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SARS-CoV-2, the etiological agent of the COVID-19 pandemic, is a member of the Coronaviridae family. It is an enveloped virus with ion channels in its membrane, the most characterized of which is the E protein. Therefore, in an attempt to identify blockers of the E channel, we screened a library of 2839 approved-for-human-use drugs. Our approach yielded eight compounds that exhibited appreciable activity in three bacteria-based channel assays. Considering the fact that the E channel is the most conserved of all SARS-CoV-2 proteins, any inhibitor of its activity may provide an option to curb the viral spread. In addition, inhibitors can also enhance our ability to understand the exact role played by the E protein during the infectivity cycle. Finally, detailed electrophysiological analyses, alongside in vitro and in vivo studies will be needed to establish the exact potential of each of the blockers identified in our study.

The etiological agent of the COVID-19 pandemic is SARS-CoV-2. As a member of the Coronaviridae, the enveloped pathogen has several membrane proteins, of which two, E and 3a, were suggested to function as ion channels. In an effort to increase our treatment options, alongside providing new research tools, we have sought to inhibit the 3a channel by targeted drug repurposing. To that end, using three bacteria-based assays, we screened a library of 2839 approved-for-human-use drugs and identified the following potential channel-blockers: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine and Fludarabine. The stage is now set for examining the activity of these compounds in detailed electrophysiological studies and their impact on the whole virus with appropriate biosafety measures.

It is difficult to overstate the impact that COVID-19 had on humankind. The pandemic’s etiological agent, SARS-CoV-2, is a member of the Coronaviridae, and as such, is an enveloped virus with ion channels in its membrane. Therefore, in an attempt to provide an option to curb the viral spread, we searched for blockers of its E protein viro-porin. Using three bacteria-based assays, we identified eight compounds that exhibited activity after screening a library of ca. 3000 approved-for-human-use drugs. Reassuringly, analysis of viral replication in tissue culture indicated that most of the compounds could reduce infectivity to varying extents. In conclusion, targeting a particular channel in the virus for drug repurposing may increase our arsenal of treatment options to combat COVID-19 virulence.Significance StatementThe goal of our study was to expand the treatment arsenal against COVID-19. To that end, we have decided to focus on drug therapy, and as a target - the E protein, an ion channel in the virus. Ion channels as a family are excellent drug targets, but viral channels have been underexploited for pharmaceutical point intervention. To hasten future regulatory requirements and focus the chemical search space, we screened a library of ca. 3000 approved-for-human-use drugs using three independent bacteria-based assays. Our results yielded eight compounds, which were subsequently tested for antiviral activity in tissue culture. Gratifyingly, most compounds were able to reduce viral replication, and as such, both validate our approach and potentially augment our anti-COVID tool kit.

The development of drug resistance has long plagued our efforts to curtail viral infections in general and influenza in particular. The problem is particularly challenging since the exact mode of resistance may be difficult to predict, without waiting for untreatable strains to evolve. Herein, a different approach is taken. Using a novel genetic screen, we map the resistance options of influenza's M2 channel against its aminoadamantane antiviral inhibitors. In the process, we could identify clinically known resistant mutations in a completely unbiased manner. Additionally, novel mutations were obtained, which, while known to exist in circulating viruses, were not previously classified as drug resistant. Finally, we demonstrated the approach against an anti-influenza drug that has not seen clinical use, identifying several resistance mutations in the process. In conclusion, we present and employ a method to predict the resistance options of influenza's M2 channel to antiviral agents ahead of clinical use and without medical hazard.

Motivation: Most integral membrane proteins form dimeric or oligomeric complexes. Oligomerization is frequently supported by the noncovalent interaction of transmembrane helices. It is currently not clear how many high-affinity transmembrane domains (TMD) exist in a proteome and how specific their interactions are with respect to preferred contacting faces and their underlying residue motifs. Results: We first identify a threshold of 55% sequence similarity, which demarcates the border between meaningful alignments of TMDs and chance alignments. Clustering the human single-span membrane proteome using this threshold groups � 40% of the TMDs. The homotypic interaction of the TMDs representing the 33 largest clusters was systematically investigated under standardized conditions. The results reveal a broad distribution of relative affinities. High relative affinity frequently coincides with (i) the existence of a preferred helix–helix interface and (ii) sequence specificity as indicated by reduced affinity after mutating conserved residues. Contact: langosch@tum.de Supplementary information: Supplementary data are available at Bioinformatics online.

The interactions between channels and their cognate blockers are at the heart of numerous biomedical phenomena. Herein, we unravel one particularly important example bearing direct pharmaceutical relevance: the blockage mechanism of the influenza M2 channel by the anti-flu amino-adamantyls (amantadine and rimantadine) and how the channel and, consequently, the virus develop resistance against them. Using both computational analyses and experimental verification, we find that amino-adamantyls inhibit M2's H(+) channel activity by electrostatic hindrance due to their positively charged amino group. In contrast, the hydrophobic adamantyl moiety on its own does not impact conductivity. Additionally, we were able to uncover how mutations in M2 are capable of retaining drug binding on the one hand yet rendering the protein and the mutated virus resistant to amino-adamantyls on the other hand. We show that the mutated, drug-resistant protein has a larger binding pocket for the drug. Hence, despite binding the channel, the drug remains sufficiently mobile so as not to exert a H(+)-blocking positive electrostatic hindrance. Such insight into the blocking mechanism of amino-adamantyls, and resistance thereof, may aid in the design of next-generation anti-flu agents.

