality crystals were obtained for DprE1 in complex with BTZ043, in presence of the ionic liquid tetrabutylphosphonium bromide. Diffraction data to 2.6 Å was obtained at the Swiss Light Synchrotron. The structure of the DprE1-BTZ043 complex was then solved, and showed how BTZ043 binds to DprE1 forming a covalent bond with a cysteine residue in the active site. These results were recently published in Science Translational Medicine: J. Neres et al. - Structural basis for benzothiazinone-mediated killing of Mycobacterium TB, Sci. Transl. Med. 4, 150ra121 (2012).
Synthesis and evaluation of fluorescent benzothiazinone analogues as imaging agents for M. TB
17 fluorescently-labelled benzothiazinone analogues were synthesised during this project. Several of these compounds retained significant killing activity against Mtb, considering the structural differences with BTZ043. One compound, JN29 (MIC of 6 µg/mL against M. TB) was selected for in vitro fluorescence imaging studies. Following incubation with culture medium containing JN29 at concentrations close to its MIC, M. TB bacteria became fluorescent and most exhibited higher intensity fluorescence spots at the poles. This is consistent with the expected cellular localisation of DprE1 in the cells, and therefore it is likely that JN29 selectively labels DprE1 in vitro. Further studies are currently ongoing to further explore JN29 and analogues for imaging of M. TB bacteria, in vitro and potentially in vivo.
Synthesis and evaluation of fluorescent benzothiazinone analogues as substrates for Fluorescent polarisation (FP) assays for DprE1
Some of the synthesised fluorescent benzothiazinones were evaluated as substrates for an FP assay for DprE1. One of these compounds is currently being studied with this purpose, and a novel FP assay will be available in the future, which will allow for high-throughput screening of libraries of compounds, in order to discover alternative DprE1 inhibitor structures.
Perspectives
Results from the research performed within the scope of this Marie Curie fellowship provided important information to better understand the mode of action of BTZ043, one of the most potent TB drugs under development. DprE1 is a fully validated drug target for anti-TB drug discovery, therefore the crystal structure of its complex with BTZ043, together with the biochemical characterisation of the enzyme, provide tools to discover novel inhibitors of this enzyme. Novel DprE1 inhibitors could potentially become novel TB drugs to be used in the clinic to eradicate this infectious disease.
Mtb has a thick, impermeable cell wall which shields the bacterium from environmental stress, protects them from many drugs and plays key roles in the pathogenesis of TB infections. Arabinans, lipoarabinomannan and arabinogalactan are crucial structural components of the Mtb cell wall, containing arabinose sugars. Decaprenyl-phosphoryl arabinose (DPA) is produced by Mtb and serves as the sole arabinose donor for cell wall biosynthesis. Without DPA, Mtb bacteria are not able to survive. DprE1 is one of the enzymes responsible for the biosynthesis of DPA in Mtb. This enzyme is the target of BTZ043, a potent TB drug currently in the late stages of preclinical development. The main objectives of this project were to further understand the mode of action of BTZ043 and improve the knowledge on the structure and function of DprE1. Furthermore, by designing and synthesising fluorescent derivatives of BTZ043, we aimed to develop a new assay to test potential inhibitors of DprE1 and therefore provide tools for the development of new DprE1 inhibitors.
The research work of this project took place at the laboratories of Microbial Pathogenesis (Global Health Institute, Prof. Stewart Cole) and the Laboratory of Organometallic and Medicinal Chemistry (Institute of Chemical Sciences and Engineering, Prof. Paul Dyson), at EPFL. The main results are summarised in the following sections.
Crystal structure of DprE1
In order to have a detailed view and understanding of the way through which BTZ043 binds to and inhibits DprE1, we initiated crystallisation trials for recombinant DprE1. The protein was produced in E. coli. cells transformed with a plasmid vector containing the dprE1 gene. The protein was purified and subjected to crystallisation trials, initially in 96-well plate format, where about 1 200 crystallisation solutions with different compositions.
We attempted crystallisation of DprE1 alone and in presence of various inhibitors and substrates. A crystallisation condition was found that provided protein crystals of satisfactory quality. This condition was optimised by varying the composition of its components, and screening various additives. Finally, good quality crystals were obtained for DprE1 in complex with BTZ043, in presence of the ionic liquid tetrabutylphosphonium bromide. Diffraction data to 2.6 Å was obtained at the Swiss Light Synchrotron. The structure of the DprE1-BTZ043 complex was then solved, and showed how BTZ043 binds to DprE1 forming a covalent bond with a cysteine residue in the active site. These results were recently published in Science Translational Medicine: J. Neres et al. - Structural basis for benzothiazinone-mediated killing of Mycobacterium TB, Sci. Transl. Med. 4, 150ra121 (2012).
Synthesis and evaluation of fluorescent benzothiazinone analogues as imaging agents for M. TB
17 fluorescently-labelled benzothiazinone analogues were synthesised during this project. Several of these compounds retained significant killing activity against Mtb, considering the structural differences with BTZ043. One compound, JN29 (MIC of 6 µg/mL against M. TB) was selected for in vitro fluorescence imaging studies. Following incubation with culture medium containing JN29 at concentrations close to its MIC, M. TB bacteria became fluorescent and most exhibited higher intensity fluorescence spots at the poles. This is consistent with the expected cellular localisation of DprE1 in the cells, and therefore it is likely that JN29 selectively labels DprE1 in vitro. Further studies are currently ongoing to further explore JN29 and analogues for imaging of M. TB bacteria, in vitro and potentially in vivo.
Synthesis and evaluation of fluorescent benzothiazinone analogues as substrates for Fluorescent polarisation (FP) assays for DprE1
Some of the synthesised fluorescent benzothiazinones were evaluated as substrates for an FP assay for DprE1. One of these compounds is currently being studied with this purpose, and a novel FP assay will be available in the future, which will allow for high-throughput screening of libraries of compounds, in order to discover alternative DprE1 inhibitor structures.
Perspectives
Results from the research performed within the scope of this Marie Curie fellowship provided important information to better understand the mode of action of BTZ043, one of the most potent TB drugs under development. DprE1 is a fully validated drug target for anti-TB drug discovery, therefore the crystal structure of its complex with BTZ043, together with the biochemical characterisation of the enzyme, provide tools to discover novel inhibitors of this enzyme. Novel DprE1 inhibitors could potentially become novel TB drugs to be used in the clinic to eradicate this infectious disease.