Synthesis and in vitro antiplasmodial activities of fluoroquinolone analogs.

Fluoroquinolone analogs were synthesized by simple alkylation followed by click chemistry and evaluated for their antimalarial in vitro against chloroquine sensitive strain of Plasmodium falciparum while ciprofloxacin was used as standard. Our results showed that the compound 12 was found most active with IC(50) value of 1.33 µg/mL while ciprofloxacin showed IC(50) = 8.81 µg/mL. Therefore, screening of either known or unknown quinolone/fluoroquinolone analogs are worthwhile to find more potent antimalarial drugs which might prove useful in the treatment of mild or severe malaria in human either alone or in combination with existing antimalarial drugs.

Nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica and construction of a conjugative shuttle vector.

The complete nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica DSM 43387 was determined. Sequence analysis revealed that pA387 is 30,157 bp long and has a G+C content of 71.74%. To obtain a minimal transferable replicon capable of self-replication, a 2,176 bp fragment of pA387 was cloned, and we demonstrated that this fragment is sufficient for autonomous replication. The replication region of pA387 exhibited no significant homology to any known replication proteins available in databases. Putative maintenance and transfer functions were identified on pA387. The predicted products of open reading frames, ORF 2 and ORF 12, resembled the plasmid stabilizing proteins, a DNA resolvase and a ParA protein, respectively. The putative translational products of ORF 15 and ORF 16 showed similarity to known bacterial conjugation proteins, TraG and TraA, respectively. A conjugative Escherichia coli -Amycolatopsis shuttle-cloning vector was constructed by using the pA387 replicon and designated pSETRL1. Shuttle vector pSETRL1 successfully transformed Amycolatopsis mediterranei DSM 40773 and Amycolatopsis orientalis NBRC 12806 by conjugation and electroporation, and is likely to be a useful vector in Amycolatopsis research.

Proposal to reclassify [Sphingomonas] xenophaga Stolz et al. 2000 and [Sphingomonas] taejonensis Lee et al. 2001 as Sphingobium xenophagum comb. nov. and Sphingopyxis taejonensis comb. nov., respectively.

The sphingomonad group contains bacterial isolates that are quite diverse in terms of their phylogenetic, ecological and physiological properties. Thus, the genus Sphingomonas was divided into four distinct genera, Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis on the basis of 16S rRNA gene sequence phylogenetic analysis, signature nucleotides, fatty acid profiles and polyamine patterns and this classification is currently widely accepted. In this study, a complete analysis of the 16S rRNA gene sequences of all the members of the group of sphingomonads encompassed in the genera Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis was inferred by using tree-making algorithms. [Sphingomonas] xenophaga DSM 6383T was found to form a distinct clade with the members of the genus Sphingobium, whereas [Sphingomonas] taejonensis DSM 15583T forms a clade with the members of the genus Sphingopyxis. The respective positions of these strains were also supported by the data for signature nucleotides, 2-hydroxy fatty acid profiles, polyamine patterns and the nitrate reduction properties of the strains. We therefore propose the reclassification of [Sphingomonas] xenophaga and [Sphingomonas] taejonensis as Sphingobium xenophagum comb. nov. (type strain DSM 6383T = CIP 107206T) and Sphingopyxis taejonensis comb. nov. (type strain DSM 15583T = KCTC 2884T = KCCM 41068T), respectively.

Antibodies to a conserved-motif peptide sequence of the Plasmodium falciparum thrombospondin-related anonymous protein and circumsporozoite protein recognize a 78-kilodalton protein in the asexual blood stages of the parasite and inhibit merozoite invasion in vitro.

Athrombospondin-related anonymous protein (TRAP) of the human malaria parasite Plasmodium falciparum shares highly conserved amino acid sequence motifs with the circumsporozoite protein of all plasmodia sequenced so far, as well as with unrelated proteins like thrombospondin and properdin. Although it was first described as an asexual blood stages protein, there has been some controversy about its expression in these stages. Pursuant to our interest in the conserved sequences within the malaria antigens, we synthesized an 18-residue peptide (18-mer) representing a conserved motif of TRAP and raised polyclonal antibodies against it. In an immunoblot assay in which we probed proteins from the asexual blood stages of the parasite, we found that this antibody recognized predominantly a 78-kDa protein in the whole parasite lysate. Furthermore, in another immunoblot, the recombinant TRAP constructs containing the conserved-motif sequence were distinctly recognized by the antipeptide antibodies, whereas a construct lacking the motif sequence was not, suggesting that the antibodies specifically cross-reacted with a protein which might be a TRAP-like protein present in the asexual blood stages of the parasite. Also, in an immunofluorescence assay, this antibody brightly stained the acetone-fixed trophozoites of the parasite. Most significantly, anti-18-mer immunoglobulin G, as well as antipeptide antibody against a smaller (nonamer) construct representing the most conserved motif within the 18-mer, inhibited the merozoite invasion of erythrocytes in a dose-dependent manner. These results provide evidence of the expression of TRAP or a TRAP-like protein in the asexual blood stages of the parasite and of a possible role of the conserved motifs in the parasite-host cell interaction during the process of invasion.

In vitro selection of Plasmodium falciparum lines resistant to dihydrofolate-reductase inhibitors and cross resistance studies.

