Our proposal aims to target the Leishmania kinome for anti-parasitic drug development. This focused strategy will allow fast and cost-efficient progress beyond the state-of-the-art towards the identification of novel inhibitors for the following reasons:
(i) Leishmania protein kinases are validated as drug targets. Based on protein homology, the Leishmania major kinome contains 179 genes that encode for putative homologues to eukaryotic protein kinases (ePKs) and 17 that encode for atypical protein kinases [14]. A potentially important role of protein kinase networks in Leishmania virulence/fitness has been suggested based on biochemical and genetic evidence thus validating these signaling molecules as potential drug targets. For example, L.mexicana null mutants lacking the MAP kinase homolog LmxMPK1 were unable to establish mammalian infection [15], and deletion of the Leishmania protein-tyrosine phosphatase LPTP1 or over-expression of a mammalian homolog in L.donovani affected amastigote differentiation and virulence [16, 17]. Leishmania phosphoprotein abundance and phosphorylation pattern show dramatic changes during amastigote differentiation, which indicates the presence of stage-specific kinase and phosphatase activities [18, 19]. This hypothesis has been recently confirmed by consortium members (partner 1) for the Leishmania MAP kinase homolog LmaMPK10. The endogenous protein was constitutively expressed in both L.donovani promastigotes and axenic amastigotes but was phosphorylated specifically in amastigotes [6]. These data revealed the presence of a stage-specific kinase cascade composed at least of a higher order kinase that uses LmaMPK10 as substrate, which in turn may phosphorylate its down-stream targets. One major aim of LEISHDRUG is to define and exploit the unique structural features of LmaMPK10 and related LmaMPK7 for drug development (WP2, WP3, WP6). Identification of parasite-specific structural elements with druggability will provide important progress beyond the state-of-the-art.
(ii) We use innovative approaches for drug screening and target identification. We will use a highly multi-disciplinary strategy for anti-leishmanial drug development that has not been applied previously on parasitic systems. First, we will use a visual screening to discover compounds capable of killing intracellular Leishmania amastigotes without deteriorating the macrophage host cell (WP1). This approach for the first time enables the identification of inhibitors in situ under physiological conditions and simultaneously allows for the assessment of anti-parasitic activity and host cell toxicity. Adaptation of this unique and powerful approach to medium/high through put screening condition by developing appropriate robotics and automated high-content image processing will provide a major breakthrough in anti-microbial research.
Second, we will use a complementary target-based strategy utilizing recombinant Leishmania kinases in combination with small molecule inhibitor libraries. The novelty here lies in the process of target identification. We will apply a functional screen that will allow us to identify any parasite molecule with phosphotransferase activity in the Leishmania proteome (WP5). This approach is supported by in silico screens using novel bioinformatic tools that were developed and successfully applied by consortium members (WP6). These tools will be used to narrow down the list of potential compounds and identify the most promising targets among the Leishmania kinases. The identification of novel protein kinase families with limited or no homology to mammalian proteins will establish an important milestone towards the development of highly parasite-specific therapeutic tools.
Third, we will exploit phosphoproteomic data obtained by the consortium to develop a peptide-based strategy to anti-parasitic treatment. We will utilize cell penetrating peptides (CPPs) to deliver phosphopeptides that are recognized by Leishmania kinases, thus competing with the endogenous substrate and interfering with signaling processes relevant for intracellular parasite survival and virulence/fitness (WP3). Utilizing CPPs as universal delivery tools and targeting vital parasite signaling pathways by substrate mimics will open novel therapeutic possibilities beyond the current state-of-the-art.
(iii) Our approach benefits from the pharmaceutical interest on host protein kinases. The pharmaceutical industry has a growing interest in developing protein kinase inhibitors. Our project will be able to exploit this interest and will benefit from commercially available protein kinase inhibitor libraries, well established robust protein kinase assays suitable for high throughput screening, and structural data for a variety of host protein kinases available for comparative structure analysis and structure predictions of parasite kinases. Compared with other validated parasite drug targets, our focus on the Leishmania kinome allows us to build on prior knowledge allowing for highly cost and time efficient drug development.
In conclusion, LEISHDRUG will deliver considerable progress beyond the state-of-the-art utilizing strategies that combine
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