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. 2019 Jul 19;10(1):3226.
doi: 10.1038/s41467-019-11239-0.

Antimalarial activity of primaquine operates via a two-step biochemical relay

Grazia Camarda  1 , Piyaporn Jirawatcharadech  1 , Richard S Priestley  1   2 , Ahmed Saif  1   3 , Sandra March  4 , Michael H L Wong  5 , Suet Leung  5 , Alex B Miller  4 , David A Baker  6 , Pietro Alano  7 , Mark J I Paine  8 , Sangeeta N Bhatia  4 , Paul M O'Neill  5 , Stephen A Ward  1 , Giancarlo A Biagini  9
Affiliations

Antimalarial activity of primaquine operates via a two-step biochemical relay

Grazia Camarda et al. Nat Commun. .

Abstract

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

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Conflict of interest statement

S.N.B. is a co-founder of Ascendance, which commercially manufactures and distributes micropatterned co-cultures. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Structures of PQ and OH-PQm and their activity against P. falciparum liver stages and gametocytes. a Structures of compounds used in this study. b, f Dose dependent reduction in exoerythrocytic forms (EEFs) numbers at day 3.5 post infection with NF54 sporozoites in YEM (poor metaboliser, black line) and NON (extensive metaboliser, dashed blue line) hepatocyte lots. b Primaquine (PQ). c 5-hydroxy-primaquine (5-HPQ). d 5-quinoneimine (PQQI). e 5,6-dihydroxy-primaquine (5,6-DPQ). f 6-hydroxy-5-quinoneimine (6OHPQQI). Viability is expressed as mean percentage of vehicle control ± S.D. of two independent experiments performed in triplicates. For each drug, IC50 values are reported for the poor metaboliser, YEM, and extensive metaboliser, NON, hepatocyte lots. g Coupled in vitro metabolism-GC-LUC assay. PQ and PQ metabolites (30 μM) were reacted with (blue squares) or without (red circles human liver microsomes (HLM) prior to dilution to 10 μM (nominal parental compound concentration) in a GC-LUC assay with mature gametocytes. Viability was measured after 72 h and expressed as mean percentage of control (no drug) viability ± S.D. (n = 3, each in triplicate). h as in g, but with (blue squares) or without (red circles) CYP2D6 (n = 2, 5 total replicates). For paroxetine inhibition, CYP2D6 was pre-incubated with 10 μM paroxetine for 15 min prior to compounds (black triangles, n = 2, 4 total replicates); i as in g, but without (red circles) or with (blue squares) recombinant human CPR, or with huCPR in the presence of 10 mM sodium pyruvate (brown diamonds) with huCPR in the absence of NAPD+ (black triangles). NoNADP+ n = 1 in duplicate; all other huCPR conditions n = 4 in duplicate
Fig. 2
Fig. 2
Metabolism-mediated generation of H2O2. H2O2 production was measured as catalase-mediated oxygen release after compound incubation (30 μM) with a human CPR, b human CPR + pyruvate, c human liver microsomes, d CYP2D6-expressing baculosomes, e mouse bone marrow extracts or f red blood cell extracts. The x axis was adjusted by defining the addition of catalase as t = 0, and the corresponding y axis value defined as 0 nmol mL−1. The average of two (PQ and 5,6-DPQ) and five (5-HPQ and MeOH) independent measurements are shown
Fig. 3
Fig. 3
Dose-response viability of late stage gametocytes treated with PQ and OH-PQm upon reaction with human CPR. Compounds at concentrations ranging from 30 μM to 11.7 nM were incubated with human CPR and diluted 1:3 for GC-LUC assays (n = 2, each in triplicate). IC50s (±S.E.M.) were calculated from nonlinear regression (curve fit) in GraphPad Prism
Fig. 4
Fig. 4
Schematic representation of primaquine mode of action. The results presented in this work support a two-step biochemical relay mechanism for PQ mode of action. In Step 1, PQ is converted into hydroxylated metabolites (OH-PQm) through the CPR/CYP2D6 metabolic complex. In Step 2, metabolites then undergo spontaneous oxidation to quinoneimines (O = PQm) with concomitant generation of H2O2. Human CPR then receives two electrons from NADPH and orchestrates one-electron transfers via FAD/FMN cofactors to quinoneinimes, thus reducing them back to the hydroxyl forms and perpetuating a catalytic cycle which brings about H2O2 accumulation at sites of metabolic transformation (liver, bone marrow and possibly others). Plasmodium parasites present at these locations are then killed by H2O2 action
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