Plasmodium falciparum parasites are killed by a transition state analogue of purine nucleoside phosphorylase in a primate animal model

Abstract

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.

Figure 1. Purine and polyamine metabolisms in P. falciparum-infected human erythrocytes.

Purine pathway: AMP, adenosine 5′-monophosphate; ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; IMP, inosine 5′-monophosphate; XMP, xanthosine 5′-monophosphate; GMP, guanosine 5′-monophosphate; MTA, methylthioadenosine; MTI, methylthioinosine; AdS, adenylosuccinate; hADA, human adenosine deaminase; hPNP, human purine nucleoside phosphorylase; hHGPRT, human hypoxanthine-guanine phosphoribosyl transferase; hAK, human adenosine kinase; hAMPDA, human adenosine 5′-monophosphate deaminase; hAPRT, human adenine phosphoribosyl transferase; Pf ADA, P. falciparum adenosine deaminase; Pf PNP, P. falciparum purine nucleoside phosphorylase; Pf HGXPRT, P. falciparum hypoxanthine-guanine-xanthine phosphoribosyl transferase; Pf AMPDA, P. falciparum adenosine 5′-monophosphate deaminase; Pf IMPDH, P. falciparum inosine 5′-monophosphate dehydrogenase; Pf GMPs, P. falciparum guanosine 5′-monophosphate synthase; Pf AdSS, adenylosuccinate synthase; Pf AdSL, adenylosuccinate lyase. Polyamine pathway: AdoMet, S-adenosylmethionine; AdoHC, S-adenosylhomocysteine; HC, homocysteine; Met, methionine; dcAdoMet, decarboxylated S-adenosylmethionine; Pf SpdSyn, P. falciparum spermidine synthase; Pf ODCAdoMetDC, P. falciparum ornithine decarboxylase/S-adenosylmethionine decarboxylase; Pf MetTfase, P. falciparum methyltransferase(s); Pf AHC, P. falciparum S-adenosyl homocysteinase; Pf MetSyn, P. falciparum methionine synthase; Pf AdoMetSyn, P. falciparum S-adenosylmethionine synthase. The metabolically favored direction is indicated with bold arrows on reversible steps. The metabolic step inhibited by BCX4945 is indicated in red. Nucleoside/nucleobase transporters are indicated on each membrane: human erythrocyte nucleoside transporter (brown), P. falciparum NT1 transporter (blue) and yet-to-be characterized adenosine 5′-monophosphate transporter (purple).

Figure 2. BCX4945 inhibits PNP to block inosine salvage.

(A) Chemical structure and effect of BCX4945 on in vitro growth of different P. falciparum strains. Parasites were incubated in the presence of the indicated concentrations of BCX4945 for 72 h at 1% hematocrit, followed by DNA quantitation. IC₅₀ values were calculated from fits (Origin software) to the overall response curve. The graph is constructed by individual point connections. (B, C) Counts per minute (cpm) levels of [³H]inosine and [³H]hypoxanthine metabolically incorporated into purine derivatives. P. falciparum infected-red blood cells in schizont and trophozoite stages were metabolically labeled with [³H]inosine or [³H]hypoxanthine in the absence or presence of 10 µM of BCX4945. Labeled inosine (INO-S) and hypoxanthine (HX-S) present in the supernatant. Means ± s.d. from triplicates are represented.

Figure 3. Oral administration of BCX4945 inhibits PNP and clears P. falciparum from infected Aotus monkeys.

(A) Parasitaemia in infected untreated monkey (IUM, n = 1) or infected treated monkeys (ITM, n = 3). The arrow indicates mefloquine treatment to cure the infected untreated control monkey. Grey bar on the x-axis indicates days of treatment. (B) Blood cell PNP activity was assayed from untreated (n = 1) and treated monkeys (n = 3, means ± s.d.). Each sample was analyzed in triplicate. (C) Samples from blood cells and plasma were analyzed by UPLC-MS/MS. The (−4) time point indicates blood was taken before the monkey was infected. The (0) time point indicates that blood was drawn before the treatment started. Day 1 reflects parasitaemia or metabolite levels 24 h after the first dose and the metabolic effect of two BCX4945 doses within 24 h (also for days 2 to 5). Days 12 and 18 are counted from the start of treatment.

Figure 4. Stereo view crystal structure of BCX4945 and phosphate bound to hPNP and PfPNP.

Crystal structures of hPNP-BCX4945-PO₄ (A) and PfPNP-BCX4945-PO₄ (B) were determined to 2.3 and 2.0 Å resolution, respectively. BCX4945 (grey) active site residues (yellow), residues from adjacent subunits (green) and phosphate (orange) are indicated. Hydrogen bonds are indicated as black dashed lines. The ion-pair interaction between the ribocation mimic and phosphate is indicated as a magenta dashed line with the distances indicated.