Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodiumfalciparumblood forms (W2 clone, chloroquine-resistant) oftamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl(ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 humanhepatoma cells. Surprisingly with these series, results indicate that the biologicalactivity of ruthenocifens is better than that of ferrocifens and other tamoxifen-likecompounds. The synthesis of a new metal-based compound is also described. It wasshown, for the first time, that ruthenocifens are good antiplasmodial prototypes.Further studies will be conducted aiming at a better understanding of their mechanismof action and at obtaining new compounds with better therapeutic profile.     

Plasmodium falciparum Carbonic Anhydrase is a Possible Target for Malaria Chemotherapy

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the α class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the α-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having α-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

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.     

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.     

微生物介导的恶性疟原虫PfMSP-1疫苗的研制现状

恶性疟原虫引起的恶性疟是一种严重危害人类健康的寄生虫病,采用疫苗防治该病是当前研究的热点领域之一。PfMSP-1抗原是一种有效的疫苗候选分子,鼠伤寒沙门菌、卡介苗、酵母菌、根癌农杆菌、嗜热四膜菌、腺病毒、牛痘病毒和杆状病毒等微生物经过改造后均可成为有希望的疫苗载体。本文综述了重组鼠伤寒沙门菌（rSt-PfMSP-1和rSt-PfMSP-119）、重组BCG疫苗（rBCG-PfMSP-1c和rBCG-PfMSP-119）、重组酵母菌（rPp-PfMSP-1）、重组根癌农杆菌（rAt-PfMSP-142、rAt-PfMSP4/5和rAt-Pf38）、重组嗜热四膜菌（rTt-PfMSP-119）、重组腺病毒（rAd5-PfMSP-142和rChAd63-PfMSP-1）、重组牛痘病毒（rVV-PfMSP-1和rVV-PfHGFSP）以及重组杆状病毒（rBDES-PfMSP-119）的构建及其免疫机制的研制现状。     

Differentiation of Gametocytes in Microcultures of Human Blood Infected with Plasmodium falciparum*

SYNOPSIS. Gametocytes differentiated from ring-stage parasites in microcultures of human blood infected with Plasmodium falciparum. Immature gametocytes could be distinguished morphologically from late asexual trophozoites after ～ 40 h of culture. Differentiation into crescentic forms took several days and the incorporation of [³H]-isoleucine by developing gametocytes was demonstrated. About 1% of red cells contained gametocytes at the maximum densities attained. Differentiation of gametocytes occurred either directly from rings placed in culture or from the progeny of subsequent cycles of schizogony and invasion in vitro. The latter occurrence was confirmed by the development of gametocytes in marker fetal red cells added to cultures, although fetal red cells provide a less favorable environment than those with HbA for growth of the parasites.     

多表位恶性疟疾疫苗M.RCAg-1在大肠杆菌表达的条件优化和大规模培养

通过以培养基配方、IPTG浓度、金属离子复合液浓度、镁离子浓度、表达时间、接种量、诱导时间点等发酵的重要条件对重组蛋白表达量影响的研究,确定多表位恶性疟疾疫苗M.RCAg-1蛋白最佳表达条件为以改良TB培养基培养、最优Mg2+,诱导剂IPTG和金属离子复合液浓度分别为10mmol/L,0.5mmol/L,6μl/ml,接种量为10%,表达时间为4.5h,将优化后的参数用于50L发酵罐进行连续3批中试规模的发酵,最终收获菌体湿重平均为31.8±1.78g/L,目的蛋白表达量可占菌体总蛋白的50%左右,试验确定了恶性疟疾多表位随机组合蛋白M.RCAg-1在大肠杆菌中的最优表达条件,该条件能够适合大规模培养需要。     

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

重组裂殖子表面蛋白质1特异性抗体有效抑制恶性疟原虫体外生长

42kD恶性疟原虫裂殖子表面蛋白质 1C末端片段 (MSP1 42 )是当今重要的疟疾疫苗候选抗原。为获得大量构象正确的MSP1 42重组蛋白进行疫苗有效性试验 ,在毕氏酵母系统中分泌表达了MSP1 42重组蛋白。通过与一组特异性识别构象表位的单抗反应 ,该重组蛋白在重要构象表位上与天然蛋白质一致。由该蛋白质诱生的抗体能有效地抑制恶性疟原虫的体外生长 ,这些结果为进一步开展MSP1 42重组蛋白疫苗有效性试验提供了基础     

Isolation and functional characterization of Plasmodium falciparum merozoite antigens

Merozoite surface proteins are thought to play an important role during the invasion of red blood cells by merozoites. In this article the strategies for the chromatographic isolation and for the functional and molecular characterisation of isolated antigens from freshly harvested Plasmodium falciparum merozoites from cultures are described.     

A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras

Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt “CVMNK” genotype in codons 72-76.     

