There are over 20 pathogenic species of the genus Leishmania that are the causative agent for leishmaniasis. With each species causing a disease with different symptoms ranging from mild to severe cutaneous lesions to visceral leishmaniasis that is fatal if left untreated. Leishmania is spread by the bite of the phlebotomine sandfly. Inside the sandfly the parasite grows as promastigotes that are then transmitted to the mammalian host upon feeding of the sandfly vector. Inside the mammalian host the cells are engulfed by macrophages. The acidic environment of the phagosome combined with the increased temperature of the host induce differentiation to the amastigote stage. Amastigotes not only survive within the macrophage but proliferate. Leishmaniasis is mainly described as a disease of poverty since it infects people in mainly impoverished regions of the world. There are no vaccines available for Leishmania and the drugs used to treat it are either toxic or are too expensive for the resource poor victims of the disease, also drug resistance to some of the drugs is starting to be a problem in the field. Due to this, new drugs are needed for the treatment of leishmaniasis. Drug development against Leishmania is further complicated by the lack of conventional tools such as RNAi and condition gene knockouts. As a class, protein kinases provide an excellent class of new potential drug targets. Their ATP-binding site, while evolutionarily conserved, still contain enough diversity between protein kinases such that small molecule inhibitors can be isolated to a specific protein kinase. Over 37 drugs have been approved for use by the FDA targeting protein kinase. A vast majority of protein kinases tolerate mutation of a bulky amino acid at the back of the ATP-binding site. This amino acid referred to as the gatekeeper residue when changed to a smaller amino acid such as glycine or alanine opens up the back of the ATP-binding site such that it can bind a class of ATP-analogs containing large chains called bumped kinase inhibitors (BKIs). BKIs will selectively interact with the mutant protein since the other protein kinases all contain larger gatekeeper residues that exclude the BKI from the ATP binding site. These mutant proteins are referred to as being analog sensitive (AS). We propose generating AS-alleles for a number protein kinase in Leishmania, based on a list of known essential kinases in the related pathogen Trypanosoma brucei, and then replacing the wild-type endogenous genes with the AS-alleles. We will then identify a BKI that can bind the AS- allele using either an in vivo growth assay or an in vitro binding assay. Strains expressing AS-protein kinases will then be differentiated into amastigotes and infected into macrophages. We will then determine the essentiality of the protein kinase by adding the BKI to the infected macrophages and looking for growth defected of the amastigotes. Upon completion of this project we will have proven the technologies required to interrogate the kinome of Leishmania for new drug targets.
Leishmaniasis is a neglected pathogen with over a million cases reported each year, and with no vaccine available and drug resistance emerging for the drugs used for treatment, new drugs are need. Protein kinases have shown to be excellent drug targets for the development of new chemotherapeutic targets for cancer. We propose using the technologies developed for analyzing protein kinases in mammalian cells, namely the use of analog sensitive alleles of protein kinases, to begin the mining of the Leishmania kinome for new potential drug targets.
