Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) catalyzes an essential step in purine salvage for parasite growth. 4'-Deaza-1'- Aza-2'-Deoxy-1'-(9-Methylene)-Immucillin-G (DADMe- ImmG) is a transition state analog inhibitor of this enzyme, and P. falciparum infections in an Aotus primate malaria model can be cleared by oral administration of DADMe-ImmG. P. falciparum cultured under increasing DADMe-ImmG drug pressure exhibited PfPNP gene amplification, increased protein expression and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (∼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function o... More
Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) catalyzes an essential step in purine salvage for parasite growth. 4'-Deaza-1'- Aza-2'-Deoxy-1'-(9-Methylene)-Immucillin-G (DADMe- ImmG) is a transition state analog inhibitor of this enzyme, and P. falciparum infections in an Aotus primate malaria model can be cleared by oral administration of DADMe-ImmG. P. falciparum cultured under increasing DADMe-ImmG drug pressure exhibited PfPNP gene amplification, increased protein expression and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (∼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function of PfPNP. We hypothesized that M183L subunits may form mixed oligomers of native and mutant PfPNP monomers to give hybrid hexameric enzymes with properties conferring DADMe-ImmG resistance. To test this hypothesis, we designed PfPNP constructs that covalently linked native and the catalytically weak M183L mutant subunits. Engineered hybrid PfPNP yielded trimer-of-dimer hexameric protein with alternating native and catalytically weak M183L subunits. This hybrid PfPNP gave near-native K values for substrate but the affinity for DADMe-ImmG and catalytic efficiency were both reduced approximately 9-fold relative to a similar construct of native subunits. Contact between the relatively inactive M183L and native subunits are responsible for altered properties of the hybrid protein. Thus, gene amplification of PfPNP provides adequate catalytic activity while resistance to DADMe-ImmG occurs in the hybrid oligomer to promote parasite survival. Coupled with the slow development of drug resistance, this resistance mechanism highlights the potential for DADMe-ImmG use in antimalarial combination therapies.