Reduced variation around drug-resistant dhfr alleles in African Plasmodium falciparum

We have measured microsatellite diversity at 26 markers around the dhfr gene in pyrimethamine-sensitive and -resistant parasites collected in southeast Africa. Through direct comparison with diversity on sensitive chromosomes we have found significant loss of diversity across a region of 70 kb around the most highly resistant allele which is evidence of a selective sweep attributable to selection through widespread use of pyrimethamine (in combination with sulfadoxine) as treatment for malaria. Retrospective analysis through four years of direct and continuous selection from use of sulfadoxine-pyrimethamine as first-line malaria treatment on a Plasmodium falciparum population in KwaZulu Natal, South Africa, has revealed how recombination significantly narrowed the margins of the selective sweep over time. A deterministic model incorporating selection coefficients measured during the same interval indicates that the transition was toward a state of recombination-selection equilibrium. We compared loss of diversity around the same resistance allele in two populations at either extreme of the range of entomological inoculation rates (EIRs), namely, under one infective bite per year in Mpumalanga, South Africa, and more than one per day in southern Tanzania. EIRs determine effective recombination rates and are expected to profoundly influence the dimensions of the selective sweep. Surprisingly, the dimensions were broadly consistent across both populations. We conclude that despite different recombination rates and contrasting drug selection histories in neighboring countries, the region-wide movement of resistant parasites has played a key role in the establishment of resistance in these populations and the dimensions of the selective sweep are dominated by the influence of high initial starting frequencies.     

Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria

We have adopted a proteomic strategy to investigate the actions of the two active components of the new antimalarial CoArtem, artemether and lumefantrine, following pharmacologically relevant drug exposure in the human malaria parasite Plasmodium falciparum. Both drugs induced profound alterations in the parasite's proteome. Moreover, the pattern of proteome alteration was specific for the drug used. The two drugs induced opposing effects on key glycolytic enzymes while exerting similar influence of the expression of stress response proteins. These initial results demonstrate the power of this approach in the study of pleiomorphic mechanisms of drug action.     

恶性疟原虫红内期Pf332抗原基因未知序列的测定及分析

测定恶性疟原虫红内期Pf332抗原 (Ag332 )基因的未知序列 ,并进行序列分析 .根据非洲恶性疟原虫Palo alto株Pf332基因的G1片段序列 ,设计 1对引物 ,从中国恶性疟原虫海南株 (FCC1 HN)基因组DNA中扩增出P332 1片段 .Pf332基因中经常出现SVTEEI短肽的编码序列 ,据此分别设计非特异的正、反义寡核苷酸引物 (NSP1、NSP2 ) ,应用低严谨PCR(LSPCR)分别扩增出P332 1邻近的未知序列片段P332 up1和P332 dow1.根据恶性疟原虫Palo alto株Pf332基因G1片段上、下游的G9和C1片段序列以及测定的P332 up1和P332 dow1序列 ,分别设计 2对特异引物继续扩增邻近的未知序列片段P332 up2和P332 dow2 .根据P332 dow2片段的 3'端序列 ,设计 2条特异引物分别与非特异引物NSP2行LSPCR和巢式PCR ,扩增出P332 dow2邻近的未知序列片段P332 dow3.对获得的Pf332基因片段进行序列测定 ,并用分子生物学软件辅助进行序列分析 .序列测定和拼接结果显示 ,共获得了连续 6 14 4bp的恶性疟原虫FCC1 HN株Pf332基因序列 .序列分析表明 ,所获得的 614 4bp序列位于Pf332基因的编码区内 ,不含内含子 ,编码 2 0 4 8个氨基酸残基 ,包含 5个氨基酸残基重复区 .对恶性疟原虫FCC1 HN株Pf332基因 6 14 4bp序列的测定和分析 ,为获得Pf332全基因     

Meiotic recombination, cross-reactivity, and persistence in Plasmodium falciparum

We incorporate a representation of Plasmodium falciparum recombination within a discrete-event model of malaria transmission. We simulate the introduction of a new parasite genotype into a human population in which another genotype has reached equilibrium prevalence and compare the emergence and persistence of the novel recombinant forms under differing cross-reactivity relationships between the genotypes. Cross-reactivity between the parental (initial and introduced) genotypes reduces the frequency of appearance of recombinants within three years of introduction from 100% to 14%, and delays their appearance by more than a year, on average. Cross-reactivity between parental and recombinant genotypes reduces the frequency of appearance to 36% and increases the probability of recombinant extinction following appearance from 0% to 83%. When a recombinant is cross-reactive with its parental types, its probability of extinction is influenced by cross-reactivity between the parental types in the opposite manner; that is, its probability of extinction after appearance decreases. Frequencies of P. falciparum outcrossing are mediated by frequencies of mixed-genotype infections in the host population, which are in turn mediated by the structure of cross-reactivity between parasite genotypes. The three leading hypotheses about how meiosis relates to oocyst production lead to quantitative, but no qualitative, differences in these results.     

Probing the structure of falcipain-3, a cysteine protease from Plasmodium falciparum: comparative protein modeling and docking studies

Increasing resistance of malaria parasites to conventional antimalarial drugs is an important factor contributing to the persistence of the disease as a major health threat. The ongoing search for novel targets has resulted in identification and expression of several enzymes including cysteine proteases that are implicated in hemoglobin degradation. Falcipain-2 and falcipain-3 are considered to be the two principal cysteine proteases in this degradation, and hence, are potential drug targets. A homology model of falcipain-3 was built and validated by various structure/geometry verification tools as well as docking studies of known substrates. The correlation coefficient of 0.975 between interaction energies and K(m) values of these substrates provided additional support for the model. On comparison with the previously reported falcipain-2 homology model, the currently constructed falcipain-3 structure showed important differences between the S2 pockets that might explain the variations in the K(m) values of various substrates for these enzymes. Further, docking studies also provided insight into possible binding modes and interactions of ligands with falcipain-3. Results of the current study could be employed in de novo drug design leading to development of new antimalarial agents.