thapsigargin and Malaria

Recent reports of increased tolerance to artemisinin derivatives--the most recently adopted class of antimalarials--have prompted a need for new treatments. The spirotetrahydro-beta-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.

Topics: Adenosine Triphosphatases; Animals; Antimalarials; Cell Line; Drug Discovery; Drug Resistance; Erythrocytes; Female; Genes, Protozoan; Humans; Indoles; Malaria; Male; Mice; Models, Molecular; Mutant Proteins; Mutation; Parasitic Sensitivity Tests; Plasmodium berghei; Plasmodium falciparum; Plasmodium vivax; Protein Synthesis Inhibitors; Protozoan Proteins; Rats; Rats, Wistar; Spiro Compounds

Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H(+)-PPase and V-H(+)-ATPase in these organelles.

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Egtazic Acid; Erythrocytes; Inorganic Pyrophosphatase; Ionomycin; Kinetics; Macrolides; Malaria; Male; Mice; Mice, Inbred BALB C; Monensin; Nigericin; Organelles; Plasmodium berghei; Proton Pumps; Pyrophosphatases; Thapsigargin; Vacuoles

Topics: Animals; Calcium; Circadian Rhythm; Dose-Response Relationship, Drug; Estrenes; Female; Malaria; Melatonin; Mice; Mice, Inbred BALB C; Pineal Gland; Plasmodium chabaudi; Plasmodium falciparum; Pyrrolidinones; Thapsigargin; Tryptamines