The influenza M2 H+ channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed Ks measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system.

Human malaria parasites, including the most lethal Plasmodium falciparum , are increasingly resistant to existing antimalarial drugs. One remarkable opportunity to selectively target P. falciparum stems from the unique AT-richness of its genome (80% A/T, relative to 60% in human DNA). To rationally explore this opportunity, we used drugs (adozelesin and bizelesin) which distinctly target AT-rich minisatellites and an in silico approach for genome-wide analysis previously experimentally validated in human cells [Woynarowski JM, Trevino AV, Rodriguez KA, Hardies SC, Benham CJ. AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs. J Biol Chem 2001;276:40555–66]. Both drugs demonstrate a potent, rapid and irreversible inhibition of the cultured P. falciparum (50% inhibition at 110 and 10 ± 2.3 pM, respectively). This antiparasital activity reflects most likely drug binding to specific super-AT-rich regions. Relative to the human genome, the P. falciparum genome shows 3.9- and 7-fold higher frequency of binding sites for adozelesin and bizelesin, respectively. The distribution of these sites is non-random with the most prominent clusters found in large unique minisatellites [median size 3.5 kbp of nearly pure A/T, with multiple converging repeats but no shared consensus other than (A/T) n ]. Each of the fourteen P. falciparum chromosomes contains only one such “super-AT island” located within ∼3–7.5 kbp of gene-free and nucleosome-free loci. Important functions of super-AT islands are suggested by their exceptional predicted potential to serve as matrix attachment regions (MARs) and a precise co-localization with the putative centromeres. Conclusion Super-AT islands, identified as unique domains in the P. falciparum genome with presumably crucial functions, offer therapeutically exploitable opportunity for new antimalarial strategies.

The ever evolving resistance of the most virulent malaria parasite, Plasmodium falciparum , to antimalarials necessitates the continuous development of new drugs. Our previous analysis of the antimalarial activities of the hemolytic antimicrobial peptides dermaseptins and their acylated derivatives implicated the importance of hydrophobicity and charge for drug action. Following these findings, an oligoacyllysine (OAK) tetramer designed to mimic the characteristics of dermaseptin was synthesized and assessed for its antimalarial activity in cultures of P. falciparum . The tetramer inhibited the growth of different plasmodial strains at low micromolar concentrations (mean 50% inhibitory concentration [IC 50 ], 1.8 μM). A structure-activity relationship study involving eight derivatives unraveled smaller, more potent OAK analogs (IC 50 s, 0.08 to 0.14 μM). The most potent analogs were the most selective, with selectivity ratios of 3 orders of magnitude. Selectivity was strongly influenced by the self-assembly properties resulting from interactions between hydrophobic OAKs, as has been observed with conventional antimicrobial peptides. Further investigations performed with a representative OAK revealed that the ring and trophozoite stages of the parasite developmental cycle were equally sensitive to the compound. A shortcoming of the tested compound was the need for long incubation times in order for it to exert its full effect. Nevertheless, the encouraging results obtained in this study regarding the efficiency and selectivity of some compounds establish them as leads for further development.

Arylpiperazines in which the terminal secondary amino group is unsubstituted were found to display a mefloquine-type antimalarial behavior in being significantly more potent against the chloroquine-resistant (W2 and FCR3) strains of Plasmodium falciparum than against the chloroquine-sensitive (D10 and NF54) strains. Substitution of the aforementioned amino group led to a dramatic drop in activity across all strains as well as abolition of the preferential potency against resistant strains that was observed for the unsubstituted counterparts. The data suggest that unsubstituted arylpiperazines are not well-recognized by the chloroquine resistance mechanism and may imply that they act mechanistically differently from chloroquine. On the other hand, 4-aminoquinoline-based heteroarylpiperazines in which the terminal secondary amino group is also unsubstituted, were found to be equally active against the chloroquine-resistant and chloroquine-sensitive strains, suggesting that chloroquine cross-resistance is not observed with these two 4-aminoquinolines. In contrast, two 4-aminoquinoline-based heteroarylpiperazines are positively recognized by the chloroquine resistance mechanism. These studies provide structural features that determine the antimalarial activity of arylpiperazines for further development, particularly against chloroquine-resistant strains.

In most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K+ permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies. (Blood. 2005; 105:4853-4860)

To understand the osmotic stability of a Plasmodium falciparum-infected red blood cell, whose membrane permeability becomes highly increased during parasite growth, we developed an integrated mathematical model of the homeostasis of an infected red cell. The model encoded the known time courses of red cell membrane permeabilisation and of haemoglobin digestion, as well as alternative options for parasite volume growth. Model simulations revealed that excess haemoglobin digestion, by reducing the colloid-osmotic pressure within the host red cell, is essential to preserve the osmotic stability of the infected cell for the duration of the parasite asexual cycle. We present here experimental tests of the model predictions and discuss the available evidence in the context of the interpretations provided by the model.

