White John, Dhingra Satish K, Deng Xiaoyi, El Mazouni Farah, Lee Marcus C S, Afanador Gustavo A, Lawong Aloysus, Tomchick Diana R, Ng Caroline L, Bath Jade, Rathod Pradipsinh K, Fidock David A, Phillips Margaret A
Departments of Chemistry and Global Health , University of Washington , 36 Bagley Hall, 400 15th Avenue NE , Seattle , Washington 98195 , United States.
Department of Microbiology & Immunology , Columbia University Irving Medical Center , 701 West 168th Street, HHSC 1502 , New York , New York 10032 , United States.
ACS Infect Dis. 2019 Jan 11;5(1):90-101. doi: 10.1021/acsinfecdis.8b00211. Epub 2018 Nov 13.
Malaria is one of the most challenging human infectious diseases, and both prevention and control have been hindered by the development of Plasmodium falciparum resistance to existing therapies. Several new compounds with novel mechanisms are in clinical development for the treatment of malaria, including DSM265, an inhibitor of Plasmodium dihydroorotate dehydrogenase. To explore the mechanisms by which resistance might develop to DSM265 in the field, we selected for DSM265-resistant P. falciparum parasites in vitro. Any of five different amino acid changes led to reduced efficacy on the parasite and to decreased DSM265 binding to P. falciparum DHODH. The DSM265-resistant parasites retained full sensitivity to atovaquone. All but one of the observed mutations were in the DSM265 binding site, and the remaining C276F was in the adjacent flavin cofactor site. The C276F mutation was previously identified in a recrudescent parasite during a Phase IIa clinical study. We confirmed that this mutation (and the related C276Y) accounted for the full level of observed DSM265 resistance by regenerating the mutation using CRISPR/Cas9 genome editing. X-ray structure analysis of the C276F mutant enzyme showed that conformational changes of nearby residues were required to accommodate the larger F276 residue, which in turn led to a restriction in the size of the DSM265 binding pocket. These findings underscore the importance of developing DSM265 as part of a combination therapy with other agents for successful use against malaria.
疟疾是最具挑战性的人类传染病之一，疟原虫对现有疗法产生耐药性阻碍了疟疾的预防和控制。几种具有新作用机制的新化合物正在进行治疗疟疾的临床开发，包括二氢乳清酸脱氢酶抑制剂DSM265。为了探索在野外可能对DSM265产生耐药性的机制，我们在体外筛选出了对DSM265耐药的恶性疟原虫。五种不同的氨基酸变化中的任何一种都会导致对寄生虫的疗效降低，并减少DSM265与恶性疟原虫二氢乳清酸脱氢酶（DHODH）的结合。对DSM265耐药的寄生虫对阿托伐醌仍保持完全敏感性。观察到的突变中除一个外都位于DSM265结合位点，其余的C276F位于相邻的黄素辅因子位点。C276F突变先前在一项IIa期临床研究中的复发病例寄生虫中被鉴定出来。我们通过使用CRISPR/Cas9基因组编辑技术重现该突变，证实了这种突变（以及相关的C276Y）导致了观察到的DSM265耐药性的全部水平。对C276F突变酶的X射线结构分析表明，附近残基的构象变化是为了容纳更大的F276残基，这反过来又导致了DSM265结合口袋大小的限制。这些发现强调了将DSM265开发为与其他药物联合治疗方案的一部分以成功对抗疟疾的重要性。
