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Identification and Validation of Small- Gatekeeper Kinases as Drug Targets in Giardia lamblia

TitleIdentification and Validation of Small- Gatekeeper Kinases as Drug Targets in Giardia lamblia
Publication TypeJournal Article
Year of Publication2016
AuthorsHennessey KM
Secondary AuthorsSmith TR, Xu JW, Alas GCM, Ojo KK, Merritt EA, Paredez AR
JournalPLOS Neglected Tropical Diseases
Date Published11/2016
Type of ArticleResearch Article

Giardiasis is widely acknowledged to be a neglected disease in need of new therapeutics
to address toxicity and resistance issues associated with the limited available treatment
options. We examined seven protein kinases in the Giardia lamblia genome that are predicted
to share an unusual structural feature in their active site. This feature, an expanded
active site pocket resulting from an atypically small gatekeeper residue, confers sensitivity
to ªbumpedº kinase inhibitors (BKIs), a class of compounds that has previously shown good
pharmacological properties and minimal toxicity. An initial phenotypic screen for biological
activity using a subset of an in-house BKI library found that 5 of the 36 compounds tested
reduced trophozoite growth by at least 50% at a concentration of 5 μM. The cellular localization
and the relative expression levels of the seven protein kinases of interest were determined
after endogenously tagging the kinases. Essentiality of these kinases for parasite
growth and infectivity were evaluated genetically using morpholino knockdown of protein
expression to establish those that could be attractive targets for drug design. Two of the
kinases were critical for trophozoite growth and attachment. Therefore, recombinant
enzymes were expressed, purified and screened against a BKI library of >400 compounds
in thermal stability assays in order to identify high-affinity compounds. Compounds with
substantial thermal stabilization effects on recombinant protein were shown to have good
inhibition of cell growth in wild-type G. lamblia and metronidazole-resistant strains of G.
lamblia. Our data suggest that BKIs are a promising starting point for the development of
new anti-giardiasis therapeutics that do not overlap in mechanism with current drugs.

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