It is generally accepted that intraerythrocytic malaria parasites digest hemoglobin to supply the amino acids needed for the synthesis of their own proteins. This view has never been quantitatively tested. In this investigation we have measured the degradation of hemoglobin and the increase in parasite protein content as a function of parasite maturation in cultures of Plasmodium falciparum. Defined parasite stages were obtained either from tightly synchronized cultures or from asynchronous cultures after density-fractionation. We showed that both hemoglobin digestion and total parasite protein content increased with parasite maturation, from the early trophozoite stage onwards, although the total protein content of the parasite remained significantly lower than that of other eukaryotes. The parasite digested up to 65% of the host cell's hemoglobin but utilized only up to about 16% of the amino acids derived from hemoglobin digestion. This large discrepancy is profoundly puzzling particularly in view of the need to detoxify the cell from the large quantities of ferriprotoporphyrin IX and iron released during hemoglobin digestion.

HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to possess ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K(+) transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K(+) concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers.

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum , in vitro , irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC 50 ), assessed by parasite incorporation of [ 3 H]hypoxanthine, were between 0.2 and 1.1 μM. CLT concentrations of 2 μM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (≈48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 μM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZAAZBAZCTHPSCB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZBAZA approximately PSAZC approximately CBMB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.

Our previous analysis of antiplasmodial properties exhibited by dodecanoyl-based oligo-acyl-lysyls (OAKs) has outlined basic attributes implicated in potent inhibition of parasite growth and underlined the critical role of excess hydrophobicity in hemotoxicity. To dissociate hemolysis from antiplasmodial effect, we screened >50 OAKs for in vitro growth inhibition of Plasmodium falciparum strains, thus revealing the minimal requirements for antiplasmodial potency in terms of sequence and composition, as confirmed by efficacy studies in vivo . The most active sequence, dodecanoyllysyl-bis(aminooctanoyllysyl)-amide (C 12 K-2α 8 ), inhibited parasite growth at submicromolar concentrations (50% inhibitory concentration [IC 50 ], 0.3 ± 0.1 μM) and was devoid of hemolytic activity (12 K-2α 8 demonstrated sustainable high concentrations in blood (e.g., 0.1 mM at 25 mg/kg of body weight). In Plasmodium vinckei -infected mice, C 12 K-2α 8 significantly affected parasite growth (50% effective dose [ED 50 ], 22 mg/kg) but also caused mortality in 2/3 mice at high doses (50 mg/kg/day × 4).

The ability to discriminate between highly similar substrates is one of the remarkable properties of enzymes. For example, transporters and channels that selectively distinguish between various solutes enable living organisms to maintain and control their internal environment in the face of a constantly changing surrounding. Herein, we examine in detail the selectivity properties of one of the most important salt transporters: the bacterial Na+/H+ antiporter. Selectivity can be achieved at either the substrate binding step or in subsequent antiporting. Surprisingly, using both computational and experimental analyses synergistically, we show that binding per se is not a sufficient determinant of selectively. All alkali ions from Li+ to Cs+ were able to competitively bind the antiporter's binding site, whether the protein was capable of pumping them or not. Hence, we propose that NhaA's binding site is relatively promiscuous and that the selectivity is determined at a later stage of the transport cycle.

The DNA of malarial parasites is significantly richer in A and T than that of mammalian cells. Antibiotics which bind to the minor groove of B-DNA with a preference for AT-rich sequences, such as distamycin A, netropsin, 4'-6-diamidino-2-phenylindole (DAPI) and bis-benzimide (Hoechst 33258) were found to inhibit the growth and propagation of Plasmodium falciparum in culture. Distamycin A readily inhibited nucleic acid and protein synthesis and was more toxic to the ring stage than to the trophozoite stage in various parasite strains, irrespective of their susceptibility to chloroquine. Distamycin A, netropsin, DAPI and Hoechst 33258 were considerably more toxic to parasites than to mammalian cells, while chromomycin A3 and mithramycin A, which bind preferentially to GC-rich sequences, were either equally toxic or more harmful to mammalian cells. These results suggest that the mere difference in DNA base composition of parasites and host cells may account for the selective toxicity of minor groove ligands. Distamycin A, DAPI and Hoechst 33258 were also found to be more toxic to Saccharomyces cerevisiae grown on glycerol than to yeast cells grown on glucose, consistent with the preferential binding of these ligands to the relatively AT-rich mitochondrial DNA of yeast cell. These results underscore the generality of selective toxicity of minor groove binders endowed by the DNA base composition.

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.

