Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti-malarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

Isolation and in vitro antimalarial activity of hexane extract from Thai Picrasma javanica B1 stembark.

The in vitro antimalarial activities against Plasmodium falciparum K1 of four extracts from the stembark of Picrasma javanica B1; ie water, methanol, chloroform and hexane extracts were studied using a modification of the [3H]hypoxanthine incorporation method. It was found that the hexane extract showed in vitro antimalarial activity with IC50 of 3.3 microg/ml. The extract was further fractionated using quick column chromatography, resulting in ten fractions. Fraction V was the most effective against P. falciparum K1 with IC50 of 4.4 microg/ml. Further isolation of fraction V using a column chromatographic technique provided six fractions. According to 1H- and 13C-NMR spectra, it could be concluded that the major compound in fraction V-3 was beta-sitosterol. Unfortunately, the antimalarial activity of beta-sitosterol could not be determined because of its low solubility in DMSO. However, fractions V-2 and V-4 still showed in vitro antimalarial activities with IC50 of 2.8 and 3.4 microg/ml, respectively. The further fractionation of these two active fractions could lead to promising candidates as antimalarial agents.

C-16 artemisinin derivatives and their antimalarial and cytotoxic activities: syntheses of artemisinin monomers, dimers, trimers, and tetramers by nucleophilic additions to artemisitene.

Nucleophilic additions of lithium keto and ester enolates and mono- and bifunctional Grignard reagents to artemisitene provided C-16-derived artemisinin monomers, dimers, trimers, and tetramers whose antimalarial and cytotoxic activities have been evaluated.

Bioxanthracenes from the insect pathogenic fungus. Cordyceps pseudomilitaris BCC 1620. I. Taxonomy, fermentation, isolation and antimalarial activity.

Eleven bioxanthracenes and two monomers, six novel in nature, were isolated from the insect pathogenic fungus Cordyceps pseudomilitaris BCC 1620. Growth optimization of the strain led to the improvement of bioxanthracenes production. The bioxanthracenes were evaluated for their antimalarial activity and cytotoxicity.

Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance.

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.

Development of a lead inhibitor for the A16V+S108T mutant of dihydrofolate reductase from the cycloguanil-resistant strain (T9/94) of Plasmodium falciparum.

The Ala16Val+Ser108Thr (A16V+S108T) mutant of the Plasmodium falciparum dihydrofolate reductase (DHFR) is a key mutant responsible for cycloguanil-resistant malaria due to steric interaction between Val-16 and one of the C-2 methyl groups of cycloguanil. 4,6-Diamino-1,2-dihydrotriazines have been prepared, in which both methyl groups of cycloguanil are replaced by H or by H and an alkyl or phenyl group, and their inhibition constants against wild-type and mutant DHFR determined. The S108T mutation is considered to decrease cycloguanil binding further through the effect on the orientation of the p-chlorophenyl group. By moving the p-chloro-substituent to the m-position in the chlorophenyl group, the activity against the A16V+S108T mutant enzyme is improved, and this effect is reinforced by the p-chloro substituent in the 3, 4-dichlorophenyl group. A lead compound has been found with inhibitory activity similar to that of cycloguanil against the wild-type DHFR and about 120-fold more effective than cycloguanil against the A16V+S108T mutant enzyme. The activity of this compound against P. falciparum clone (T9/94 RC17) which harbors the A16V+S108T DHFR is about 85-fold greater than cycloguanil.

Antimycobacterial and antiplasmodial cyclodepsipeptides from the insect pathogenic fungus Paecilomyces tenuipes BCC 1614.

Bioassay-guided fractionation of the crude extract from the insect pathogenic fungus Paecilomyces tenuipes BCC 1614 led to the isolation and identification of two antimycobacterial and antiplasmodial cyclodepsipeptides, beauvericin and beauvericin A.

Inactivation of artemisinin by thalassemic erythrocytes.

Plasmodium falciparum infecting alpha-thalassemic erythrocytes (Hb H or Hb H/Hb Constant Spring) is resistant to artemisinin derivatives. Similar resistance, albeit at a much lower level, is shown by the parasite infecting beta-thalassemia/Hb E erythrocytes. The resistance is due to host-specific factors, one of which is the higher uptake of the drugs by thalassemic erythrocytes than normal erythrocytes, due to binding with Hb H. In addition to higher drug binding, incubation of artemisinin with alpha-thalassemic erythrocytes resulted in preferential inactivation of the drug. Both thalassemic and normal erythrocytes have the capability to inactivate the drug. Addition of serum can protect against inactivation by normal erythrocytes, but not by thalassemic erythrocytes. Incubation with either the hemolysate or the membrane fraction from these erythrocytes also resulted in preferential inactivation of the drug. The drug was also inactivated by purified Hb H. It is concluded that the ineffectiveness of artemisinin derivatives against P. falciparum infecting thalassemic erythrocytes is due partly to competition of the host cell components for binding with the drugs, and partly to inactivation of the drugs by the cell components.

