Montgomery Jemma A, Alday P Holland, Choi Ryan, Khim Monique, Staker Bart L, Hulverson Matthew A, Ojo Kayode K, Fan Erkang, Van Voorhis Wesley C, Doggett J Stone
Division of Infectious Diseases, Portland VA Medical Center, Portland, Oregon 97239, United States.
Division of Infectious Diseases, Oregon Health & Science University, Portland, Oregon 97239, United States.
ACS Infect Dis. 2025 Jun 13;11(6):1552-1562. doi: 10.1021/acsinfecdis.5c00051. Epub 2025 May 23.
A subset of bumped kinase inhibitors (BKIs) has been optimized to inhibit the calcium dependent protein kinase 1 (CDPK1; TGGT1_301440) of and pathogenic species. Extensive preclinical development of BKIs has identified BKI-1748 as a highly effective lead compound for toxoplasmosis. Phenotypic and therapeutic effects of BKIs have suggested that BKIs may have targets other than CDPK1. The mechanism of BKI-1748 action was further investigated using a forward genetic screen of chemical mutagenesis, selection of BKI-1748 resistant clones, and whole genome sequence analysis. Resistant clones were found to have single nucleotide changes in the ATP binding site of the mitogen-activated protein kinase-like 1 gene (MAPKL1; TGGT1_312570). One of the mutations found in the ATP binding pocket, Leu162Gln, was introduced into a wild-type strain, resulting in 2 to 6-fold resistance to BKI-1748 and a set of structurally varied BKIs. A strain with a CDPK1 gatekeeper substitution, Gly128Met, was also created and demonstrated a similar degree of resistance. The combination of MAPKL1 and CDPK1 substitutions together in a single strain resulted in a substantial increase in resistance to BKIs, up to 157-fold. The identification of MAPKL1 in addition to CDPK1 as a target of BKIs provides a greater understanding of the BKI mechanism of action that is important for further therapeutic development and suggests a high genetic barrier to meaningful drug resistance for this promising class of compounds.
一组经优化的激酶抑制剂（BKIs）能够抑制疟原虫和致病弓形虫的钙依赖性蛋白激酶1（CDPK1；TGGT1_301440）。BKIs广泛的临床前研究已确定BKI-1748是治疗弓形虫病的高效先导化合物。BKIs的表型和治疗效果表明，其靶点可能不止CDPK1一个。利用化学诱变正向遗传筛选、BKI-1748抗性克隆的筛选以及全基因组序列分析，进一步研究了BKI-1748的作用机制。发现抗性克隆在疟原虫丝裂原活化蛋白激酶样1基因（MAPKL1；TGGT1_312570）的ATP结合位点存在单核苷酸变化。在ATP结合口袋中发现的一个突变Leu162Gln被引入野生型菌株，导致其对BKI-1748和一组结构各异的BKIs产生2至6倍的抗性。还构建了一个CDPK1守门人替换为Gly128Met的菌株，其也表现出相似程度的抗性。在单个菌株中同时存在MAPKL1和CDPK1替换，导致对BKIs的抗性大幅增加，高达157倍。除CDPK1外，MAPKL1也被确定为BKIs的靶点，这有助于更深入地理解BKIs的作用机制，对进一步的治疗开发具有重要意义，并表明这类有前景的化合物产生有意义耐药性的遗传屏障较高。
