Rossarin Suwanarusk

Spiroindolones, a Potent Compound Class for the Treatment of Malaria

Antimalarial Drug Candidate Spiroindolones were discovered as promising antimalarial drug candidates through a high-throughput screening approach that should be applicable to a range of neglected infectious diseases. Rottmann et al. (p. 1175; see the Perspective by Wells) present the preclinical profile for an optimized spiroindolone drug candidate, NITD609. They obtained evidence for a decrease in drug sensitivity in strains of the malaria parasite Plasmodium falciparum bearing amino acid mutations in the P-type ATPase, indicating possible mechanisms of action and/or resistance. High-throughput screening has offered up an oral antimalarial drug and pointers to its mechanism of action. Recent reports of increased tolerance to artemisinin derivatives—the most recently adopted class of antimalarials—have prompted a need for new treatments. The spirotetrahydro-β-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration. Spiroindolones rapidly inhibit protein synthesis in P. falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows pharmacokinetic properties compatible with once-daily oral dosing and has single-dose efficacy in a rodent malaria model.

On the Cytoadhesion of Plasmodium vivax–Infected Erythrocytes

BACKGROUND Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Targeting Plasmodium PI(4)K to eliminate malaria

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.Summary Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here, we identify a lipid kinase, phosphatidylinositol 4-kinase (PI4K), as the target of imidazopyrazines, a novel antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens, P. falciparum and P. vivax, and inhibit liver stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI4K, altering the intracellular distribution of phosphatidylinositol 4-phosphate. Collectively, our data define PI4K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Chloroquine Resistant Plasmodium vivax: In Vitro Characterisation and Association with Molecular Polymorphisms

Background Treatment failure of chloroquine for P. vivax infections has reached high levels in the eastern provinces of Indonesia, however, in vitro characterization of chloroquine resistance and its associated molecular profile have yet to be determined. Methods Using a modified schizont maturation assay we investigated the in vitro chloroquine susceptibility profile and molecular polymorphisms of P. vivax isolates collected from Papua, Indonesia, where high levels of clinical chloroquine treatment failure have been reported, and from Thailand, where chloroquine treatment is generally effective. Results The geometric mean chloroquine IC50 for P. vivax isolates from Papua (n = 145) was 312 nM [95%CI: 237–411 nM] compared to 46.8 nM [95%CI: 34.7–63.1 nM] from Thailand (n = 81); p<0.001. Correlating with the known clinical efficacy of the area, a cut off for chloroquine resistance was defined as 220nM, a level exceeded in 13.6% (11/81) of Thai isolates and 65% (94/145) of Papuan isolates; p<0.001. Several sequence polymorphisms in pvcrt-o and pvmdr1, and difference in pvmdr1 copy number were identified. A Y976F mutation in pvmdr1 was present in 96% (123/128) of Papuan isolates and 25% (17/69) of Thai isolates; p<0.001. Overall, the geometric mean chloroquine IC50 in isolates with the Y976F mutation was 283 nM [95%CI: 211–379], compared to 44.5 nM [95%CI: 31.3–63.4] in isolates with the wild type; p< 0.001. Pvmdr1 amplification occurred in 23% (15/66) of Thai isolates compared to none (0/104) of Indonesian isolates (p<0.001), but was not associated with increased chloroquine resistance after controlling for geographical location. Conclusions In vitro susceptibility testing of P. vivax discriminates between populations with differing levels of clinical efficacy of chloroquine. The pvmdr1 polymorphism at Y976F may provide a useful tool to highlight areas of emerging chloroquine resistance, although further studies defining its clinical correlates are needed.

The Deformability of Red Blood Cells Parasitized by Plasmodium falciparum and P. vivax

Red blood cells (RBCs) must deform considerably during their multiple passages through the microvasculature and the sinusoids of the spleen. RBCs infected with Plasmodium falciparum (Pf-IRBCs) become increasingly rigid as they mature but avoid splenic clearance by sequestering in venules and capillaries. In contrast, RBCs infected with P. vivax (Pv-IRBCs) do not sequester. We compared the effects of P. vivax and P. falciparum infection on RBC deformability in a laminar shear flow system. Pf-IRBCs became more rigid as the parasite matured, but equivalent maturation of Pv-IRBCs resulted in a doubling of flexibility. Coincidentally, the IRBC surface area increased from 56.7+/-1.3 microm2 to 74.7+/-0.6 microm2 to 90.9+/-1.1 microm2 in ring-, trophozoite-, and schizont-stage Pv-IRBCs, respectively, whereas Pf-IRBCs did not increase in size. P. vivax increases the deformability of IRBCs and thereby avoids splenic entrapment.