Identification of hemoglobin degradation products in Plasmodium falciparum.

Malaria parasites break down human hemoglobin to its constituent amino acids by cysteine and aspartic proteinases. However, no one has previously been able to identify hemoglobin cleavage products in intact parasites. When isolated parasites were subjected to non-denaturing polyacrylamide gels electrophoresis, a unique protein band was found which contains heme and reacts with anti-human hemoglobin antibodies. This protein does not appear to represent oxidized or glycosylated hemoglobin, and is present in isolated parasites but not in the cytosol of infected or uninfected erythrocytes. When this band was eluted and subjected to SDS polyacrylamide gel electrophoresis, three bands were seen on Western blots. The proteins in these bands contain proteins with the N-terminal sequences of alpha- and beta-globin chains but molecular masses of only 13.2-13.4 kDa. These data suggest that hemoglobin alpha- and beta-chains are initially cleaved within the parasite phagolysosome to release peptides of 15-17 and 23-25 amino acids from the C-termini of alpha- and beta-globin chains, respectively. Production of the hemoglobin breakdown products was inhibited by E-64, a cysteine proteinase inhibitor, suggesting the involvement of a cysteine proteinase in an early step of hemoglobin degradation.

Artemisinin neurotoxicity: neuropathology in rats and mechanistic studies in vitro.

Despite the wide use of artermisinin and its derivatives, concerns have been raised about their potential neurotoxicity. Accordingly, studies were undertaken on rats treated with high doses of arteether and on mouse neuroblastoma cells (Neu2a) treated with 3H-dihydroartemisinin. Rats uniformly developed neurologic symptoms following intramuscular administration of 50 mg/kg/day of arteether for 5-6 days. Acute neuronal necrosis associated with vacuolization and focal axonal swelling in the neuropil was observed in specific areas of the brain, especially the vestibular nuclei and red nuclei. Scattered swollen neurons were also evident in the cerebellar nuclei and the reticular formation. No neurologic symptoms, neuronal nuclei necrosis, nor gliosis was observed in rats administered 25 or 30 mg/kg/day for six or eight days. In vitro, Neu2a cells took up much less 3H-dihydroartemisinin than Plasmodium falciparum-infected red blood cells when incubated under identical conditions for 4 hr with 4.2 microM 3H-dihydroartemisinin. This selective uptake may explain why the artemisinin derivatives are selectively toxic to malaria parasites. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels run from 3H-dihydroartemisinin-treated cells showed that neuronal proteins with molecular weights of 27, 32, 40, and 81 kD were alkylated, although not nearly as strongly or rapidly as the P. falciparum proteins. The results indicate that while artemisinin derivatives have neurotoxic effects in rats and alkylate proteins in neuroblastoma cells, these effects only occur at high doses or after prolonged exposure.

Artemisinin and the antimalarial endoperoxides: from herbal remedy to targeted chemotherapy.

Artemisinin and its derivatives are endoperoxide-containing compounds which represent a promising new class of antimalarial drugs. In the presence of intraparasitic iron, these drugs are converted into free radicals and other electrophilic intermediates which then alkylate specific malaria target proteins. Combinations of available derivatives and other antimalarial agents show promise both as first-line agents and in the treatment of severe disease.

The mode of action of the antimalarial artemisinin and its derivatives.

1. Atremisinin (qinghaosu) is a sesquiterpene endoperoxide derived from a plant which was used in Chinese herbal medicine for thousands of years. 2. Artemisinin and its derivatives have potent antimalarial activity, and are now being used clinically in much of the world. 3. The artemisinin derivatives have an unusual mode of action involving the iron-catalyzed generation of a carbon-centered free radical followed by the alkylation of malaria-specific proteins.

Mechanism-based development of new antimalarials: synthesis of derivatives of artemisinin attached to iron chelators.

Various derivatives of artemisinin covalently linked to iron chelators were synthesized, and their antimalarial activities were evaluated. Although results show no indication that the presence of an iron chelator in the vicinity of artemisinin potentiates its action, the linked compounds prepared still retain comparable activities to that of artemisinin.

Alterations and pathology of thalassemic red cells: comparison between alpha- and beta-thalassemia.

Two main types of thalassemia have been categorized according to defective production of the globin gene ie alpha-thalassemia and beta-thalassemia. We report different red cell abnormalities between these two types. The study included 139 thalassemic patients including 91 patients with hemoglobin (Hb) H disease (52 cases with the classical genotype and 39 cases with Hb Constant Spring) and 48 were beta-thalassemia/Hb E disease. The deformability index of thalassemic red cells measured by laser diffractometer was significantly lower than that of normal red cells. Increased susceptibility of the thalassemic red cells to monocyte phagocytosis was markedly noted. Few sialic acid molecules were scattered on red cell surface of thalassemic red cells. Reticulocytes with delayed maturation stage were also observed in thalassemia indicating enhanced release from the bone marrow. The alpha-thalassemic red cells had relatively better deformability, increased susceptibility to phagocytosis, reduced sialic acid content and greater degree irregular distribution of sialic acid on red cell surface as compared to beta-thalassemic red cells. The alpha-type with hemoglobin Constant Spring (Hb CS) had increased percentage of reticulocyte and young reticulocyte (high fluorescent intensity) as compared to beta-thalassemic red cells. The different abnormalities between alpha- and beta-thalassemic red cells may lead to different mechanism of red cell destruction and different severity of the disease.