Malaria parasites demonstrably increase the permeability of the membrane of the erythrocyte in which they develop and propagate. New permeability pathways (NPPs) generated by parasite activity and identified in the erythrocyte membrane are held responsible for these changes. Here, we present a novel analysis of hemolysis curves of infected cells in iso-osmotic solutions of solutes that penetrate selectively into infected cells, as a function of parasite development. The analysis yields three parameters: the t1/2 of lysis (reciprocally related to permeability), the maximal lysis, and a parameter that expresses the variation of the cell population. Different developmental stages of the parasite were obtained either by sampling synchronized cultures with time or by the fractionation of asynchronous cultures on a Percoll–sorbitol density gradient. While the results confirm previous reports on the stage-dependent evolution of NPPs, they also reveal that the evolution of NPPs is not synchronous: NPPs evolve differentially throughout the ring stage and only at the mid-trophozoite stage they are fully deployed in the majority of the infected cells, but not in all. This leads to desynchronization in the culture and to less than the maximal possible rate of multiplication.

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by 4-aminoquinolines. As a result FP accumulates in the membrane fraction and associates with enzymes of infected cells in parallel with parasite killing. Free FP is degraded by reduced glutathione (GSH). This degradation is inhibited by chloroquine (CQ) and amodiaquine (AQ) but not by quinine (Q) or mefloquine (MQ). Increased GSH levels in Plasmodium falciparum-infected cells confer resistance to CQ and vice versa, and sensitize CQ-resistant Plasmodium berghei by inhibiting the synthesis of glutathione. Some drugs are known to reduce GSH in body tissues when used in excess, either due to their pro-oxidant activity or their ability to form conjugates with GSH. We show that acetaminophen, indomethacin and disulfiram were able to potentiate the antimalarial action of sub-curative doses of CQ and AQ in P. berghei- or Plasmodium vinckei petteri-infected mice, but not that of Q and MQ. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. Although these results imply that alteration in GSH are involved, measurement of total glutathione either in uninfected or P. berghei-infected mice, treated with these drugs did not reveal major changes. In conclusion, experimental evidences provided in this study suggest that some off the counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.

Esters of amino acids are known to penetrate into cells by simple diffusion. Subsequently, they are hydrolyzed by hydrolases to release the parent amino acid. Due to the abundance of hydrolases in phagolysosomes, amino acids accumulate, there because the rate of influx and hydrolysis exceed the rate of amino acid efflux through specific carriers. The osmotic effect of this accumulation results in the disruption of the organelles. This mechanism has been demonstrated to be responsible for the killing of Leishmania amastigotes by amino acid esters. In this investigation, it is shown that all esters tested, including alcohol esters, N-acetyl esters and the esters of some dipeptides, inhibit the growth of Plasmodium falciparum in culture. Inhibition is time-dependent and, in some cases, ring-stage parasites are more sensitive than trophozoites. Similar to the findings with Leishmania, alcohol esters of Glu, Leu, Met, Phe and Trp are more toxic to Plasmodium whereas Ala, Gly, His and Ile are much less noxious. Esters caused the release of acridine orange that selectively accumulates in the phagolysosome-like food vacuole of the parasite, attesting the ostensible destruction of this organelle by osmotic lysis. The toxicity of the N-acetyl esters is probably associated in part to their ability to inhibit cytosolic proteases. Since excess of amino acids can also inhibit proteolysis, the effect of free amino acids on parasite growth was also tested. Of the 19 odd amino acids tested, only three, namely Cys, His and Trp, were found to be toxic to the parasites at millimolar concentrations and the reasons for their possible specific toxicity are discussed.

Transglutaminase was identified in malaria parasites by immunofluorescence microscopy using alpha-transglutaminase antiserum. Functional enzyme was demonstrated in vivo and in vitro using labeled polyamines that become incorporated into protein substrates through TGase activity. In Plasmodium falciparum intraerythrocytic parasites, transglutaminase activity was stage-dependent: it was weak in ring-forms but much stronger in trophozoites and schizonts. High levels of activity were detected in P. gallinaceum zygotes and ookinetes and in capsules of oocysts developing on mosquito midguts. Unlike most known transglutaminases, the enzymatic activity in Plasmodium was Ca(2+)-independent. Furthermore, levels of activity were similar at 37 and 26 degrees C. Parasite transglutaminase may be responsible for the modification of erythrocytic cytoskeleton in infected cells and it may facilitate the construction of oocyst capsules by cross-linking mosquito-derived basement membrane components with Plasmodium-derived proteins.

During the digestion of its host cell hemoglobin, large amounts of toxic ferriprotoporphyrin IX (FPIX) are generated in the intraerythrocytic malaria parasite. FPIX is detoxified either by being polymerized into hemozoin inside the food vacuole, or through its degradation by glutathione in the cytosol. Chloroquine is able to complex with FPIX, thus inhibiting both processes and thereby generating receptors for its own uptake. These leads to the accumulation of FPIX in the membrane fraction of infected cells that results in membrane permeabilization and disruption of cation homeostasis and concluded in parasite death. Several unresolved questions, such as the site of FPIX:chloroquine complex formation, the role of pH gradient in drug accumulation and resistance, the role of Pgh-1 in resistance, the mode of action of reversers and the involvement of proteins and their mutants in resistance, are discussed. Copyright 1999 Harcourt Publishers Ltd.

