Antiplasmodial activity of the natural product compounds alstonine and himbeline.

Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue. Despite a decline in malaria related deaths over the last decade, overall progress has plateaued. Key challenges to malaria prevention and control include the lack of a broadly effective vaccine and parasite drug resistance, including to the current gold standard artemisinin combination therapies (ACTs). New drugs with unique modes of action are therefore a priority for both the treatment and prevention of malaria. Unlike treatment drugs which need to kill parasites quickly to reduce or prevent clinical symptoms, compounds that kill parasites more slowly may be an option for malaria prevention. Natural products and natural product derived compounds have historically been an excellent source of antimalarial drugs, including the artemisinin component of ACTs. In this study, 424 natural product derived compounds were screened for in vitro activity against P. falciparum in assays designed to detect slow action activity, with 46 hit compounds identified as having >50% inhibition at 10 µM. Dose response assays revealed nine compounds with submicromolar activity, with slow action activity confirmed for two compounds, alstonine and himbeline (50% inhibitory concentration (IC50) 0.17 and 0.58 µM, respectively). Both compounds displayed >140-fold better activity against P. falciparum versus two human cell lines (Selectivity Index (SI) >1,111 and > 144, respectively). Importantly, P. falciparum multi-drug resistant lines showed no cross-resistance to alstonine or himbeline, with some resistant lines being more sensitive to these two compounds compared to the drug sensitive line. In addition, alstonine displayed cross-species activity against the zoonotic species, P. knowelsi (IC50 ~1 µM). Outcomes of this study provide a starting point for further investigations into these compounds as antiplasmodial drug candidates and the investigation of their molecular targets.

Synthesis, biological characterisation and structure activity relationships of aromatic bisamidines active against Plasmodium falciparum.

Malaria is one of the most significant tropical diseases and remains a major challenge due to the lack of a broadly effective vaccine and parasite resistance to current drugs. This means there is a need for new drug candidates with novel modes of action. Aromatic bisamidines, such as furamidine (DB75), were initially developed as anti-Trypanosoma agents however as clinical trials with furamidine highlighted potential side effects they were not pursued further in that setting. Despite apparent cytotoxicity liabilities the potency of furamidine against Plasmodium falciparum makes it a promising scaffold for the development of new anti-Plasmodium agents with improved selectivity. In this study a bisamidine compound series based on furamidine was synthesized by introducing modifications at the furan core structure and terminal amidine groups. The activity of the derived compounds was tested in vitro against drug sensitive and resistant P. falciparum lines and a human cell line (HEK293 cells) to generate anti-Plasmodium structure-activity relationships and to provide preliminary selectivity data.

HIV and malaria co-infection: interactions and consequences of chemotherapy.

The global epidemiology of HIV/AIDS and malaria overlap because a significant number of HIV-infected individuals live in regions with different levels of malaria transmission. Although the consequences of co-infection with HIV and malaria parasites are not fully understood, available evidence suggests that the infections act synergistically and together result in worse outcomes. The importance of understanding chemotherapeutic interactions during malaria and HIV co-infection is now being recognized. We know that some antimalarial drugs have weak antiretroviral effects; however, recent studies have also demonstrated that certain antiretroviral agents can inhibit malaria-parasite growth. Here, we discuss recent findings on the impact of HIV/AIDS and malaria co-infection and the possible roles of chemotherapy in improving the treatment of these diseases.

Stronger activity of human immunodeficiency virus type 1 protease inhibitors against clinical isolates of Plasmodium vivax than against those of P. falciparum.

