Glycogen Synthase Kinase 3 Is a Potential Drug Target for African Trypanosomiasis Therapy

Abstract

Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 "short" (Tb10.161.3140) than for T. brucei GSK-3 "long" (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei. Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found.

High Throughput Screening against the Peroxidase Cascade of African Trypanosomes Identifies Antiparasitic Compounds That Inactivate Tryparedoxin

“…Structurally related compounds have been described as irreversible inhibitors of human glycogen synthase kinase 3 (GSK3-␤) (43). The cysteine residue that is putatively affected is conserved in the two GSK3 homologs of T. brucei (44). Both compounds 10 and 11 readily inhibited the peroxidase system in vitro but did not reveal any specificity for the parasite compared with HeLa cells in the cell-based assays.…”

Induced Resistance to Methionyl-tRNA Synthetase Inhibitors in Trypanosoma brucei Is Due to Overexpression of the Target

“…DNA was isolated from three different clones from 1312-resistant, 1433-resistant, and wild-type parasites using standard procedures (16). Each DNA sample (6 g per digestion) was digested with two different sets of restriction digestion enzymes (NsiI/EagI and NsiI/SacII) purchased from New England BioLabs.…”

“…At time zero, two 1433-resistant and two wild-type clones (10 3 cells/ml) with and without 1.9 M (ϳ10 times the baseline IC 50 ) 1433 were cultured in 5 ml in T-25 flasks. Total parasites were quantified at designated time points using the PerkinElmer ATPlite kit as previously described (16).…”

New Chemical Scaffolds for Human African Trypanosomiasis Lead Discovery from a Screen of Tyrosine Kinase Inhibitor Drugs

“…This result exceeded expectations because the drug was optimized against a human EGFR type 2 (HER2) (54); it was neither optimized against a trypanosomal protein nor tested in a phenotypic screen against T. brucei. Although T. brucei lacks HER2, lapatinib binds four trypanosome protein kinases (22), three of which are essential for the viability of the parasite (55)(56)(57). Therefore, it is conceivable that the drug modulates the activity of one or more of those enzymes in killing and controlling the trypanosome infection.…”