The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC 50 ) in the micromolar range. Among these, the substituted S4 analog K 4 K 20 -S4 was the most potent (IC 50 = 0.2 μM), while its shorter version, K 4 -S4(1–13)a, retained a considerable potency (IC 50 = 6 μM). Both K 4 K 20 -S4 and K 4 -S4(1–13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K 4 K 20 -S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K 4 K 20 -S4, the most hemolytic dermaseptin. The same trend was observed with K 4 -S4(1–13)a. The d isomers of K 4 K 20 -S4 or K 4 -S4(1–13)a were as active as the l counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.

We have shown previously that chloroquine and amodiaquine inhibit the glutathione-dependent degradation of ferriprotoporphyrin IX (FP). We have also demonstrated that treatment of human erythrocytes infected with Plasmodium falciparum with chloroquine or amodiaquine results in a dose- and time-dependent accumulation of FP in the membrane fraction of these cells in correlation with parasite killing. High levels of membrane FP are known to perturb the barrier properties of cellular membranes, and could thereby irreversibly disturb the ion homeostasis of the parasite and cause parasite death. We here report on the effect of various 4-aminoquinolines, as well as pyronaridine, halofantrine and some bis-quinolines, on glutathione-mediated destruction of FP in aqueous solution, when FP was bound non-specifically to a protein, and when it was dissolved in human erythrocyte ghost membranes. We showed that all drugs were capable of inhibiting FP degradation in solution. The inhibitory efficacy of some drugs declined when FP was bound non-specifically to protein. Quinine and mefloquine were unable to inhibit the degradation of membrane-associated FP, in line with their inability to increase membrane-associated FP levels in malaria-infected cells following drug treatment. The discrepancy between chloroquine and amodiaquine on the one hand, and quinine and mefloquine on the other, is discussed in terms of the particular location of drugs and FP in the phospholipid membrane, and may suggest differences in the mechanistic details of the antimalarial action of these drugs.

The intraerythrocytic malaria parasite digests considerable amounts of its host cell cytosol, which consists mostly of hemoglobin. In order to avert the toxicity of ferriprotorphyrin IX (FP) thus produced, it is generally accepted that FP is polymerized to the non-toxic hemozoin. Investigating the fate of FP in cultured Plasmodium falciparum -infected human red blood cells, revealed a straight correlation between amounts of digested hemoglobin and hemozoin, but the latter contained less FP than produced. The efficacy of FP polymerization is stage-dependent, increasing with parasite maturation. Different strains display dissimilar efficacy in hemozoin production. Unpolymerized FP possibly exits the food vacuole and is degraded by glutathione, thus accounting for the low levels of free FP found in infected cells. 4-aminoquinoline antimalarials demonstrably form complexes with FP and inhibit hemozoin production in vitro. Chloroquine, amodiaquine, quinine and mefloquine were found to inhibit hemozoin production in intact infected cells, but only the first two drugs caused a dose-dependent accumulation of FP in the membrane fraction of infected cells that correlated well with parasite killing, due to the permeabilization of membranes to ions. This differential effect is explained by the ability of chloroquine and amodiaquine to inhibit the degradation of membrane-associated FP by glutathione and the incapacity of quinine and mefloquine to do so. This discrepancy implies that the antimalarial mode of action of chloroquine and amodiaquine is different in its mechanistic details from that of quinine and mefloquine and is compatible with the diametric sensitivity of most strains to chloroquine and mefloquine and the disparate interaction of these drugs with enhancers of their antimalarial action.

We propose here a new and detailed model for the antimalarial action of chloroquine (CQ), based on the its ability to inhibit degradation of heme by glutathione. Heme, which is toxic to the malaria parasite, is formed when the intraerythrocytic malaria parasite ingests and digests inside its food vacuole its host cell cytosol, which consists mainly of hemoglobin. The parasite protects itself against the toxicity of heme by polymerizing some of it to insoluble hemozoin (HZ). We show here that in Plasmodium falciparum at the trophozoite stage only ca. 30% of the heme is converted into hemozoin. We suggest that nonpolymerized heme exits the food vacuole and is subsequently degraded by glutathione, as has been shown before for uninfected erythrocytes. Marginal amounts of free heme could be detected in the membrane fraction of infected cells but nowhere else. It is well established that CQ and amodiaquine (AQ) accumulate in the parasite's food vacuole and inhibit heme polymerization, thereby increasing its efflux out of the food vacuole. We found that these drugs competitively inhibit the degradation of heme by glutathione, thus allowing heme to accumulate in membranes. Incubation of intact infected cells with CQ and AQ results in a marked increase in membrane-associated heme in a dose- and time-dependent manner, and a relationship exists between membrane heme levels and the extent of parasite killing. Heme has been shown to disrupt the barrier properties of membranes and to upset ion homeostasis in CQ-treated malaria-infected cells. In agreement with the predictions of our model, increasing the cellular levels of glutathione leads to increased resistance to CQ, whereas decreasing them results in enhanced sensitivity to the drug. These results insinuate a novel mechanism of drug resistance.

SUMMARYThe balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malaria combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their β-O-glucosidic bond, become potent pro-oxidants. In this work we show that the glucosides inhibit the growth ofPlasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of β-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD-erythrocytes. These results suggest that G6PD-infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD-erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD-bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD-genotype.

