Identification and Mechanistic Understanding of Dihydroorotate Dehydrogenase Point Mutations in Plasmodium falciparum that Confer in Vitro Resistance to the Clinical Candidate DSM265.
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.
['White|John|J|', 'Dhingra|Satish K|SK|', 'Deng|Xiaoyi|X|', 'El Mazouni|Farah|F|', 'Lee|Marcus C S|MCS|', 'Afanador|Gustavo A|GA|', 'Lawong|Aloysus|A|', 'Tomchick|Diana R|DR|', 'Ng|Caroline L|CL|', 'Bath|Jade|J|', 'Rathod|Pradipsinh K|PK|', 'Fidock|David A|DA|', 'Phillips|Margaret A|MA|0000-0001-5250-5578']
D000595:Amino Acid Sequence D000818:Animals D000962:Antimalarials D001665:Binding Sites D064113:CRISPR-Cas Systems D018360:Crystallography, X-Ray D000090722:Dihydroorotate Dehydrogenase D004351:Drug Resistance D000072669:Gene Editing D006801:Humans D016778:Malaria, Falciparum D044925:Oxidoreductases Acting on CH-CH Group Donors D010963:Plasmodium falciparum D017354:Point Mutation D011743:Pyrimidines D014230:Triazoles
dihydroorotate dehydrogenase Plasmodium malaria drug resistance gene editing