Identification and Validation of Small-Gatekeeper Kinases as Drug Targets in Giardia lamblia.
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 "bumped" 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.
Department of Biology, University of Washington, Seattle, Washington, United States of America.
Kelly M
KM
Hennessey
Division of Allergy and Infectious Diseases, Center for Emerging and Re-emerging Infectious Disease (CERID), University of Washington, Seattle, Washington, United States of America.
Tess R
TR
Smith
Department of Biology, University of Washington, Seattle, Washington, United States of America.
Jennifer W
JW
Xu
Department of Biology, University of Washington, Seattle, Washington, United States of America.
Germain C M
GC
Alas
Division of Allergy and Infectious Diseases, Center for Emerging and Re-emerging Infectious Disease (CERID), University of Washington, Seattle, Washington, United States of America.
Kayode K
KK
Ojo
Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
Ethan A
EA
Merritt
Department of Biology, University of Washington, Seattle, Washington, United States of America.
Alexander R
http://orcid.org/0000-0002-9298-3264
AR
Paredez
D020816:Amino Acid Motifs; D000981:Antiprotozoal Agents / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain; D055808:Drug Discovery; D016829:Giardia lamblia / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005873:Giardiasis / Q000469:parasitology*; D006801:Humans; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000981:Antiprotozoal Agents; D047428:Protein Kinase Inhibitors; D015800:Protozoan Proteins; D011494:Protein Kinases