[Show abstract][Hide abstract] ABSTRACT: Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy.
[Show abstract][Hide abstract] ABSTRACT: At least 53 distinct isoforms of PfCRT protein are expressed in strains or isolates of Plasmodium falciparum malarial parasites from around the globe. These parasites exhibit a range of sensitivities to chloroquine (CQ) and other drugs. Mutant PfCRT is believed to confer cytostatic CQ resistance (CQRCS) by transporting CQ away from its DV target (free heme released upon hemoglobin digestion). One theory is that variable CQ transport catalyzed by these different PfCRT isoforms is responsible for the range of CQ sensitivities now found for P. falciparum. Alternatively, additional mutations in drug-selected parasites, or additional functions of PfCRT, might complement PfCRT mediated CQ transport in conferring the range of observed resistance phenotypes. To distinguish between these possibilities we recently optimized a convenient method for measuring PfCRT mediated CQ transport, involving heterologous expression in Saccharomyces cerevisiae. Here, we use this method to quantify drug transport activity for 45 / 53 of the naturally occurring PfCRT isoforms. Data show that variable levels of CQR likely depend upon either additional PfCRT functions or additional genetic events, including perhaps changes that influence DV membrane potential. The data also suggest that the common K76T PfCRT mutation that is often used to distinguish a P. falciparum CQR phenotype is not, in and of itself, a fully reliable indicator of CQR status.
[Show abstract][Hide abstract] ABSTRACT: Elucidating mechanisms of antimalarial drug resistance accelerates development of improved diagnostics and the design of new, effective malaria therapy. Recently, several studies have emphasized that chloroquine (CQ) resistance (CQR) can be quantified in two very distinct ways, depending on whether sensitivity to the growth inhibitory effects or parasite-kill effects of the drug are being measured. It is now clear that these cytostatic and cytocidal CQR phenotypes are not equivalent, and recent genetic, cell biological, and biophysical evidence suggests how the molecular mechanisms may overlap. These conclusions have important implications for elucidating other drug resistance phenomena and emphasize new concepts that are essential for the development of new drug therapy.
No preview · Article · Feb 2014 · Trends in Parasitology
[Show abstract][Hide abstract] ABSTRACT: Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L(-1)), dihydroartemisinic acid (70.0±0.3 mg L(-1)), arteannuin B (1.3±0.0 mg L(-1)), isovitexin (105.0±7.2 mg L(-1)) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B's potentiation of artemisinin.
[Show abstract][Hide abstract] ABSTRACT: Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR(CS)) as quantified by CQ IC50 shift, account for only 10-20% of cytocidal CQR (CQR(CC)) as quantified by CQ LD50 shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR(CS) vs CQR(CC) phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD50. Candidate genes in these loci are consistent with a role for autophagy in CQR(CC), leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR(CC). Taken together, the data show that an unusual autophagy - like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD50 doses (but not IC50 doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR(CC) phenotype.
[Show abstract][Hide abstract] ABSTRACT: Drug combination therapy is the frontline of malaria treatment. There is an ever-accelerating need for new, efficacious combination therapies active against drug resistant malaria. Proven drugs already in the treatment pipeline, such as the quinolines, are important components of current combination therapy and also present an attractive test bank for rapid development of new concepts.
The efficacy of several drug combinations versus chloroquine-sensitive and chloroquine-resistant strains was measured using both cytostatic and cytocidal potency assays.
These screens identify quinoline and non-quinoline pairs that exhibit synergy, additivity, or antagonism using the fixed-ratio isobologram method and find tafenoquine -- methylene blue combination to be the most synergistic. Also, interestingly, for selected pairs, additivity, synergy, or antagonism defined by quantifying IC50 (cytostatic potency) does not necessarily predict similar behaviour when potency is defined by LD50 (cytocidal potency). These data further support an evolving new model for quinoline anti-malarials, wherein haem and haemozoin are the principle target for cytostatic activity, but may not be the only target relevant for cytocidal activity.
