Chambers Jeremy W, Fowler Matthew L, Morris Meredith T, Morris James C
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.
Mol Biochem Parasitol. 2008 Apr;158(2):202-7. doi: 10.1016/j.molbiopara.2007.12.013. Epub 2008 Jan 3.
Glycolysis is essential to the parasitic protozoan Trypanosoma brucei. The first step in this metabolic pathway is mediated by hexokinase, an enzyme that transfers the gamma-phosphate of ATP to a hexose. The T. brucei genome (TREU927/4 GUTat10.1) encodes two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. Our previous efforts have revealed that TbHK2 is an important regulator of TbHK1 in procyclic form parasites. Here, we have found through RNAi that TbHK1 is essential to the bloodstream form parasite. Silencing the gene for 4 days reduces cellular hexokinase approximately 60% and leads to parasite death. Additionally, we have found that the recombinant enzyme is inhibited by lonidamine (IC(50)=850 microM), an anti-cancer drug that targets tumor hexokinases. This agent also inhibits HK activity from whole parasite lysate (IC(50)=965 microM). Last, lonidamine is toxic to cultured bloodstream form parasites (LD(50)=50 microM) and procyclic form parasites (LD(50)=180 microM). Interestingly, overexpression of TbHK1 protects PF parasites from lonidamine. These studies provide genetic evidence that TbHK1 is a valid therapeutic target while identifying a potential molecular target of the anti-trypanosomal agent lonidamine.
糖酵解对于寄生原生动物布氏锥虫至关重要。该代谢途径的第一步由己糖激酶介导，这是一种将ATP的γ-磷酸基团转移至己糖的酶。布氏锥虫基因组（TREU927/4 GUTat10.1）编码两种己糖激酶（TbHK1和TbHK2），它们在氨基酸水平上有98%的同一性。我们之前的研究表明，在原循环型寄生虫中，TbHK2是TbHK1的重要调节因子。在此，我们通过RNA干扰发现，TbHK1对于血流型寄生虫至关重要。使该基因沉默4天会使细胞己糖激酶减少约60%，并导致寄生虫死亡。此外，我们发现重组酶受到氯尼达明（IC(50)=850微摩尔）的抑制，氯尼达明是一种靶向肿瘤己糖激酶的抗癌药物。该药物也抑制全寄生虫裂解物中的HK活性（IC(50)=965微摩尔）。最后，氯尼达明对培养的血流型寄生虫（LD(50)=50微摩尔）和原循环型寄生虫（LD(50)=180微摩尔）有毒性。有趣的是，TbHK1的过表达可保护原循环型寄生虫免受氯尼达明的影响。这些研究提供了遗传学证据，表明TbHK1是一个有效的治疗靶点，同时确定了抗锥虫药物氯尼达明的一个潜在分子靶点。