Recent studies using laboratory clones have demonstrated that several antiretroviral protease inhibitors (PIs) inhibit the growth of Plasmodium falciparum at concentrations that may be of clinical significance, especially during human immunodeficiency virus type 1 (HIV-1) and malaria coinfection. Using clinical isolates, we now demonstrate the in vitro effectiveness of two HIV-1 aspartic PIs, saquinavir (SQV) and ritonavir (RTV), against P. vivax (n = 30) and P. falciparum (n = 20) from populations subjected to high levels of mefloquine and artesunate pressure on the Thailand-Myanmar border. The median 50% inhibitory concentration values of P. vivax to RTV and SQV were 2,233 nM (range, 732 to 7,738 nM) and 4,230 nM (range, 1,326 to 8,452 nM), respectively, both within the therapeutic concentration range commonly found for patients treated with these PIs. RTV was fourfold more effective at inhibiting P. vivax than it was at inhibiting P. falciparum, compared to a twofold difference in SQV sensitivity. An increased P. falciparum mdr1 copy number was present in 33% (3/9) of isolates and that of P. vivax mdr1 was present in 9% of isolates (2/22), but neither was associated with PI sensitivity. The inter-Plasmodium sp. variations in PI sensitivity indicate key differences between P. vivax and P. falciparum. PI-containing antiretroviral regimens may demonstrate prophylactic activity against both vivax and falciparum malaria in HIV-infected patients who reside in areas where multidrug-resistant P. vivax or P. falciparum is found.

Potent antimalarial activity of histone deacetylase inhibitor analogues.

The malaria parasite Plasmodium falciparum has at least five putative histone deacetylase (HDAC) enzymes, which have been proposed as new antimalarial drug targets and may play roles in regulating gene transcription, like the better-known and more intensively studied human HDACs (hHDACs). Fourteen new compounds derived from l-cysteine or 2-aminosuberic acid were designed to inhibit P. falciparum HDAC-1 (PfHDAC-1) based on homology modeling with human class I and class II HDAC enzymes. The compounds displayed highly potent antiproliferative activity against drug-resistant (Dd2) or drug sensitive (3D7) strains of P. falciparum in vitro (50% inhibitory concentration of 13 to 334 nM). Unlike known hHDAC inhibitors, some of these new compounds were significantly more toxic to P. falciparum parasites than to mammalian cells. The compounds inhibited P. falciparum growth in erythrocytes at both the early and late stages of the parasite's life cycle and caused altered histone acetylation patterns (hyperacetylation), which is a marker of HDAC inhibition in mammalian cells. These results support PfHDAC enzymes as being promising targets for new antimalarial drugs.

Synergistic interactions of the antiretroviral protease inhibitors saquinavir and ritonavir with chloroquine and mefloquine against Plasmodium falciparum in vitro.

The antimalarial activity of several antiretroviral protease inhibitor combinations was investigated. Data demonstrate that ritonavir and saquinavir behave synergistically with chloroquine and mefloquine. These data, and interactions with pepstatin-A, E-64, and bestatin, suggest that human immunodeficiency virus protease inhibitors do not target digestive-vacuole plasmepsins.

Characterization of the effect of retinol on Plasmodium falciparum in vitro.

A preliminary study from our laboratory found retinol (vitamin A alcohol) to have in vitro activity against Plasmodium falciparum at concentrations close to those in normal human serum (1-3 microM). To characterize the antimalarial potential of retinol in more detail, the 3D7 and K1 laboratory strains of P. falciparum were maintained in continuous culture and [3H]hypoxanthine incorporation and microscopy were used to assess the effect of retinol against asexual stages of the parasite life-cycle. Losses of retinol and retinol-associated hemolysis were also quantified in the in vitro culture system. There were retinol losses of >50% but no hemolysis was observed with added retinol concentrations up to 100 microM. All stages of parasite development showed comparable sensitivity to retinol including merozoite invasion (range of mean IC50 values 10.1-21.4 microM after adjustment for losses). Retinol pre-treatment of uninfected RBC did not inhibit merozoite invasion. Retinol treatment was associated with increased vacuolization within the parasite food vacuole and evidence of parasite membrane rupture. These appearances were similar to those seen with quinoline and artemisinin compounds. Although these data do not support a role for acute retinol supplementation in the treatment of falciparum malaria, they add to knowledge regarding potential antimalarial therapies and justify assessment of more potent synthetic retinoids and their metabolites.

In vitro antimalarial activity of retinoids and the influence of selective retinoic acid receptor antagonists.

