Pharmacophoric studies of in vitro inhibition of Plasmodium falciparum growth

Malaria continues to be a scourge in India and the situation has been compounded by the emergence of resistant strains of Plasmodium falciparum which is the primary cause of fatality in this disease. Therefore, there is an urgent need to develop newer drugs. Molecular modeling and pharmacophoric determination have become predominant methods today in the design and synthesis of newer and more effective drugs. Many Plasmodium specific enzymes and proteins involved in crucial biochemical pathways have been identified and their structures have been determined by X-ray crystallography. These enzymes and proteins are excellent targets for newer antimalarial agents. Bisphosphonates have shown potent inhibitory activity against Plasmodium farneysl diphosphate synthase (FPPS) enzyme, which is vital to the protein prenylation pathway of the organism. In this study, a set of 26 bisphosphonate inhibitors, synthesized by Oldfield et al [J Med Chem (2008) 51, 7827-7833] were subjected to rigorous 3D-QSAR studies using the PHASE computational package. In vitro Plasmodium growth inhibition rather than direct enzyme inhibition was considered in the study for a more realistic approach. Good statistical correlations were obtained for the pharmacophoric model as revealed by the regression values, indicating good stability of the model. Three hydrogen bond acceptors and a hydrogen bond donor defined the pharmacophore from the present study. This pharmacophore, AAAD (A = Hydrogen bond acceptor and D = Hydrogen bond donor) was put through a search-run for matching structures from the SPECS database yielding four matching structures, which could function as starting points for more novel and potent antimalarials.     

Toxic effect of isolated glycophorin A on the in vitro growth of Plasmodium falciparum

We have examined the inhibitory potencies of glycophorin A, a mixture of glycophorins B and C, chymotryptic fragments of GpA, desialylated GpA, alkaliborohydride treated GpA, and the O-linked tetrasaccharide isolated from GpA on the invasion of human red blood cells by synchronous Plasmodium falciparum (strain FCB). 50% inhibition of invasion, as measured by 3H-hypoxanthine incorporation into parasites, was achieved at 14 and 155 microM for GpA and GpA-CH1, respectively. We have noticed, however, that isolated GpA exhibits a toxic effect on the intraerythrocytic growth of the parasite whereas the chymotryptic fragment (amino acid residues 1-64 of GpA) does not. Thus the inhibitory potency of isolated GpA during erythrocyte invasion by the merozoite should be regarded as the result of both an inhibitory and a toxic effect. The inhibitory effect should be attributed to the carbohydrate-rich outer portion of GpA carrying clusters of neuraminic acid. The toxic effect should be attributed to the hydrophobic region of GpA which might be capable of inserting into the membrane of free merozoites and/or erythrocytes. Our data suggest that results previously obtained with glycoprotein inhibitors carrying hydrophobic portions may have to be questioned.     

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.     

Analysis of Plasmodium falciparum growth in culture using acridine orange and flow cytometry

The growth of Plasmodium falciparum in cultures of human red blood cells was studied using acridine orange to stain RNA and DNA, followed by flow cytometric analysis. The cycle of the parasite is characterized by a period of growth, prior to initiation of DNA synthesis, in which a significant increase in red fluorescence is observed, with only a small change in green fluorescence. Following this phase, which is formally similar to the G1 period in mammalian cells, initiation of DNA synthesis is characterized by increases in green fluorescence. Sorting of cells from several regions of the two-dimensional display shows that the distribution of morphological stages correlates with differences in red and green fluorescence. The effect of aphidicolin on the growth cycle of the parasite was also studied.     

Quantification of multiple infections of Plasmodium falciparum in vitro

ABSTRACT: BACKGROUND: Human malaria infections caused by the parasite Plasmodium falciparum often contain more than one genetically distinct parasite. Despite this fact, nearly all studies of multiple strain P. falciparum infections have been limited to determining relative densities of each parasite within an infection. In light of this, new methods are needed that can quantify the absolute number of parasites within a single infection. METHODS: A quantitative PCR (qPCR) method was developed to track the dynamic interaction of P. falciparum infections containing genetically distinct parasite clones in cultured red blood cells. Allele-specific primers were used to generate a standard curve and to quantify the absolute concentration of parasite DNA within multi-clonal infections. Effects on dynamic growth relationships between parasites under drug pressure were examined by treating mixed cultures of drug sensitive and drug resistant parasites with the anti-malarial drug chloroquine at different dosing schedules. RESULTS: An absolute quantification method was developed to monitor the dynamics of P. falciparum cultures in vitro. This method allowed for the observation of competitive suppression, the reduction of parasites numbers due to the presence of another parasite, and competitive release, the improved performance of a parasite after the removal of a competitor. These studies demonstrated that the presence of two parasites led to the reduction in density of at least one parasite. containing both a drug resistant and drug sensitive parasites resulted in an increased proportion of the drug resistant parasite. Moreover, following drug treatment, the resistant parasite experienced competitive release by exhibiting a fitness benefit greater than simply surviving drug treatment, due to the removal of competitive suppression by the sensitive parasite. CONCLUSIONS: The newly developed assay allowed for the examination of the dynamics of two distinct clones in vitro; both competitive suppression and release were observed. A deeper understanding of the dynamic growth responses of multiple strain P. falciparum infections, with and without drug pressure, can improve the understanding of the role of parasite interactions in the spread of drug resistant parasites, perhaps suggesting different treatment strategies.     

Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo‐4

Mutations in the multidrug resistance transporter of Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host–parasite system have never been analysed. The fluorochrome Fluo‐4 is a well‐documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo‐4 fluorescence uptake as a measure of compartmental Fluo‐4 concentration accumulation in the different compartments of the host–parasite system. We performed a ‘reverse Fluo‐4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca²⁺ fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.     

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Stage-dependent inhibition of chloroquine on Plasmodium falciparum in vitro

Morphological observation of the life cycle of the malaria parasite, Plasmodium falciparum, in highly synchronous cultures after an exposure to therapeutic concentrations of chloroquine in ring, trophozoite and schizont stages, respectively, were carried out in order to determine the influence of chloroquine on the growth of the different stages of the malarial parasites. It was found that chloroquine could not affect merozoite invasion of the erythrocytes; the ring stage was more sensitive to chloroquine than the trophozoite and schizont stages; and chloroquine in therapeutic concentrations prevented only the transformation of rings to trophozoites and could not affect the transformations of trophozoites to schizonts and schizonts to new rings. The determination of the IC50 of chloroquine showed that the IC50 of trophozoites was about 6 times as high as that of rings.     

Plasmodium falciparum: assessment of in vitro growth by [3H]hypoxanthine incorporation

To evaluate rapidly Plasmodium falciparum growth in Vitro, [3H]hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, [3H]hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting [3H]hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro.     

The production of antigens by Plasmodium falciparum in vitro

La, Lb, R¹ and S-antigens in extracts of human and Aotus monkey blood infected with Plasmodium falciparum were inactivated by several proteolytic enzymes but were not affected by nucleases and selected carbohydrases. S-antigens also survived oxidation by periodate under conditions which inactivated a known carbohydrate-associated antigen. It was concluded that the L, R and S-antigens are proteins.Autoradiographic studies of extracts of parasitized red cells which had been maintained in vitro in the presence of radioactive amino acids, showed that various La, Lb and R-antigens were labelled but S-antigens were not. The incorporation of labelled amino acids into antigens derived from parasitized mature mammalian red cells was taken as evidence that these antigens were of parasite origin whcreas the unlabelled S-antigens might be of host origin.     

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.     

Mechanisms of in vitro resistance to dihydroartemisinin in Plasmodium falciparum

The recent reports of artemisinin (ART) resistance in the Thai-Cambodian border area raise a serious concern on the long-term efficacy of ARTs. To elucidate the resistance mechanisms, we performed in vitro selection with dihydroartemisinin (DHA) and obtained two parasite clones from Dd2 with more than 25-fold decrease in susceptibility to DHA. The DHA-resistant clones were more tolerant of stressful growth conditions and more resistant to several commonly used antimalarial drugs than Dd2. The result is worrisome as many of the drugs are currently used as ART partners in malaria control. This study showed that the DHA resistance is not limited to ring stage, but also occurred in trophozoites and schizonts. Microarray and biochemical analyses revealed pfmdr1 amplification, elevation of the antioxidant defence network, and increased expression of many chaperones in the DHA-resistant parasites. Without drug pressure, the DHA-resistant parasites reverted to sensitivity in approximately 8 weeks, accompanied by de-amplification of pfmdr1 and reduced antioxidant activities. The parallel decrease and increase in pfmdr1 copy number and antioxidant activity and the up and down of DHA sensitivity strongly suggest that pfmdr1 and antioxidant defence play a role in in vitro resistance to DHA, providing potential molecular markers for ART resistance.     

Prenylated chalcones isolated from Crotalaria genus inhibits in vitro growth of the human malaria parasite Plasmodium falciparum

A prenylated chalcone 2 named crotaorixin, has been isolated from the aerial parts of the Crotalaria orixensis. Its structure has been established by extensive 1D and 2D NMR measurements. In vitro antimalarial activity of crotaorixin as well as few prenylated chalcones isolated from C. medicagenia and C. ramosissima were evaluated at three concentrations (50, 10 and 2 microg/ml) against Plasmodium falciparum (Strain NF-54). Compound 3 has exhibited 100% inhibition of schizont maturation at 2 microg/ml concentration.     

Initial extracellular development in vitro of merozoites of Plasmodium falciparum

Late schizonts from continuous cultures of P. falciparum were concentrated over Percoll, inoculated to various experimental media at the rate of about 20 X 10(6) per 0.5 ml of medium, and incubated in a candle jar at 37 degrees for 1 day. Controls in standard culture medium showed a heavy invasion with young rings in the previously uninfected red cells introduced with the inoculum of schizonts. In a medium of high potassium content containing a 33% extract of human erythrocytes, this invasion was inhibited and many free merozoites were present. If, however, this same medium was supplemented with both ATP, as the dipotassium salt at 1.6 mM, and sodium pyruvate at 3.6 mM, there appeared large numbers of extracellular forms resembling young rings. Examination of these by electron microscopy shows that they are indeed merozoites that have begun to differentiate extracellularly. This suggests that the trigger for differentiation of merozoites may not depend on the process of entry into a red cell but rather on specific factors within the red cell.     

Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.