Drug Target Deconvolution in Wolbachia: a Chemical Proteomic Route to Discovering Novel Antibacterial Targets
Lead Research Organisation:
University of Liverpool
Department Name: Chemistry
Abstract
Lymphatic filariasis and onchocerciasis are tropical parasitic worm infectious diseases that are leading causes of global disability. The control and elimination of these diseases is hampered by the lack of safe and effective drugs. Targeting an essential bacterial symbiont of the worm (Wolbachia) leads to death of the adult worms - an important advance over currently used treatments. The industrial and academic Anti-Wolbachia (AWOL) consortium has developed a Phase I candidate molecule (Tylosin A (TylAMacTM)) and an independent pre-clinical candidate (AWZ1066) that demonstrate cidal activity in Wolbachia. AWOL also discovered that fusidic acid, which is an antibiotic already used against skin infections, also demonstrates anti-Wolbachia activity. However, the specific targets of these compounds within the bacteria are unknown. Target deconvolution is necessary for determining the drug's mechanism of action, which could be utilised in further optimisation and provide tools to study potential adverse effects and resistance pathways.
In phenotype-based drug discovery, a variety of different techniques can be used to identify protein targets. Affinity chromatography purification, which is a type of chemical proteomics, is known to be the most widely employed target deconvolution technique in current research. In this approach, compounds are attached to solid support, smaller 'click chemistry' generated affinity tags or tags in combination with photoreactive groups (Fig. 1). The incorporation of photoreactive groups is beneficial as it induces the binding between the ligand and target protein. This can be achieved either by modifying the hit with a small photoreactive group and a reporter group, which is required for isolation, or by using 'all-in-one' functional groups that contain both components. During the deconvolution process, drug-loaded affinity matrices are used to isolate target proteins, non-binders are removed by washing and the hit-target complex is eluted using an excess of the active ligand which replaces the immobilised molecules. The protein-ligand complex can then be analysed by mass spectroscopy allowing identification of the target.
This project will utilise chemical biology techniques to identify the protein targets of AWZ1066, TylAMacTM and fusidic acid with the intention of validating this approach for the discovery of novel bacterial targets and tools to monitor resistance. Photoaffinity probe molecules will be designed and prioritised for synthesis through modelling of existing compound (>200) data sets to ensure good biological activity, using routes already established for these chemotypes. Once optimisation of the probes and confirmation of their activity against Wolbachia are completed, an affinity purification approach, following incubation with lysates will be performed in order to assess specific and non-specific binding and thus determine the proteins of interest. These enriched proteins will be identified and semi-quantified by a fully automated LC-MS/MS protocol complemented by bioinformatics, validating for the first time this target identification approach in Wolbachia. Initial modelling of TylAMacTM will be performed against the known bacterial ribosomal targets of the broader macrolide class with a view to homology based studies using a Wolbachia model post target identification.
In phenotype-based drug discovery, a variety of different techniques can be used to identify protein targets. Affinity chromatography purification, which is a type of chemical proteomics, is known to be the most widely employed target deconvolution technique in current research. In this approach, compounds are attached to solid support, smaller 'click chemistry' generated affinity tags or tags in combination with photoreactive groups (Fig. 1). The incorporation of photoreactive groups is beneficial as it induces the binding between the ligand and target protein. This can be achieved either by modifying the hit with a small photoreactive group and a reporter group, which is required for isolation, or by using 'all-in-one' functional groups that contain both components. During the deconvolution process, drug-loaded affinity matrices are used to isolate target proteins, non-binders are removed by washing and the hit-target complex is eluted using an excess of the active ligand which replaces the immobilised molecules. The protein-ligand complex can then be analysed by mass spectroscopy allowing identification of the target.
This project will utilise chemical biology techniques to identify the protein targets of AWZ1066, TylAMacTM and fusidic acid with the intention of validating this approach for the discovery of novel bacterial targets and tools to monitor resistance. Photoaffinity probe molecules will be designed and prioritised for synthesis through modelling of existing compound (>200) data sets to ensure good biological activity, using routes already established for these chemotypes. Once optimisation of the probes and confirmation of their activity against Wolbachia are completed, an affinity purification approach, following incubation with lysates will be performed in order to assess specific and non-specific binding and thus determine the proteins of interest. These enriched proteins will be identified and semi-quantified by a fully automated LC-MS/MS protocol complemented by bioinformatics, validating for the first time this target identification approach in Wolbachia. Initial modelling of TylAMacTM will be performed against the known bacterial ribosomal targets of the broader macrolide class with a view to homology based studies using a Wolbachia model post target identification.
Organisations
People |
ORCID iD |
| Gemma Nixon (Primary Supervisor) | |
| Monika Lisauskaite (Student) |