A cloned Plasmodium falciparum line was subjected to in vitro drug pressure, by employing a relapse protocol, to select progressively resistant falciparum lines to pyrimethamine and cycloguanil, the two dihydrofolate-reductase (DHFR) inhibitor antimalarial drugs. The falciparum lines resistant to pyrimethamine were selected much faster than those resistant to cycloguanil. In 348 days of selection/cultivation, there was 2,400-fold increase in IC50 value to pyrimethamine, whereas only about 75-fold decrease in sensitivity to cycloguanil was registered in 351 days. Pyrimethamine-resistant parasites acquired a degree of cross resistance to cycloguanil and methotrexate, another DHFR inhibitor, but did not show any cross resistance to some other groups of antimalarial drugs. The highly pyrimethamine-resistant line was not predisposed for faster selection to cycloguanil resistance. Resistance acquired to pyrimethamine was stable. The series of resistant lines obtained form a good material to study the 'evolution' of resistance more meaningfully at molecular level.

Cure with cisplatin (II) or murine malaria infection and in vitro inhibition of a chloroquine-resistant Plasmodium falciparum isolate.

Antiplasmodium properties of cisplatin [cis-platinum (II) diamine dichloride], a neoplastic drug, have been assessed in in vivo and in vitro model systems of malarial parasite. A well-tolerated dose of 6 mg/kg body weight of the compound cured the mice infected with Plasmodium berghei and the amount of cisplatin required for in vitro inhibition (IC50) of a chloroquine-resistant Plasmodium falciparum isolate was smaller than either chloroquine or quinine. The minimum inhibitory concentration (MIC) needed to prevent the in vitro multiplication of asexual blood parasites was 30 ng/ml. Late ring and trophozoite stages of the erythrocytic cycle were the most susceptible, whereas schizont and early ring stages were the least sensitive to the toxic effect of cisplatin. Multiple smaller doses were more effective in curing malaria in mice than a single large dose. In a few of the mice treated with a single intraperitoneal large dose of 6 mg/kg body weight, there was a delay in appearance of parasitemia but most of them recovered completely but slowly. This compound exerts its toxicity mainly by randomly damaging and cross-linking DNA strands as shown by Southern hybridization with a synthetic oligonucleotide probe, which is a repeat sequence in the falciparum genome. The report clearly demonstrates the antimalarial potentials of this compound and suggests a closer evaluation of this and other related compounds, specially in combination with antimalarial drugs to probe their synergistic properties.

In vitro gametocytocidal activity of artemisinin and its derivatives on Plasmodium falciparum.

Artemisinin and six of its derivatives were evaluated for their in vitro gametocytocidal and erythrocytic schizontocidal properties on an Indian isolate of Plasmodium falciparum. One of the metabolic derivatives, DADF-dihydroartemisinin (NIH02), was found to possess gametocytocidal and erythrocytic schizontocidal properties similar to those of artemisinin. Gametocytes of this isolate were highly susceptible to the toxic effect of NIH02 (IC50 = 6.6 ng/ml) and younger stages were more sensitive. This is the first report about the in vitro gametocytocidal properties of a derivative of artemisinin.

Characterization of Plasmodium falciparum cloned lines with respect to gametocyte production in vitro, infectivity to Anopheles mosquitoes, and transmission to Aotus monkeys.

The production of gametocytes in vitro and their subsequent infectivity to mosquitoes by 3 cloned lines of Plasmodium falciparum were studied. 2 of the cloned lines, Honduras I-clone B3 and Indochina III-clone W2, produced mature gametocytes (stage V) that were infective to Anopheles mosquitoes. The third clone, Sierra Leone I-clone D6, produced gametocytes, the majority of which did not develop beyond stage III. Fully mature gametocytes of Sierra Leone I-clone D6 were not infective to mosquitoes. Sporozoites collected from An. freeborni infected with Honduras I-clone B3 were used in transmission studies. Two of three Aotus monkeys were infected after prepatent periods of 19 and 20 d, respectively. This study supports previous reports that cloned lines of P. falciparum contain the full genetic capacity to produce morphologically mature gametocytes. The transmission to Aotus monkeys has also conclusively established that biologically competent gametocytes of both sexes are produced by clones.

Variations in the organization of repetitive DNA sequences in the genomes of Plasmodium falciparum clones.

Repetitive DNA in cultured Plasmodium falciparum was examined by restriction digestion and transfer hybridization, using cloned repetitive DNA probes. The arrangement of repetitive DNA was unstable: after 6 months culture of a cloned population, variations could be detected. Significant differences can be seen between clones derived from a single isolate, and are even more marked between isolates of diverse geographical origin. No cross-species conservation was seen for the sequences examined, and no relationship was observed between their representation in the P. falciparum genome and the potential for sexual differentiation.

Gametocyte-forming and non-gametocyte-forming clones of Plasmodium falciparum.

Three clones have been prepared from the Honduras I/CDC strain of Plasmodium falciparum by a method of microscopic selection. One of these (HB-2) does not form gametocytes whereas the others (HB-1 and HB-3) do. All three are as resistant to pyrimethamine as the original line, and all three form knobs on the erythrocyte surface.