AbstractTrypanosomatid parasites are the causative agents of neglected tropical diseases for which current therapies are inadequate. As primitive eukaryotic organisms, they also represent a useful model system to investigate fundamental cellular biology while studies of potential drug targets endeavour to develop new drug molecules. Aspects of both of these areas are explored in this thesis.
Microtubules are polymers of tubulin and are essential in eukaryotes for cell division, motility and maintenance of cell morphology. Five tubulin-binding cofactors (TBC, named A-E) are proteins implicated in the folding, polymerisation and processing of tubulin, the major component of the trypanosomatid cytoskeleton. At the initiation of this study, there was no structural information available for any trypanosomatid TBC. We therefore sought to investigate these proteins by X-ray crystallography and assess their potential tubulin-interaction capabilities to support the current functional model. The crystal structure of tubulin-binding cofactor A (TBCA) from Leishmania major is presented, determined using diffraction data to 1.9 Å resolution. Prior to tubulin polymerisation, TBCA forms a complex with ß-tubulin in the pathway to aß-tubulin heterodimerisation. It maintains a soluble pool of ß-tubulin and can prevent premature polymerisation. This is a short helical protein, similar in structure to published homologues. The similarities and some distinct local features that may impact on ß-tubulin binding are discussed. In particular, the surface properties of a prominent bend in the helix bundle represents an area that may be capable of interacting with its tubulin partner.
Tubulin-binding cofactor C (TBCC) is implicated in stimulating the hydrolysis of GTP bound to ß-tubulin prior to release of the assembly-competent aß-tubulin heterodimer from a supercomplex between TBCC, TBCD, TBCE and both tubulin subunits. Full-length recombinant Trypanosoma brucei and Leishmania major tubulin-binding TBCC were degraded and crystallisation could not be achieved. However, crystals of a truncated TBCC construct were obtained. Despite efforts to optimise crystallisation and diffraction data, the structure was not solved for inclusion in this thesis. Instead, homologous structures were analysed and a potential tubulin interaction site is suggested based on the proposed GTPase-stimulating activity of TBCC and the similarity with the human protein, Retinitis Pigmentosa 2 (RP2), predicted to contain a domain with similar fold. Progress towards the soluble recombinant expression of the other cofactors also lays the foundation for future investigations into trypanosomatid TBC structure and function.
Pteridine reductase 1 (PTR1), an enzyme unique to trypanosomatids, is the subject of Part II of this thesis. PTR1 is a broad-spectrum NADPH-dependent reductase, catalysing the two-stage reduction of biopterin to dihydrobiopterin and tetrahydrobiopterin and that of folate to dihydrofolate and tetrahydrofolate. As such, it can provide a bypass mechanism for the reduction of folates, reducing the therapeutic action of traditional antifolate molecules in these organisms. Inhibition of PTR1 is therefore desirable from a drug discovery viewpoint. The crystal structure of Leishmania donovani PTR1 was determined using data extending to 2.5 Å resolution with a view to generating ligand-complex structures and providing a model for inhibitor design. This structure was found to contain a disordered active site, with several loop regions not modelled or relocated. A sulfate molecule from the crystallisation mixture binds in the cofactor phosphate binding-site and the sequential binding of cofactor before substrate or inhibitor can not occur. Although this crystal form was considered unsuitable for further studies, it provides the only structure of PTR1 in the absence of cofactor.
With an established crystallisation protocol, Trypanosoma brucei PTR1 then forms the basis of a collaborative investigation of over 100 novel potential inhibitory molecules. Kinetic evaluation, isothermal titration calorimetry (ITC) and co-crystallisation were applied to generate ligand-binding profiles of pyrrolopyrimidine derivatives. Several interesting binding features were identified from the 24 ligand complex structures obtained. These include the discovery of two covalent inhibitors, confirming the reactivity of a non-conserved active site cysteine, and molecules that are able to bind simultaneously at two locations within the active site pocket, exploiting hydrogen-bonding interactions with key catalytic and other nearby residues. The thermodynamic binding profiles of seven inhibitors also provide insight into the enthalpic and entropic contributions to ligand binding. We assessed the suitability of ITC for this system and while a high attrition rate was observed, chemical substitutions were able to enhance the binding entropy. These studies have strengthened our understanding of the structure-activity relationship between PTR1 and inhibitors, offering opportunities to develop new molecules that focus on increasing the potency generated by favourable enthalpy alongside improving the drug-like properties.
|Date of Award||2013|
|Supervisor||Bill Hunter (Supervisor)|