Leishmaniasis is an endemic disease in tropical and subtropical regions of over 80 countries with more than 2 million new cases occurring annually www.who.int/tdr/diseases/leish/diseaseinfo.htm. Receptor for activated C kinase 1 (RACK1), a ubiquitous and highly conserved scaffold protein, binds to proteins involved in many signaling processes leading to cell survival, growth and differentiation. We have successfully generated short peptide regulators that either disrupt or enhance the interactions of RACK1 with these signaling proteins and thus regulate their biological effects. Little is known about the cellular function of the Leishmania homolog of RACK1, Leishmania homolog of receptors for activated C kinase, LACK. We believe that LACK, due to its highly conserved nature, is also a key scaffolding protein involved in essential signaling processes of the Leishmania parasite. We propose a novel approach, utilizing our well established methods of peptide design, to identify and characterize LACK- binding partners and to target protein-protein interactions between LACK and these Leishmaniasis- signaling enzymes. These peptides may serve as leads for the development of new therapeutics for the treatment of Leishmaniasis. Specifically, in this early feasibility proposal, we plan to: 1. Identify and characterize Leishmania signaling proteins that are LACK-binding partners (LACK-BP). 2. Rationally design peptides that selectively inhibit the interactions between the scaffold LACK and LACK-BP and test their activity towards Leishmania sp. proliferation and in vitro infection. To identify and characterize LACK-BP, we will utilize several methodologies including protein overlays (Far Western), in vitro pull-down assays, and expression interaction cloning. To design peptide modulators of protein-protein interactions, we will use sequences in RACK-BP that are required for RACK binding and identify homologous sequences in LACK-BP. Additionally, we will identify evolutionary conserved sequences in homologs of RACK-BP and use structural modeling to look for regions of homology for peptide design. Finally, we will identify short peptides derived from LACK that are likely to disrupt the binding and hence the signaling of LACK-BPs. The peptide modulators will be tethering to cell-permeable vectors and tested for their biological activity on proliferation of Leishmania promastigotes in culture and on Leishmania-infected murine macrophages. The peptides developed as part of the proposed project are likely to exert significant biological effects. Importantly, identification of LACK-binding proteins and peptides that interfere with their biological activities can help guide the development of new therapeutics for patients suffering from Leishmania infection. PROJECT NARRATIVE: Two million new cases of Leishmania infection are reported annually, with a resultant death rate of 3%. The current treatment for Leishmaniasis, chemotherapy, is highly toxic and ineffective. Our proposed studies will use a new approach to identify key players involved in Leishmania viability, allowing us to identify and create highly specific therapeutics that disrupt these cellular processes. This therapeutic approach is likely to provide a less toxic, highly specific and effective way of treating Leishmaniasis.
|Qvit, Nir; Schechtman, Deborah; Pena, Darlene Aparecida et al. (2016) Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors. Int J Parasitol Drugs Drug Resist 6:74-84|