Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biological Sciences. / Includes bibliographical references (leaves 64-69).

Undergraduate honors paper--Mount Holyoke College, 2007. Program in Biochemistry. / Includes bibliographical references (leaves 54-58).

Trypanosome alternative oxidase (TAO) is the sole terminal oxidase responsible for the aerobic respiration of the parasite T. b. brucei. Specific strains of this parasite cause the neglected tropical disease Human African trypanosomiasis (HAT), and thus TAO is an interesting target for the potential treatment of this disease. Inhibition of TAO with the natural product inhibitors colletochlorin B or ascofuranone has been shown to clear infections of T. b. brucei in mice at high concentrations. However, these natural product inhibitors contain undesirable chemical functionality and have poor physicochemical properties, preventing adequate drug exposure to effectively treat HAT. Robust protocols for the expression and purification of recombinant TAO were developed, which enabled the development of biochemical assays to identify inhibitors of TAO function. Single point inhibition screening of the Medicines Malaria Venture 'kinetoplastid collection' of 400 compounds identified a range of micro-molar inhibitors of TAO. A program of chemical optimisation was carried out around the natural product inhibitor colletochlorin B, with the aim to improve the physicochemical properties and retain inhibitory potency against TAO. The structure activity relationships generated over the course of this exploration identified a dependency on high lipophilicity to retain potent TAO inhibition. The TAO inhibitors synthesised were also assessed for parasite growth inhibition and mammalian cell cytotoxicity to correlate inhibition data with cellular efficacy, in collaboration with Novartis. The physicochemical properties of these novel compounds showed improvement over the natural product colletochlorin B and prompted further assessment of leading compounds in advanced parasite kill kinetic and parasite clearance assays at Novartis. The data generated in these assays for compounds synthesised in this thesis determined that TAO inhibition results in a trypanostatic response, and not a preferred trypanocidal response in T. b. brucei.

No description available.

This thesis deconstructs the social, ecological, and colonial elements of the 1900-1910 Human African Trypanosomiasis (African Sleeping Sickness) epidemic which affected British Uganda and Belgian Congo. This paper investigates the epidemic’s medical history, and the subsequent social control policies which sought to govern the actions of the indigenous population. In addition, this paper argues that the failure to understand and respect the region’s ecological conditions and local knowledge led to disease outbreaks in epidemic proportions. Retroactive policies sought to inflict western medical practices on a non-western population, which resulted in conflict and unrest in the region. In the Belgian Congo, colonial authorities created a police state in which violence and stringent control measures were used to manage the local population. In Uganda, forced depopulation in infected regions destabilized local economies. This thesis compares and contrasts the methods used in these regions, and investigates the effects of Germ Theory on Sleeping Sickness policy and social perceptions during the colonial period in Africa.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds have the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds ave the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biological Sciences. / Includes bibliographical references (leaves 51-53).

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Biological Sciences. / Includes bibliographical references (leaves 46-50).

The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the ‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis respectively. In their human infective form in the bloodstream all three trypanosomatid parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6- bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set of structural and regulatory differences compared to the mammalian host enzyme. These differences, coupled with the availability of trypanosomatid PFK crystal structures present an opportunity for the structure-based design of specific inhibitors against the enzyme. Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’) were designed against T. brucei PFK with the aim of developing novel treatments against Human African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to trypanosomatid PFKs. We show that the compounds are specific; neither competing with the natural substrates ATP or F6P nor inhibiting the human PFK enzyme. We describe the development and testing of highly potent and specific low molecular weight PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage I HAT in mice models. We describe the tight, 1:1 binding of these compounds with trypanosomatid PFKs, and the thermodynamic characteristics of binding through various biophysical assays. We also show the unprecedented characterisation of the reverse PFK reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and inhibition by the drug-like compounds against trypanosomatid PFKs.