Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

Abstract

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 purine nucleoside phosphorylase (PNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. cultured under incremental DADMe-ImmG drug pressure initially exhibited increasedPNP gene copy number and protein expression. At increased drug pressure, additional PNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. MutantPNPs 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 PNP. Crystal structures of native and mutatedPNPs 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 (2 clonal selection). Transition-state analog inhibitors against PNP are slow to induce resistance and may have promise in malaria therapy.

Enzymatic Transition States and Drug Design

“…When P. falciparum was cultured under incremental DADMe-ImmG drug pressure, drug resistance developed slowly. 439 Initial phases of drug resistance were caused by an increase in Pf PNP gene copy number (3–4-fold) and corresponding PNP protein expression. Increased drug pressure caused additional Pf PNP gene copies (up to 14-fold gene copy number), some of which contained point mutations Met183Leu (M183L) or Val181Asp (V181D) at catalytic site residues (Figure 82).…”

“…Clones have distinct boundaries for gene amplification, each containing the coding region for the PfPNP gene. Reproduces with permission from ref 439. Copyright 2018 National Academy of Sciences.…”

Transition-State Analogues of Campylobacter jejuni 5′-Methylthioadenosine Nucleosidase

“…The effect of HexS-DADMe-ImmA (HTDIA), BuS-DADMe-ImmA (BTDIA), 2-pyrazines-DADMe-ImmA (PTDIA), 5′-deoxy-5′-Pro-DADMe-ImmA (PDIA), and MeSDADMe-ImmA (MTDIA) on C. jejuni growth was used to establish IC values using liquid culture media. 46 In brief, C. jejuni cultures (200 μ L/well in a 96-well plates) with an initial optical density of 0.005 absorbance units ( λ = 600 nm) were grown in the presence of increasing concentrations of each compound. The transition-state analogue inhibitor concentrations were kept at a series of 3-fold dilutions, ranging from 1 nM to 20 μ M. Wells containing no inhibitor served as controls for their respective plates.…”

A resistant mutant of Plasmodium falciparum purine nucleoside phosphorylase uses wild-type neighbors to maintain parasite survival

“…DADMe-ImmG is a picomolar transition state analog (TSA) of PNP, which inhibits with a K d of 670 pM and has an IC 50 of 160 nM for parasites cultured in human erythrocytes ( 13 , 19 ). The crystal structure of Pf PNP in complex with DADMe-ImmG (PDB ID: 3PHC) shows hydrogen bond and ion-pair interactions with active site residues, phosphate, and structural waters ( Fig.…”