[Show abstract][Hide abstract] ABSTRACT: A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify
the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite
proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the
antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins
interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.
Full-text · Article · Jun 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The function of P. falciparum chloroquine resistance transporter (PfCRT) can be quantified using a S. cerevisiae model system (Baro, N. K., Pooput C and Roepe P.D. Biochemistry. 50, 6701 - 6710). We further optimize this system to distinguish PfCRT isoforms found in P. falciparum strains and isolates from across the globe. We create and express 13 naturally occurring pfcrt alleles associated with a range of chloroquine resistant (CQR) phenotypes. Using galactose induction of PfCRT we quantify PfCRT and chloroquine (CQ) dependent yeast growth inhibition, and [3H]-CQ transport specifically due to a given PfCRT isoform. Surprisingly, we find poor correlation between these parameters vs CQ IC50 observed in strains of malaria harboring the same isoforms. This suggests that increased CQ transport due to PfCRT mutation is necessary, but not sufficient, for the range of CQ IC50 observed in globally distributed CQR P. falciparum isolates.
[Show abstract][Hide abstract] ABSTRACT: Historically, the most successful molecular target for antimalarial drugs has been heme biomineralization within the malarial parasite digestive vacuole. Heme released from catabolized host red blood cell hemoglobin is toxic, so malarial parasites crystallize heme to nontoxic hemozoin. For years it has been accepted that a number of effective quinoline antimalarial drugs (e.g. chloroquine, quinine, amodiaquine) function by preventing hemozoin crystallization. However, recent studies over the past decade have revealed a surprising molecular diversity in quinoline-heme molecular interactions. This diversity shows that even closely related quinoline drugs may have quite different molecular pharmacology. This paper reviews the molecular diversity and highlights important implications for understanding quinoline antimalarial drug resistance and for future drug design.
No preview · Article · Apr 2013 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Ezrin is a key regulator of osteosarcoma metastasis. Our earlier work has established that NSC305787 can directly bind to ezrin and inhibit its activity, which results in reduced metastasis. The objective of this study was to evaluate common antimalarial drugs including quinine, quinidine, mefloquine, chloroquine and amodiaquine that share significant structural features with lead compound NSC305787 for their potential to inhibit ezrin function. Compounds were evaluated on inhibiting chemotaxis of osteosarcoma cells and inhibiting cancer cell invasion of HUVEC monolayer, both of which were measured by electric impedance based methods. Although tested compounds share structural similarities with NSC305787, they were ineffective in binding to ezrin and inhibiting its biochemical and biological functions. The current results will guide us in designing and synthesis of novel improved molecules that will specifically target and inhibit ezrin function.
[Show abstract][Hide abstract] ABSTRACT: Bioassay-guided fractionation of an ethanol extract of the leaves and inflorescence of Mallotus oppositifolius collected in Madagascar led to the isolation of the two new bioactive dimeric phloroglucinols mallotojaponins B (1) and C (2), together with the known mallotophenone (3). The structures of the new compounds were determined on the basis of spectroscopic evidence, including their 1D- and 2D-NMR spectra, mass spectrometry, and an X-ray crystal structure. Compounds 1 and 2 showed potent antimalarial activity against chloroquine-resistant Plasmodium falciparum, with IC(50) values of 0.75 ± 0.30 and 0.14 ± 0.04 μM, while 3 was inactive in this assay. Compounds 1-3 also displayed strong antiproliferative activity against the A2780 human ovarian cancer cell line (IC(50) 1.10 ± 0.05, 1.3 ± 0.1 and 6.3 ± 0.4 μM, respectively).
