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Probing the molecular mechanism of action of novel antimalarial falcipain 2 inhibitor endoperoxide pro-drugs in isolated parasite digestive vacuoles

Reference	BB/C006321/1
Principal Investigator / Supervisor	Prof. Paul ONeill
Co-Investigators / Co-Supervisors	Dr Patrick Bray, Professor Stephen Ward
Institution	University of Liverpool
Department	Chemistry
Funding type	Research
Value (£)	201,206
Status	Completed
Type	Research Grant
Start date	01/03/2005
End date	29/02/2008
Duration	36 months

Abstract

As HIV AIDS gains new (and deserved) attention and funds, it will be ironic if Western governments overlook malaria, an equally important and rapidly accelerating epidemic that has killed and disabled even more people in the past twenty years. Malaria is often dismissed as an ancient, largely vanquished affliction but its parasites have mutated into forms ever more resistant to the commonly available drugs. In much of sub-Saharan Africa, malaria deaths have doubled in the past decade. As a tractable solution to resistance development we have developed novel protease inhibitor peroxide hybrid pro-drugs that encapsulate and refine the concept of combination chemotherapy. Specifically, we have proven that under ferrous mediated conditions, our prototype antimalarial endoperoxides degrade by a designed cascade to produce two potentially active antimalarial compounds, a carbon centred radical that will almost certainly be toxic to the parasite and a chalcone molecule that is likely to inhibit the major malaria trophozoite cysteine protease enzymes. Using LCMS techniques, we have already proven that chalcone release takes place in the parasite digestive vacuole. In spite of the initial success of this research, we have yet to elucidate the molecular targets of the transient carbon radical species and formally demonstrate the inhibition of cysteine protease activity. These two important goals will be vital components of this proposal (see below). The selective parasite reductive cleavage of the endoperoxide pro-drug to an enone system and a secondary C-centred radical might suggest that this approach is relatively limited in the type of inhibitor that can be embedded within the pro-drug. On the contrary, mechanistic studies on the ferrous mediated decompositions of the 1,2,4-trioxane pharmacophore, has revealed that these systems readily degrade to carbonyl species in tandem with free radical production. The implications of this observation are that, in theory, any carbonyl containing antimalarial can be incorporated into an endoperoxide pro-drug unit by appropriate chemical design. The approach outlined in this proposal serves as a paradigm for a potentially generic approach to combination chemotherapy, we intend to focus on peroxides incorporating novel peptidic and peptidomimetic cysteine protease (falcipain 2) inhibitors, compounds with antimalarial effects at the level of the parasite digestive vacuole. The objectives of the proposal are: 1. To synthesise a series of carbonyl containing peptide and peptidomimetic inhibitors using standard amino acid coupling techniques. To prepare trioxane and trioxolane based pro-drugs from protease inhibitors using established techniques developed at Liverpool. To assay pro-drugs for antimalarial activity using routine techniques. 2. To determine the products of ferrous mediated degradation of these novel compounds and calculate carbonyl yield. To investigate the bioactivation of these endoperoxides in biomimetic haem-membrane model systems that are being developed in BBSRC Grant BBS/B/05508. 3. To spin-trap the C-centred radicals obtained during Fe(II) mediated degradation using the spin traps DBNBS and DMPO in collaboration with the National EPSRC Service Centre, Manchester. To extend studies to cell-culture EPR techniques and perform spin-tapping experiments in isolated parasite digestive vacuoles. 4. To employ dynamic confocal imaging to reveal the location of protease inhibition and free radical generation. 5. To track for the first time, the biological fate of the secondary carbon centred radical liberated from one of our prototype ECPI pro-drugs using a proteomic approach. This integrated interdisciplinary research will reveal conclusively, the molecular interactions responsible for bioactivation of these pro-drug molecules and will enable us to appraise this novel and potentially valuable approach to the chemotherapy of malaria.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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