Determinants of In Vitro Drug Susceptibility Testing of Plasmodium vivax

ABSTRACT In Papua, Indonesia, the antimalarial susceptibility of Plasmodium vivax (n = 216) and P. falciparum (n = 277) was assessed using a modified schizont maturation assay for chloroquine, amodiaquine, artesunate, lumefantrine, mefloquine, and piperaquine. The most effective antimalarial against P. vivax and P. falciparum was artesunate, with geometric mean 50% inhibitory concentrations (IC50s) (95% confidence intervals [CI]) of 1.31 nM (1.07 to 1.59) and 0.64 nM (0.53 to 0.79), respectively. In contrast, the geometric mean chloroquine IC50 for P. vivax was 295 nM (227 to 384) compared to only 47.4 nM (42.2 to 53.3) for P. falciparum. Two factors were found to significantly influence the in vitro drug response of P. vivax: the initial stage of the parasite and the duration of the assay. Isolates of P. vivax initially at the trophozoite stage had significantly higher chloroquine IC50s (478 nM [95% CI, 316 to 722]) than those initially at the ring stage (84.7 nM [95% CI, 45.7 to 157]; P < 0.001). Synchronous isolates of P. vivax and P. falciparum which reached the target of 40% schizonts in the control wells within 30 h had significantly higher geometric mean chloroquine IC50s (435 nM [95% CI, 169 to 1,118] and 55.9 nM [95% CI, 48 to 64.9], respectively) than isolates that took more than 30 h (39.9 nM [14.6 to 110.4] and 36.9 nM [31.2 to 43.7]; P < 0.005). The results demonstrate the marked stage-specific activity of chloroquine with P. vivax and suggest that susceptibility to chloroquine may be associated with variable growth rates. These findings have important implications for the phenotypic and downstream genetic characterization of P. vivax.

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax

Currently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.

Amplification of pvmdr1 associated with multidrug-resistant Plasmodium vivax.

BACKGROUND Multidrug-resistant strains of Plasmodium vivax are emerging in Southeast Asia. METHODS In vitro drug susceptibility and pvmdr1 genotype were determined in P. vivax field isolates from Indonesia and Thailand. RESULTS Increased pvmdr1 copy number was present in 21% of isolates from Thailand (15/71) and none from Indonesia (0/114; P < .001). Compared with Indonesian isolates, the median IC(50) of Thai isolates was lower for chloroquine (36 vs. 114 nmol/L; P < .001) but higher for amodiaquine (34 vs. 13.7 nmol/L; P = .032), artesunate (8.33 vs. 1.58 nmol/L; P < .001), and mefloquine (111 vs. 9.87 nmol/L; P < .001). In 11 cryopreserved Thai isolates, those with increased pvmdr1 copy number had a higher IC(50) for mefloquine (78.6 vs. 38 nmol/L for single-copy isolates; P = .006). Compared with isolates with the wild-type allele, the Y976F mutation of pvmdr1 was associated with reduced susceptibility to chloroquine (154 nmol/L [range, 4.6-3505] vs. 34 nmol/L [range, 6.7-149]; P < .001) but greater susceptibility to artesunate (1.8 vs. 9.5 nmol/L; P = .009) and mefloquine (14 vs. 121 nmol/L; P < .001). CONCLUSIONS Amplification of pvmdr1 and single-nucleotide polymorphisms are correlated with susceptibility of P. vivax to multiple antimalarial drugs. Chloroquine and mefloquine appear to exert competitive evolutionary pressure on pvmdr1, similar to that observed with pfmdr1 in Plasmodium falciparum.

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes

Plasmodium vivax merozoites only invade reticulocytes, a minor though heterogeneous population of red blood cell precursors that can be graded by levels of transferrin receptor (CD71) expression. The development of a protocol that allows sorting reticulocytes into defined developmental stages and a robust ex vivo P vivax invasion assay has made it possible for the first time to investigate the fine-scale invasion preference of P vivax merozoites. Surprisingly, it was the immature reticulocytes (CD71(+)) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71(-)), principally found in the peripheral blood, were rarely invaded. Invasion assays based on the CD71(+) reticulocyte fraction revealed substantial postinvasion modification. Thus, 3 to 6 hours after invasion, the initially biomechanically rigid CD71(+) reticulocytes convert into a highly deformable CD71(-) infected red blood cell devoid of host reticular matter, a process that normally spans 24 hours for uninfected reticulocytes. Concurrent with these changes, clathrin pits disappear by 3 hours postinvasion, replaced by distinctive caveolae nanostructures. These 2 hitherto unsuspected features of P vivax invasion, a narrow preference for immature reticulocytes and a rapid remodeling of the host cell, provide important insights pertinent to the pathobiology of the P vivax infection.