Phagocytic cells constitute the first line of defence against malarial parasites. They perform their role by delivering oxidative radicals and by phagocytosing infected red blood cells (IRBC). Phagocytosis is mediated by antibody binding to clustered band 3 antigen in the IRBC membrane and activation of the alternative complement pathway. In this study we showed that treatment of IRBC containing Plasmodium falciparum with therapeutically-relevant concentrations of antimalarial drugs considerably reduced the binding of immunoglobulin G (IgG) to, and the phagocytosis of, IRBC. Opsonization of IRBC by fresh serum before drug treatment prevented this inhibitory action of drugs. Removal of the drug restored IgG binding and the phagocytic susceptibility of IRBC in a time-dependent fashion. Direct measurement of the effect of chloroquine on the clustering of band 3 in IRBC, however, failed to reveal any disruption of the aggregation. We conclude that antimalarial drugs are able to alter, by an as yet unresolved mechanism, the affinity of IgG to clustered band 3. This affinity of IRBC seems to be determined by a dynamic process that depends on the metabolic activity of the parasite.

Intralipid ® and Ivelip ® are commercial preparations of soy-bean lipid extracts used for intravenous supplementation of lipids in various clinical conditions. They were found to inhibit the growth of Plasmodium falciparum in culture with an IC 50 of 8.07 ± 2.13 and 13.32 ± 2.05 mg.ml −1 , respectively. Intralipid ® rapidly and efficiently inhibited nucleic acid synthesis in cultured P. falciparum , exhibiting full inhibitory activity in less than 2 h. Ivelip ® injected intraperitoneally, was found by the 4-day suppressive test to be active in vivo against P. vinckei petteri within the normal recommended regimen for dietary lipid supply (0.5–4 g.kg −1 ), but it was impossible to obtain a radical cure even with very high doses (6.4 g.kg −1 ). Ivelip ® was less effective against P. berghei and P. yoelii nigeriensis . As Ivelip ® showed no interference with the antimalarial activity of chloroquine, it could be considered for use in the treatment of severe human malaria in association with 4-aminoquinolines to expedite the clearance of parasites.

Following the demonstration of the antimalarial effect of the long chain saturated alcohol n -hentriacontanoi ((CH 2 ) 29 CH 2 OH), isolated from the Bolivian endemic solanaceous plant Cuatresia sp., we have tested the effect of the C18 fatty acids oleic, elaidic, linoleic, and linoleic on malaria parasites. These fatty acids inhibited the parasitemic development in mice infected with Plasmodiurn vinckei petteri or with Plasmodium yoelii nigeriensis in a 4-day suppressive test. To gain a deeper discernment of the antimalarial mode of action, the effects of these compounds were evaluated on Plasmodiun falciparum growth in culture. Whereas n -hentriacontanol did not show any inhibition of this parasite, on the contrary, the C18 acids displayed a considerable inhibitory activity at ≤ 200 μg/ml both in intact infected cells and in free parasites. In order to understand the mechanism of their antimalarial action, several tests were performed. No hemolysis of infected cells could be observed up to 500 μg/ml. No effect on the lipid peroxidation, ATP levels, transport through the parasite-induced permeabiIity pathways, or on the phagocytosis of the infected cells could be observed. The cytotoxic effect of the fatty acids was very rapid: full inhibition of nucleic acids and protein syntheses was observed in less than 30 min. This inhibition was not relieved by the addition of deferrioxamine or FeCl 3 , indicating that fatty acids (FA) do not act by facilitating the transport of iron. Inhibition was relieved in neither the presence of erotic acid or its methyl ester, indicating that FA do not act at the mitochondrial level of pyrimidine synthesis. Inhibition was not relieved when cells were incubated in high potassium medium, implying that FA did not act by depleting the parasite of this physiologically important cation. Vitamin E had no protective effect and no evidence for Lipid peroxidation could be obtained, suggesting that the inhibitory effect of FA was not due to lipid peroxidation. These results suggest that octadecenoic acids may have a specific target(s) inside the parasite. Further investigations are warranted since soybean lipid emulsions used for parenteral nutrition show similar inhibition of parasite propagation in vivo and in culture.

Malaria constitutes one of the major health threats in the tropical and sub-tropical areas of the world. Yet, few advances were made in recent years in revealing the mode of action of the common and most economically affordable antimalarial drugs, the schizontocidal 4-aminoquinolines. Data presented indubitably repudiate the previous notions that these drugs act by either halting the feeding of the parasite on its host erythrocyte cytosol or repressing nucleic acid synthesis due to intercalation into the parasite's DNA. A novel target for drugs is outlined, i.e. they are shown to inhibit in vitro the release of iron from acidified host cell cytosol, consisting mostly of hemoglobin, a process that could provide this trace element to the parasite. Resistance to quinoline-containing drugs is the principal reason for the present resurgence of malaria. Drug-resistant parasites accumulate less of these weak base-like drugs in the acidic digestive vacuoles. A kinetic model is presented, indicating that diminishing drug accumulation is due to decreased vacuolar proton pump activity and is not a result of a putative multidrug resistance (MDR) efflux pump. Findings to date on the molecular biology of parasite mdr genes are reviewed. These indicate no correlation between gene expression or mutations and phenotypic drug resistance. Reversal of parasite drug resistance by relevant compounds in MDR cancer cells seems to involve mechanism(s) different from the inhibition of the MDR pump in cancer cells.

