gramicidin-a and Malaria

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Topics: Animals; Antimalarials; Cell Line, Tumor; Drug Discovery; Drug Evaluation, Preclinical; Drug Resistance; Erythrocytes; Humans; Imidazoles; Liver; Malaria; Mice; Mice, Inbred BALB C; Molecular Structure; Piperazines; Plasmodium; Plasmodium berghei; Plasmodium falciparum; Plasmodium yoelii; Polymorphism, Single Nucleotide; Protozoan Proteins; Random Allocation; Small Molecule Libraries; Sporozoites

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans, we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.

Topics: Animals; Bacterial Proteins; Drug Evaluation, Preclinical; Enzyme Inhibitors; Female; Gluconobacter oxydans; Gramicidin; Kinetics; Lactones; Malaria; Mice; Mice, Inbred BALB C; Mycobacterium smegmatis; NADH Dehydrogenase; Plasmodium yoelii

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.

Topics: Animals; Antimalarials; Cluster Analysis; Computational Biology; Drug Evaluation, Preclinical; Drug Resistance; Folic Acid Antagonists; Malaria; Models, Molecular; Parasites; Plasmodium falciparum; Reproducibility of Results; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase

The effect of tryptophan-N-formylated gramicidin (NFG) on the growth of Plasmodium berghei in mice was tested in three different experiments. NFG was shown to be capable of inhibiting the growth of the parasite in a dose-dependent way, although its action did not result in elimination of the parasite and was only temporary, preventing mice from early death, presumably due to cerebral malaria, but not from fatal generalized malaria. Intriguingly, a similar observation was made with two other drugs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, an inhibitor of viral and eukaryotic DNA polymerases, and the presumed topoisomerase II inhibitor, a bisquaternary quinolinium salt. A rise in the level of parasitemia after 8 days, despite continued treatment, was not due to parasite-induced reticulocytosis, as demonstrated in experiments in which this condition was induced artificially. NFG was added in the form of lipid vesicles in which the peptide had been incorporated. The inhibitory action of NFG was not modulated by the lipid composition of the vesicles. Control experiments did not demonstrate any toxicity of NFG when it was administered in lipid vesicles. The main observation is that NFG is able to inhibit the growth of a malaria parasite in vivo at concentrations that are well tolerated by the host.

Topics: Animals; Anti-Bacterial Agents; Drug Evaluation, Preclinical; Gramicidin; Malaria; Male; Mice; Mice, Inbred BALB C; Parasitemia; Plasmodium berghei

Eighteen antimicrobials commonly used in tissue culture were screened in three different protozoan test systems in order to establish their suitability for routine inclusion in protozoal cultivation systems. The human malaria parasite, Plasmodium falciparum, was inhibited by more than half the antibiotics tested at concentrations recommended for normal tissue culture use. Eight compounds were well tolerated and thus could be used prophylactically to prevent microbial contamination. These antimicrobials were the bactericidal aminoglycoside antibiotics, streptomycin, gentamicin and kanamycin, the bacteriostatic protein synthesis inhibitors, chloramphenicol and chlortetracycline and the antifungals, 5-fluorocytosine, nystatin and amphotericin B. Babesia rodhaini and Theileria parva were less sensitive than P. falciparum and tolerated all 18 compounds at concentrations well above 100 micrograms ml-1. Extension of the study to examine direct antiprotozoal action of these and other antimicrobials not normally used in culture confirmed that P. falciparum was significantly more sensitive than the other parasites. Tylosin, rifamycin, gramicidin D and valinomycin were all strongly antimalarial with IC50 values of 0.245, 1.20, 1.3 X 10(-3) and 1.9 X 10(-3) micrograms ml-1 respectively. This compares with a value of 1.35 X 10(-2) micrograms ml-1 for the standard antimalarial, chloroquine. Only valinomycin and, more particularly, gramicidin D were significantly active against B. rodhaini and T. parva. Gramicidin D was more effective, but more toxic, than the standard antiprotozoal agents tested at curing in vivo malarial and babesial infections in mice.

Topics: Animals; Anti-Bacterial Agents; Apicomplexa; Babesia; Babesiosis; Cattle; Culture Techniques; Drug Evaluation, Preclinical; Drug Resistance, Microbial; Gramicidin; Malaria; Mice; Microbial Sensitivity Tests; Plasmodium falciparum; Theileriasis