Genetic resistance to purine nucleoside phosphorylase inhibition in <i>Plasmodium falciparum</i>.

Abstract

<i>Plasmodium falciparum</i> causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and <i>P. falciparum</i> purine nucleoside phosphorylase (<i>Pf</i>PNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. <i>P. falciparum</i> cultured under incremental DADMe-ImmG drug pressure initially exhibited increased <i>Pf</i>PNP gene copy number and protein expression. At increased drug pressure, additional <i>Pf</i>PNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant <i>Pf</i>PNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing <i>Pf</i>PNP. Crystal structures of native and mutated <i>Pf</i>PNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2¹³⁶ clonal selection). Transition-state analog inhibitors against <i>Pf</i>PNP are slow to induce resistance and may have promise in malaria therapy.