Glycogen Synthase Kinase 3β Promotes the Endocytosis of Transferrin in the African Trypanosome

Human parasite Trypanosoma brucei proliferates in the blood of its host, where it takes up iron via receptor-mediated endocytosis of transferrin (Tf). Mechanisms of Tf endocytosis in the trypanosome are not fully understood. Small molecule lapatinib inhibits Tf endocytosis in T. brucei and associates with protein kinase GSK3 beta (TbGSK3 beta). Therefore, we hypothesized that Tf endocytosis may be regulated by TbGSK3 beta, and we used three approaches (both genetic and small molecule) to test this possibility. First, the RNAi knock-down of TbGSK3 beta reduced Tf endocytosis selectively, without affecting the uptake of haptaglobin-hemoglobin (Hp-Hb) or bovine serum albumin (BSA). Second, the overexpression of TbGSK3 beta increased the Tf uptake. Third, small-molecule inhibitors of TbGSK3 beta, TWS119 (IC50 = 600 nM), and GW8510 (IC50 = 8 nM) reduced Tf endocytosis. Furthermore, TWS119, but not GW8510, selectively blocked Tf uptake. Thus, TWS119 phenocopies the selective endocytosis effects of a TbGSK3 beta knockdown. Two new inhibitors of TbGSK3 beta, LY2784544 (IC50 = 0.6 mu M) and sorafenib (IC50 = 1.7 mu M), were discovered in a focused screen: at low micromolar concentrations, they prevented Tf endocytosis as well as trypanosome proliferation (GI(50)s were 1.0 and 3.1 mu M, respectively). These studies show that (a) TbGSK3 beta regulates Tf endocytosis, (b) TWS119 is a small-molecule tool for investigating the endocytosis of Tf, (c) endocytosis of GPI-anchored TfR and HpHbR are differentially regulated, and (d) the imidazopyridazine aminopyrazole scaffold of LY2784544 is attractive for a hit-to-lead optimization program in antitrypanosome drug discovery.