The quinoline-containing antimalarial drugs chloroquine, quinine and mefloquine exert an irreversible inhibitory effect on erythrocytic stages of Plasmodium falciparum grown in culture. Inhibition is time-and and concentration-dependent and the full effect is observed after 2–6 hours of exposure to the drug. Washing of infected cells after drug exposure in the presence of NH 4 Cl to accelerate drug efflux, intensifies the inhibitory effect of chloroquine, probably due to the pH-dependent release of highly concentrated drug from the acidic food vacuole of the parasite. When both antimalarials and NH 4 Cl are present in the culture, drug effect is reduced, as expected from the demonstrable alkalinization of the food vacuole and the consequent reduction in drug accumulation. The protease inhibitor leupeptin inhibits digestion of ingested host cell cytosol, and thus inhibits parasite growth, though reversibly so (Rosenthal et al, J. Clin. Invest. 82 1560–1566 (1988)). Thus, although the antimalarials also inhibit the feeding process, this is not the cause of their irreversible action. Leupeptin is found to be antagonistic to antimalarials' action, suggesting that the drugs from complexes with products of host cell digestion that are responsible for irreversible inhibition of parasite growth.

The host cell membrane of Plasmodium falciparum infected cells becomes permeabilized at the trophozoite stage. A variety of otherwise impermeant substances such as carbohydrates, polyols, amino acids and anions easily gain access to the cytosol of infected cells. Using the isotonic-hemolysis method or uptake of labeled substances, we characterized the new permeation pathways as pores of approximately 0.7 nm equivalent radius. The pores bear a positively charged character which facilitates movement of small anions and excludes cations, so that the ionic composition and osmotic properties of infected cells are not drastically altered. Substances of a molecular size similar to that of disaccharides are fully excluded. Substances of limiting size might be accommodated in the pore, provided they bear a side group of hydrophobic character. The new permeation pathways may provide a vital route for acquisition or release of essential nutrients or catabolites.

The initial rates of uptake of l -tryptophan into normal human red blood cells and into cells infected by the malarial parasite Plasmodium falciparum in vitro, were investigated. We find that transport in non-infected cells, which is mediated by the specific saturable T system and the apparently non-saturable L system (Rosenberg, Young and Ellory (1980) Biochim. Biophys. Acta 598, 375–384) is considerably enhanced by blood preservation and culture conditions. This increase is mostly due to an increase in the maximal velocity of the saturable component and of the rate constant of the linear component. Uptake is further enhanced in non-infected cells by factors released from infected cells into the culture medium and, even more so, in infected cells at the advanced stage of intraerythrocytic parasite development. At these stages the susceptibility of the transport system to the non-specific inhibitor phloretin and to the competitive inhibitor phenylalanine, is virtually lost. The effect of the parasite on l -tryptophan uptake by the host cell membrane is exerted only on the maximal velocity of the T system, which is carrying most of the substrate under physiological conditions. The possible implications of these findings to the life of the intraerythrocytic parasite are briefly discussed.

The Influenza Matrix 2 (M2) protein is the target of Amantadine and Rimantadine which block its H + channel activity. However, the potential of these aminoadamantyls to serve as anti-flu agents is marred by the rapid resistance that the virus develops against them. Herein, using a cell based assay that we developed, we identify two new aminoadamantyl derivatives that show increased activity against otherwise resistant M2 variants. In order to understand the distinguishing binding patterns of the different blockers, we computed the potential of mean force of the drug binding process. The results reveal that the new derivatives are less mobile and bind to a larger pocket in the channel. Finally, such analyses may prove useful in designing new, more effective M2 blockers as a means of curbing influenza. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.

Intraerythrocytic malaria parasites feed on their host cell cytosol. We show that human red blood cells infected with the malaria parasite Plasmodium falciparum , produce free amino adds the composition of which resembles that of globin, the most abundant red blood cell protein. The rate of amino acid production is almost equal to the rate of efflux of these acids from the infected cell. Production of amino acids increases with parasite age: the rates of production at the young ring and the mature trophozoite stages were 3.3 and 13.5 nmol/10 8 infected cells per min at 37°, respectively, compared with 0.04 nmol/10 8 cells per min in uninfected cells. The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, inhibit amino acid production at the same concentrations at which they inhibit parasite growth, but have no effect on the endogenous parasite protein degradation. We suggest that parasite feeding on host cell cytosol is the primary target for the antimalarial action of these drugs. Chloroquine accumulation, the rate of amino add production by infected cells and the inhibitory effect of the drug, were determined simultaneously at the different stages of parasite development. At all stages the rate of amino add production and chloroquine accumulation were directly related and both were inversely related to the inhibitory effidency of the drug. The lysosomotropic agents methylamine and NH 4 Cl at millimolar concentrations also inhibit amino add production, suggesting that the process is pH dependent and localized in the vacuole. Host cytosol degradation and drug accumulation both ake place in the parasite food vacuole. Our observations imply that the metabolically dependent acidification of this parasite organelle is involved in both processes.