Resistance to artemisinin of malaria parasites (Plasmodium falciparum) infecting alpha-thalassemic erythrocytes in vitro. Competition in drug accumulation with uninfected erythrocytes.

Plasmodium falciparum infecting hemoglobin (Hb)H and/or Hb Constant Spring erythrocytes has higher resistance to artemisinin in vitro than when infecting normal erythrocytes. This is due to low drug accumulation of infected erythrocytes resulting from competition with uninfected variant erythrocytes, which have a higher accumulation capacity than genetically normal cells. Drug accumulation of the parasite was shown to be saturable and dependent on metabolic energy. The 50% inhibitory concentrations (IC50's) for the parasite in HbH/Hb Constant Spring erythrocytes were decreased when normal erythrocytes were added to the infected cells, and correspondingly, the IC50's in normal erythrocytes were increased when HbH/Hb Constant Spring erythrocytes were added to the infected cells. The changes of IC50 corresponded to the variation in drug accumulation of mixtures of normal and variant erythrocytes of different compositions. The IC50's for the parasite in variant erythrocytes were also greatly decreased when the hematocrit of the culture was lowered, while the IC50's in normal erythrocytes were independent of the hematocrit. The increase in IC50 values for the parasites infecting variant erythrocytes was also related to the decrease in parasite accumulation, indicating that drug accumulation capacity of the parasite also has a role in determining drug sensitivity. Artemisinin sensitivity therefore is determined by its accessibility to the parasite, which is decreased in infected variant erythrocytes.

Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu).

Artemisinin is an important new antimalarial agent containing a bridged endoperoxide. The in vitro antimalarial activity of an artemisinin derivative, arteether, is antagonized by two iron chelators, pyridoxal benzoylhydrazone and 1,2-dimethyl-3-hydroxypyrid-4-one. Similarly, the acute toxicity of artemisinin in mice is antagonized by another chelator, deferoxamine-hydroxyethylstarch. A combination of artemisinin and hemin oxidizes erythrocyte membrane thiols in vitro, and this oxidation is also inhibited by an iron chelator. Thus, iron plays a role in the mechanisms of action and toxicity of artemisinin. The combination of artemisinin and hemin also decreases erythrocyte deformability. Iron probably catalyzes the generation of free radicals from artemisinin since alpha-tocopherol antagonizes the thiol-oxidizing activity of artemisinin and since a spin-trapped free radical signal can be seen by electron paramagnetic resonance only when artemisinin is incubated in the presence of iron.

Increased susceptibility of malaria-infected variant erythrocytes to the mononuclear phagocyte system.

The interactions of the mononuclear phagocyte system with Plasmodium falciparum-infected genetically variant erythrocytes may result in a significant protection for the host. Infected hemoglobin (Hb) EE and Hb EA erythrocytes are more susceptible to phagocytosis by monocytes than are infected Hb AA erythrocytes. The increased susceptibility to phagocytosis of infected erythrocytes was also found for a number of genetic variants involving the alpha-globin chain, namely, alpha-thal 1 trait (--/alpha alpha), alpha-thal 2 trait (-alpha/alpha alpha), Hb H (--/-alpha), Hb H/Hb Constant Spring (CS) (--/alpha CS alpha), Hb CS trait, and homozygous Hb CS erythrocytes. In addition, oxidative damage from hydrogen peroxide, produced in simulation of macrophages, led to much more effective killing of parasites in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes than in normal ones. Parasites infecting Hb H/Hb CS also showed an enhanced sensitivity to hydrogen peroxide.

Susceptibility to hydrogen peroxide of Plasmodium falciparum infecting glucose-6-phosphate dehydrogenase-deficient erythrocytes.

The susceptibility to oxidant-mediated killing of Plasmodium falciparum infecting normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes was assessed by exposure to hydrogen peroxide generated by the glucose-glucose oxidase system. The parasites infecting G6PD-deficient erythrocytes had markedly greater susceptibility to hydrogen peroxide under a variety of conditions than those infecting normal erythrocytes. In both cases, the killing effect was mediated mainly through the host cells since treatment of the erythrocytes with hydrogen peroxide did not change their relatively susceptibility. The parasites were most susceptible during maturation, especially in G6PD-deficient erythrocytes, although a reduction in parasite invasion was also observed. The role of oxidant-mediated killing in the protection of G6PD-deficient hosts from P. falciparum infection is discussed.