Center for Infectious Disease Research, Seattle, Washington, USA.
Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington, USA.
Antimicrob Agents Chemother. 2018 May 25;62(6). doi: 10.1128/AAC.00051-18. Print 2018 Jun.
In , calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (ICs) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (ECs) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 ICs and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar ECs were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.
在疟原虫中,钙依赖性蛋白激酶 1(CDPK1)是一种必需的蛋白激酶,对于入侵宿主细胞是必需的。我们已经开发了数百种 CDPK1 抑制剂,其中许多抑制剂可以阻断入侵。具有相似 50%抑制浓度(IC)的抑制剂在热转移测定中进行测试,以评估它们在细胞裂解物、完整细胞或纯化形式中稳定 CDPK1 的能力。抑制寄生虫生长的化合物在所有测定中均稳定 CDPK1。相比之下,两种表现出较差生长抑制的化合物在裂解物中稳定 CDPK1,但在细胞中不稳定。因此,细胞排除可以解释靶标作用与细胞活性之间相关性的例外情况。我们使用热转移测定法研究了两个通过在 CDPK1 抑制剂 RM-1-132 中生长独立选择的克隆中的 CDPK1,这两个克隆对该化合物的 50%有效浓度(EC)增加。A 和 C 克隆在 CDPK1 激酶结构域中分别具有独特的点突变,H201Q 和 L96P,这些残基在无活性同工型中彼此靠近。纯化的突变蛋白显示出与野生型 CDPK1 相似的 RM-1-132 IC 和热转移。仅在细胞热转移测定中观察到抑制剂稳定性降低(并且假定相互作用降低)。这突出了细胞热转移测定在证明耐药性涉及靶标结合减少(即使生化测定表明存在靶标结合)的有用性。事实上,正如在相应的药物选择寄生虫中观察到的那样,突变蛋白的过表达也观察到相似的 EC,尽管与高水平野生型酶相比,高水平的 CDPK1(H201Q)仅适度增加了耐药性。