Quinoline-containing antimalarial drugs accumulate inside the acid food vacuole of the parasite where they inhibit the digestion of ingested host cell cytosol, and consequently, parasite growth. In order to verify whether this inhibition is caused by drug-induced alkalinization of the food vacuole, we investigated the accumulation of acridine orange (AO) as a vacuolar pH probe in intact Plasmodium falciparum-infected human erythrocytes as affected by the drugs chloroquine (CQ), 7H-quinoleine (7HQ), quinine (Q) and mefloquine (MQ). It was established by various criteria that AO accumulates primarily in the acid compartment(s) of the parasite as a function of the pH difference between it and the extracellular medium. This pH gradient was dissipated by the drugs in the rank order MQ greater than CQ greater than Q greater than 7HQ. The kinetics of vacuolar alkalinization and the concentration ranges at which it was observed imply that the monoprotic drugs MQ and Q exerted their effect mostly by translocating protons across the vacuolar membrane, i.e. they could cross the membrane as a protonated species, while the diprotic drugs CQ and 7HQ raised the vacuolar pH mostly by proton trapping. Similarly, hydrophobic alkylamines raised the vacuolar pH by proton translocation, while their relatively more polar congeners and ammonia did so by proton titration. However, the alkalinizing effect of each drug was observed at a concentration which was 1-2 orders of magnitude larger than the IC50 of its antimalarial effect. These results mean that vacuolar alkalinization is not the primary effect of antiparasitic action of quinoline antimalarials.

Erythrocytes actively metabolize glucose to lactic acid. Disposal of the latter is accomplished by three passive membrane pathways: a high-affinity low capacity H+-lactate cotransport system specific for L-lactate (I), a low affinity high-capacity transport system involved physiologically in C1-HCO3 exchange (II) and simple diffusion (III). In P. falciparum infected cells, intracellular lactic acid production rises by more than 100 fold. It was our assumption that specific blockade of pathway II should raise intraerythrocytic lactic acid primarily in infected cells and thus affect in vitro parasite growth. Blockade was accomplished by 4,4′-dinitro-2, 2′-stilbene disulfonate (DNDS) or the irreversible 4,4′-diisothiocyano analog DIDS, both nonpenetrating, specific inhibitors of II and by use of phloridzin and furose-mide, two classical but less specific inhibitors of II. In in vitro culture conditions, DNDS inhibited both, system-II mediated anion transport and parasite growth (but not invasion) with ID50 of ca50μM. Similar results were obtained with phloridzin (ID5020μM) and furosemide (ID5050μM). Chemical modification of noninfected cells with DIDS inhibited anion transport (ID500.25μM), arrested growth (ID501.0μM) but did not prevent invasion.

Various lysosomotropic detergents were tested in this work on in vitro cultures of Plasmodium falciparum and are shown to be potent antimalarial agents. The order of antimalarial potency was similar to that of cell toxicity on mammalian cells in culture (Miller DK et al., J Cell Biol97, 1841–1851 (1983)). The most efficient agents, N-dodecyl-imidazole (NDI) and N-dodecyl morpholine (NDM) displayed ic 50 values of 6.7 ± 0.7 μM and 23 ± 5 μM. The mechanism of action of NDI measured at ic 50 concentrations displayed the following features: irreversible antimalarial effect after 15 min exposure of cells to drug; alkalinization of the parasite food vacuole; inhibition of protein synthesis; inhibition of host cell protein digestion by the parasite; lack of vacuolar membrane disruption; lack of effect on the rate of constitutive autoproteolysis. No biochemical or ultrastructural indications were found to support a detergent-like action of NDI and its structural congeners on the major acidic compartment of the parasite, the food vacuole. Rather, alkalinization of that compartment by weak-base accumulation properties of the amphiphilic drugs and ensuing protonophoric effect are likely to play a major role in the various parasite-associated properties affected by these drugs.

The inhibitory effect of metal chelators on intraerythrocytic malarial parasites imply that trace metal have a vital role in the biology of these organisms. In the present work X-ray fluorometry was used to study the status of zinc and iron in human red blood cells infected with Plasmodium falciparum in culture conditions. It was found that while the iron level remains constant throughout the parasite cell cycle, that of zinc increases in parallel with parasite maturation to reach a 2.3-fold higher level than that of uninfected red blood cells. Compartment analysis of infected red blood cells indicated that most of this gain was associated with the parasite and some with the host-cell membrane. Analysis of the malarial pigment showed that the zinc/iron ratio was similar to that of red blood cells, implying the this compound, which results from the digestion of host-cell cytosol, sequesters the zinc of host metalloenzymes. Dipicolinic acid (DPA), like other chelators, was found to inhibit the intracellular development of the parasite with an ED50 of 1 mM. DPA does not penetrate into normal red blood cells but readily permeates into infected cells, although it does not leach out their zinc. It is uncertain whether the inhibitory effect of DPA is exerted through alterations of host cell metabolism or by directly affecting that of the parasite. The putative receptors of zinc in the infected red blood cell are discussed.