Full-text · Article · Jan 2013 · Journal of Natural Products
[Show abstract][Hide abstract] ABSTRACT: The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against P. falciparum [Karle JM, Karle IL, Gerena L, Milhous, WK. 1992. Antimicrob. Agents Chemother. 36: 1538-1544]. We synthesized 9-epi-QN (eQN) and 9-epi-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities. Relative to QN and QD we observe a large decrease in cytostatic activity (higher IC(50)) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent with previous work. However, we observe relatively small changes in cytocidal activity (LD(50)). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency, and did not affect pH-dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed the 9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-VIS titrations show that eQN and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of zinc(II) protoporphyrin IX (ZnPIX) indicates binding of the 9-epimers to monomeric heme is via a distinct geometry. We isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UV-VIS spectroscopies. The 9-epimers produced low-fluorescent adducts with 2:1 stoichiometry (drug:FPIX) which did not survive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme interactions as a drug target for cytostatic vs. cytocidal dosages of quinoline antimalarial drugs, and further elucidate a surprising structural diversity of quinoline antimalarial drug-heme complexes.
Full-text · Article · Oct 2012 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: We report an improved, nonhazardous, high-throughput assay for in vitro quantification of antimalarial drug inhibition of β-hematin (hemozoin) crystallization performed under conditions that are
more physiological relative to previous assays. The assay uses the differential detergent solubility of crystalline and noncrystalline
forms of heme and is optimized via the use of lipid catalyst. Using this assay, we quantify the effect of pH on the crystal
growth-inhibitory activities of current quinoline antimalarials, evaluate the catalytic efficiencies of different lipids,
and test for a possible correlation between hemozoin inhibition by drugs versus their antiplasmodial activity. Consistent
with several previous reports, we found a good correlation between hemozoin inhibition potency versus cytostatic antiplasmodial
potency (50% inhibitory concentration) for a series of chloroquine (CQ) analogues. However, we found no correlation between
hemozoin inhibition potency and cytocidal antiplasmodial potency (50% lethal dose) for the same drugs, suggesting that cellular
targets for these two layers of 4-aminoquinoline drug activity differ. This important concept is also explored further for
QN and its stereoisomers in the accompanying paper (A. P. Gorka, K. S. Sherlach, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:365–374, 2013).
Full-text · Article · Oct 2012 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Programmed cell death (PCD) pathways remain understudied in parasitic protozoa in spite of the fact that they provide potential targets for the development of new therapy. The best understood PCD pathway in higher eukaryotes is apoptosis although emerging evidence also points to autophagy as a mediator of death in certain physiological contexts. Bioinformatic analyses coupled with biochemical and cell biological studies suggest that parasitic protozoa possess the capacity for PCD including a primordial form of apoptosis. Recent work in Toxoplasma and emerging data from Plasmodium suggest that autophagy-related processes may serve as an additional death promoting pathway in Apicomplexa. Detailed mechanistic studies into the molecular basis for PCD in parasitic protozoa represent a fertile area for investigation and drug development.
No preview · Article · Jul 2012 · Trends in Parasitology
[Show abstract][Hide abstract] ABSTRACT: Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN.
[Show abstract][Hide abstract] ABSTRACT: Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.
Full-text · Article · Dec 2011 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Investigation of extracts from the plant Athroisma proteiforme (Humbert) Mattf. (Asteraceae) for antimalarial activity led to the isolation of the five new sesquiterpene lactones 1–5 together with centaureidin (6). The structures of the new compounds were deduced from analyses of physical and spectroscopic data, and the absolute configuration of compound 1 was confirmed by an X-ray crystallographic study. Athrolides C (3) and D (4) both showed antiplasmodial activities with IC50 values of 6.6 (3) and 7.2 μM (4) against the HB3 strain and 5.5 (3) and 4.2 μM (4) against the Dd2 strain of the malarial parasite Plasmodium falciparum. The isolates 1–6 also showed antiproliferative activity against A2780 human ovarian cancer cells, with IC50 values ranging from 0.4 to 2.5 μM.
Full-text · Article · Oct 2011 · Journal of Natural Products