Retinol (vitamin A alcohol) may have a beneficial role in the host response to malaria in humans and previously published data have suggested that it has a direct inhibitory effect on the growth of Plasmodium falciparum in vitro. To further investigate the role of retinoids as potential antimalarial agents, we assessed the effect of all-trans-retinoic acid (RA), 9-cis-RA and 13-cis-RA, as well as retinol itself and its ester, retinyl palmitate, on 3H-hypoxanthine uptake by the laboratory-adapted strains of P. falciparum 3D7 and K1. In addition, we examined the influence of three specific RA receptor antagonists, ER 27191, Ro 415253 and AGN 194301, on retinoid-induced growth inhibition of 3D7. All-trans-RA, 9-cis-RA and 13-cis-RA in concentrations ranging from 1 x 10(-4) to 5 x 10(-10) M each had antimalarial activity, but at IC50 values (5.9 x 10(-5) to 7.9 x 10(-5) M) that were less than those of retinol (2.5 x 10(-5) to 3.2 x 10(-5) M). Retinyl palmitate had minimal effect on 3H-hypoxanthine uptake. Each of the three specific antagonists inhibited growth of 3D7 (IC50 range 1.2 x 10(-5) to 3.0 x 10(-5) M) but, in isobolographic analysis, were antagonistic to retinol (dose factor potentiation, DFP 0.46-0.79) and, in the case of Ro 415253, to all-trans-RA (DFP=0.39). Although we did not attempt to quantify losses of retinoids from the system, these data suggest that retinol has greater antimalarial activity than its RA metabolites and especially retinyl palmitate. The specific RA receptor antagonists showed paradoxical antimalarial activity but consistently antagonised the effect of retinol and all-trans-RA in isobolographic experiments. We conclude that RA metabolites may be less suitable than retinol per se as antimalarial agents and that P. falciparum might possess or acquire a RA receptor-like moiety.

In vitro growth inhibition of Plasmodium falciparum by retinol at concentrations present in normal human serum.

To assess the in vitro effect of retinol on Plasmodium falciparum, the standard isolates 3D7, D10, W2 and K1 in continuous culture were exposed to retinol added in concentrations ranging from 10(-7) to 0.1 mumol/l. Parasite growth inhibition was assessed from 3H-hypoxanthine incorporation. Triplicate experiments were performed at physiological pH and in the case of D10, additional experiments were performed at pH 7.2 and 7.6. Final media retinol concentrations were assayed using high performance liquid chromatography. Retinol inhibited growth of both asynchronous and synchronous cultures of 3D7 and D10 and asynchronous cultures of W2 and K1. IC50 values determined from assayed media concentrations ranged from 0.2 to 3.9 mumol/l and were comparable to concentrations in normal human serum (1.0-3.0 mumol/l). IC50 values for asynchronous D10 cultures at pH 7.2 were lower than at pH 7.4 or 7.6 (0.5, 3.9 and 5.0 mumol/l, respectively); results from synchronous cultures were similar. These data suggest that P. falciparum is a retinol-sensitive parasite, especially at pH levels equivalent to those in an acidotic patient. Adjunctive retinol therapy may have a role in clinical management of malaria.

The efficacy of benzimidazole drugs against Plasmodium falciparum in vitro.

The sensitivities in vitro of Plasmodium falciparum to the benzimidazoles, albendazole, thiabendazole, mebendazole, omeprazole and 2 albendazole metabolites, albendazole sulphone and albendazole sulphoxide, were investigated and compared to those of the commonly used antimalarial drugs chloroquine and quinine. Quinine and chloroquine were the most potent drugs tested (EC50 values of 8 x 10(-9)-6 x 10(-8) mol/L and 5-7 x 10(-9) mol/L, respectively). Thiabendazole, mebendazole, albendazole sulphone and albendazole sulphoxide reached maximum growth inhibitions of 13-36% at the highest concentration tested (1 x 10(-4) mol/L). Albendazole (EC50 range: not achieved-2 x 10(-6) mol/L) and omeprazole (EC50 range: 2-4 x 10(-5) mol/L) were the most effective benzimidazoles. The activity of albendazole was pH dependent, as was that of chloroquine, and variable. Albendazole has its primary mode of action on trophozoites, suggesting that the drug may target parasite tubulin polymerization. Omeprazole, although also primarily effective against trophozoites, had additional activity against schizonts and ring forms, suggesting a distinct or additional parasitic target. Given the variable activity of albendazole and its rapid metabolism in vivo into compounds with even less antimalarial activity, it appears unlikely that this benzimidazole will be useful in the treatment of malaria. The rapid activity and different stage-specific profile of the more soluble benzimidazole omeprazole warrants further investigation.