No description available.

No description available.

No description available.

Leishmania species are protozoan parasites which have a complex life cycle, which is coordinated with its cell cycle. There are 11 cyclin dependent kinases (CDKs) and 11 cyclins present in the Leishmania genome reflecting the complexity of cell cycle control in this parasite, perhaps due to the requirement for synchronisation with the life cycle. Leishmania mexicana CRK3, a cdc2-related serine/threonine protein kinase of the CDK family, is essential for transition through the G2-M-phase checkpoint of the Leishmania cell cycle. The Trypanosoma brucei homologue of CRK3, with 78% identity to L. mexicana CRK3, has been shown to form an active kinase complex with the CYC6 cyclin. Using this knowledge a putative mitotic cyclin, CYC6, from Leishmania major was identified. Monomeric CRK3 does not have protein kinase activity, but was activated in vitro with CYC6 to produce a protein kinase complex with histone H1 kinase activity. CRK3his and CYC6his were co-expressed and co-purified from Escherichia coli via metal affinity and gel filtration chromatography to obtain a 1:1 ratio of CRK3:CYC6 proteins, which formed a stable protein kinase complex. Using histone H1 as a substrate, active CRK3:CYC6 was used to develop a radiometric assay suitable for low to medium throughput compound screening and then an assay suitable for high throughput screening (HTS) using IMAPTM fluorescence polarization technology. This HTS assay was used to screen a 25,000 compound chemical library to identify hits which significantly reduced CRK3:CYC6 protein kinase activity. Two main pharmacophores with the highest potency towards CRK3:CYC6 protein kinase activity were identified from the high throughput screen. Structure Activity Relationship (SAR) analysis of the hits identified the chemical groups attached to the scaffold structures which are essential for the inhibition of CRK3:CYC6 protein kinase activity. The CRK3:CYC6 hits were subsequently counter-screened against a panel of 11 mammalian kinases including human CDK1:CYCB (the functional orthologue of CRK3:CYC6), human CDK2:CYCA and human CDK4:CYCD1 to determine their selectivity. Compound hits that were selective towards CRK3:CYC6, were tested against Leishmania in vitro. Progress towards synthesising potent and selective derivatives of the HTS hits will be discussed, with the view to evaluating their potential for the development of novel therapeutics against leishmaniasis.

Acanthamoeba castellanii is an amoeboid protozoan which causes opportunistic infections, including granulomatous encephalitis in immune-compromised patients. Haematogenous dissemination follows initial infection and the pathogen exhibits an ability to cross the blood-brain barrier (BBB). In the bloodstream and at the site of BBB penetration in the brain microvasculature A. castellanii is exposed to host humoral immunity. Here, we have provided insights into A. castellanii pathogenesis and the identity of amoeba antigens participating in immune control. We have investigated the role circulating immunoglobulin plays in preventing penetration of the BBB, and whether trophozoites can alter the efficacy of the immune response. Furthermore we have extended previously published data, demonstrating that amoeba proteases can degrade all antibody classes including physiologically-derived antibody. Nonspecific binding of polyclonal antibody was also observed, and attributed to Fc-binding activity by trophozoites. Additionally, we have examined the binding dynamics of A. castellanii under physiological conditions. BBB disruption was shown to be not directly linked to binding, instead it is reliant on secreted proteases. This study provides insights into mechanisms by which A. castellanii evades host immunity and crosses the BBB. This has the potential to enhance therapeutic strategies aimed at restoring essential disease prevention processes. In addition we have identified a number of amoeba antigens that are targets for the immune system and which may therefore be exploited through vaccination or immunotherapy.