Sarah D'Alessandro

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

OBJECTIVES Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

Synthetic spirocyclic endoperoxides: new antimalarial scaffolds

Here we report the development of a straightforward synthetic procedure for the preparation of spirocyclic endoperoxides as synthetic analogues of the natural product dihydroplakortin. The peroxides presented here are more potent antiplasmodials than dihydroplakortin itself and we proved for the first time their antimalarial activity in vivo.

Salinomycin and Other Ionophores as a New Class of Antimalarial Drugs with Transmission-Blocking Activity

ABSTRACT The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

Effects of Plasmodium falciparum-infected erythrocytes on matrix metalloproteinase-9 regulation in human microvascular endothelial cells.

OBJECTIVE To investigate the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human microvascular endothelium (HMEC-1) exposed to erythrocytes infected by different strains of Plasmodium falciparum (P. falciparum). METHODS HMEC-1 cells were co-incubated for 72 h with erythrocytes infected by late stage trophozoite of D10 (chloroquine-sensitive) or W2 (chloroquine-resistant) P. falciparum strains. Cell supernatants were then collected and the levels of pro- or active gelatinases MMP-9 and MMP-2 were evaluated by gelatin zymography and densitometry. The release of pro-MMP-9, MMP-3, MMP-1 and TIMP-1 proteins was analyzed by western blotting and densitometry. RESULTS Infected erythrocytes induced de novo proMMP-9 and MMP-9 release. Neither basal levels of proMMP-2 were altered, nor active MMP-2 was found. MMP-3 and MMP-1 secretion was significantly enhanced, whereas basal TIMP-1 was unaffected. All effects were similar for both strains. CONCLUSIONS P. falciparum parasites, either chloroquine-sensitive or -resistant, induce the release of active MMP-9 protein from human microvascular endothelium, by impairing balances between proMMP-9 and its inhibitor, and by enhancing the levels of its activators. This work provides new evidence on MMP involvement in malaria, pointing at MMP-9 as a possible target in adjuvant therapy.

Dextran-shelled oxygen-loaded nanodroplets reestablish a normoxia-like pro-angiogenic phenotype and behavior in hypoxic human dermal microvascular endothelium

In chronic wounds, hypoxia seriously undermines tissue repair processes by altering the balances between pro-angiogenic proteolytic enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) released from surrounding cells. Recently, we have shown that in human monocytes hypoxia reduces MMP-9 and increases TIMP-1 without affecting TIMP-2 secretion, whereas in human keratinocytes it reduces MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. Provided that the phenotype of the cellular environment is better understood, chronic wounds might be targeted by new oxygenating compounds such as chitosan- or dextran-shelled and 2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets (OLNs). Here, we investigated the effects of hypoxia and dextran-shelled OLNs on the pro-angiogenic phenotype and behavior of human dermal microvascular endothelium (HMEC-1 cell line), another cell population playing key roles during wound healing. Normoxic HMEC-1 constitutively released MMP-2, TIMP-1 and TIMP-2 proteins, but not MMP-9. Hypoxia enhanced MMP-2 and reduced TIMP-1 secretion, without affecting TIMP-2 levels, and compromised cell ability to migrate and invade the extracellular matrix. When taken up by HMEC-1, nontoxic OLNs abrogated the effects of hypoxia, restoring normoxic MMP/TIMP levels and promoting cell migration, matrix invasion, and formation of microvessels. These effects were specifically dependent on time-sustained oxygen diffusion from OLN core, since they were not achieved by oxygen-free nanodroplets or oxygen-saturated solution. Collectively, these data provide new information on the effects of hypoxia on dermal endothelium and support the hypothesis that OLNs might be used as effective adjuvant tools to promote chronic wound healing processes.

Primaquine-based ionic liquids as a novel class of antimalarial hits

Ionic liquids derived from active pharmaceutical ingredients may open new perspectives towards low-cost rescuing of classical drugs. Thus, we have synthesized novel ionic liquids derived from the antimalarial drug primaquine, and evaluated them in vitro against three stages of malaria parasites. Results from this unprecedented approach open a new chapter in the history of antimalarial drugs.

Chloroquine-containing organoruthenium complexes are fast-acting multistage antimalarial agents

We report the pharmacological activity of organoruthenium complexes containing chloroquine (CQ) as a chelating ligand. The complexes displayed intraerythrocytic activity against CQ-sensitive 3D7 and CQ-resistant W2 strains of Plasmodium falciparum, with potency and selectivity indexes similar to those of CQ. Complexes displayed activity against all intraerythrocytic stages, but moderate activity against Plasmodium berghei liver stages. However, unlike CQ, organoruthenium complexes impaired gametocyte viability and exhibited fast parasiticidal activity against trophozoites for P. falciparum. This functional property results from the ability of complexes to quickly induce oxidative stress. The parasitaemia of P. berghei-infected mice was reduced by treatment with the complex. Our findings demonstrated that using chloroquine for the synthesis of organoruthenium complexes retains potency and selectivity while leading to an increase in the spectrum of action and parasite killing rate relative to CQ.

Artemisone and artemiside - potent pan-reactive antimalarial agents that also synergize redox imbalance in P. falciparum transmissible gametocyte stages

The emergence of resistance toward artemisinin combination therapies (ACTs) by the malaria parasite Plasmodium falciparum has the potential to severely compromise malaria control. Therefore, the development of new artemisinins in combination with new drugs that impart activities toward both intraerythrocytic proliferative asexual and transmissible gametocyte stages, in particular, those of resistant parasites, is urgently required. ABSTRACT The emergence of resistance toward artemisinin combination therapies (ACTs) by the malaria parasite Plasmodium falciparum has the potential to severely compromise malaria control. Therefore, the development of new artemisinins in combination with new drugs that impart activities toward both intraerythrocytic proliferative asexual and transmissible gametocyte stages, in particular, those of resistant parasites, is urgently required. We define artemisinins as oxidant drugs through their ability to oxidize reduced flavin cofactors of flavin disulfide reductases critical for maintaining redox homeostasis in the malaria parasite. Here we compare the activities of 10-amino artemisinin derivatives toward the asexual and gametocyte stages of P. falciparum parasites. Of these, artemisone and artemiside inhibited asexual and gametocyte stages, particularly stage V gametocytes, in the low-nanomolar range. Further, treatment of both early and late gametocyte stages with artemisone or artemiside combined with the pro-oxidant redox partner methylene blue displayed notable synergism. These data suggest that modulation of redox homeostasis is likely an important druggable process, particularly in gametocytes, and this finding thereby enhances the prospect of using combinations of oxidant and redox drugs for malaria control.

Beyond lysozyme:antimicrobial peptides against malaria

Antimicrobial peptides (AMPs) are short amino acidic sequences with less than 100 residues. They are the components of the innate immune system not only in humans but also in plants, insects, and primitive multicellular organisms. Their role is to counteract the microorganisms, which could be potentially pathogenic for the host. AMPs active against viruses, bacteria, fungi, and parasites have been described. Among the antiparasitic AMPs reported so far, some peptides affect Plasmodium development in different phases of the biological cycle, from asexual blood stages to sexual stages in the mosquito, where AMPs can block ookinetes viability or oocyst formation. AMPs with antimalarial activity derive from different organisms, especially insects, as well as amphibians. In malaria research, AMPs have been mainly proposed for the engineering of mosquitoes or parasites to reduce or interrupt the malaria parasite transmission. In this chapter, the different classes of antimalarial AMPs (defensins, cecropins, dermaseptins) or single peptides (scorpine, melittin, gambicin) are described.