Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC₅₀ values of 12 nM and 42 nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.     

Magnetic susceptibility of iron in malaria-infected red blood cells

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0 ± 0.9) × 10^? 3 mL mol^? 1. The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88 ± 0.60) × 10^? 6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.     

Glabridin induces oxidative stress mediated apoptosis like cell death of malaria parasite Plasmodium falciparum

Plants are known as the source of novel agents for developing new antimalarial drugs. Glabridin is a polyphenolic flavonoid, a main constituent in the roots of Glycyrrhiza glabra possesses various biological activities. However, its anti-plasmodial activity is unexplored. In the present work, it is for the first time demonstrated that glabridin inhibits Plasmodium falciparum growth in vitro with an IC₅₀ 23.9 ± 0.43 μM. Glabridin showed poor cytotoxicity in vitro with an IC₅₀ 246.6 ± 0.88 μM against Vero cell line and good selectivity index (9.6). In erythrocytic cycle, trophozoite stage was found to be most sensitive to glabridin. In silico study showed that glabridin inhibits Pf LDH enzyme activity by acting on NADH binding site. Glabridin induced oxidative stress by the generation of reactive oxygen and nitrogen species. Glabridin could induce apoptosis in parasite as evidenced by the depolarization of mitochondrial membrane potential (Δψ_m), activation of caspase like proteases and DNA fragmentation. These results indicate that glabridin exhibits antiplasmodial activity and is suitable for developing antimalarial agent from a cheap and sustainable source.     

Bis-alkylamine quindolone derivatives as new antimalarial leads

Quindolone derivatives, designed to target the malaria parasite digestive vacuole and heme detoxification pathway, have been synthesized by reaction with 2-chloro-N,N-diethylethanamine. This reaction gave N,O-, N,N- and O-alkylated products containing one or two basic side-chains. The compounds were evaluated for antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum W2 strain and for cytotoxicity in HepG2 A16 hepatic cells. By incorporating alkylamine side chains and chlorine atoms in the quindolone nucleus we transformed the inactive tetracyclic parent quindolones into moderate or highly active and selective antimalarial compounds. The most active and selective compound, 5c, showed an IC₅₀ = 51 nM for P. falciparum and a selectivity ratio of 98.     

Antiplasmodial activity of targeted zinc(II)-dipicolylamine complexes

This study measured the antiplasmodial activity of nine zinc-dipicolylamine (ZnDPA) complexes against three strains of Plasmodium falciparum, the causative parasite of malaria. Growth inhibition assays showed significant activity against all tested strains, with 50% inhibitory concentrations between 5 and 600 nM and almost no toxic effect against host cells including healthy red blood cells. Fluorescence microscopy studies with a green-fluorescent ZnDPA probe showed selective targeting of infected red blood cells. The results suggest that ZnDPA coordination complexes are promising antiplasmodial agents with potential for targeted malaria treatment.     

Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target

Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9 Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.     

Antimalarial and antitubercular nostocarboline and eudistomin derivatives: Synthesis,in vitro and in vivo biological evaluation

The synthesis of nine nostocarboline derivatives with substitutions of the 2-methyl group by alkyl, aryl and functionalized residues, 10 symmetrical bis cationic dimers linking 6-Cl-norharmane through the 2-position and fifteen derivatives of the marine alkaloids eudistomin N and O is reported. These compounds were evaluated in vitro against four parasites (Trypanosoma brucei rhodesiense STIB 900, Trypanosoma cruzi Tulahuen C2C4, Leishmania donovani MHOM-ET-67/L82 axenic amastigotes, and Plasmodium falciparum K1 strain), against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc²155 and Corynebacterium glutamicum ATCC13032, and cytotoxicity was determined against L6 rat myoblast cells. Nostocarboline and derivatives displayed potent and selective in vitro inhibition of P. falciparum with weak cytotoxicity. The dimers displayed submicromolar inhibition of L. donovani and T. brucei, and nanomolar activity against P. falciparum, albeit with pronounced cytotoxicity. One dimer showed a MIC₉₉ value against M. tuberculosis of 2.5 μg/ml. The alkylated eudistomin N and O derivatives displayed activities down to 18 nM against P. falciparum for N-Me Eudistomin N. Four dimers, nostocarboline and three eudostomin derivatives were evaluated in an in vivo Plasmodium berghei mouse model. No significant activity was observed for the dimers, but a 50% reduction in parasitaemia was observed at 4 × 50 mg/kg ip for nostocarboline.     

Increased reticulocyte count from cord blood samples using hypotonic lysis

Human reticulocytes are one of the fundamental components needed to study the in vitro invasion processes of the human malaria parasite Plasmodium vivax. Additionally examinations of reticulocytes and their binding proteins are difficult in areas of the world that do not have access to advanced equipment or stem cell lines. These issues are particularly relevant to malaria vaccine candidate studies that are directed against surface proteins that the parasites use to gain entry into erythrocytes. Described here is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. Exposure of cord blood to hypotonic saline (0.2%) for 5 min selectively lyses the non-reticulocytes resulting in an average 3.6-fold increase in reticulocyte count. Our studies show that this enrichment process does not damage the hemoglobin of the remaining erythrocytes which are still capable of supporting Plasmodium falciparum invasion and growth. This economical and rapid method of enrichment could facilitate studies of in vitro laboratory culturing of other malaria parasite species which preferentially invade reticulocytes such as P. vivax.     

Borrelidin analogues with antimalarial activity: Design,synthesis and biological evaluation against Plasmodium falciparum parasites

Borrelidin, a structurally unique 18-membered macrolide, was found to express antimalarial activity against drug-resistant Plasmodium falciparum malaria parasites, with IC₅₀ value of 0.93 ng/mL. However, it also displays strong cytotoxicity against human diploid embryonic MRC-5 cells. To investigate the issue of the cytotoxicity of borrelidin, borrelidin-based analogues were synthesized and their anti-Plasmodium properties were evaluated. In this communication, we report that a novel borrelidin analogue, bearing the CH₂SPh moiety via a triazole linkage, was found to retain a potent antimalarial activity, against drug-sensitive and drug-resistant parasite strains, but possess only weak cytotoxicity against human cells.     

The anthraquinone emodin inhibits the non-exported FIKK kinase from Plasmodium falciparum

The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC₅₀ = 2 μM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function.     

Select pyrimidinones inhibit the propagation of the malarial parasite,Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC₅₀ values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.     

Synthesis and biological evaluation of new bis-indolone-N-oxides

A series of bis-indolone-N-oxides, 1a–f, was prepared from bis(ethynyl)benzenes and o-halonitroaryls and studied for their in vitro antiplasmodial activities against Plasmodium falciparum and representative strains of bacteria and candida as well as for their cytotoxicity against a human tumor cell line (MCF7). They did not cause any haemolysis (300 μg mL⁻¹). Of the synthesized bis-indolones, compound 1a had the most potent antiplasmodial activity (IC₅₀ = 0.763 μmol L⁻¹ on the FcB1 strain) with a selectivity index (CC₅₀ MCF7/IC₅₀ FcB1) of 35.6. No potency against the tested microbial strains was observed.     

Hypervalent organotellurium compounds as inhibitors of P. falciparum calcium-dependent cysteine proteases

Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQ_S 3D7 and CQ_R W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC₅₀ values were 3.7 ± 1.0 μM, 1.1 ± 0.2 μM and 0.2 ± 0.01 μM, respectively. Using an assay performed in the presence of the ER Ca^2 +-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

The correction of reaction rates in continuous fluorometric assays of enzymes

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH₂, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10^− 4 × s^− 1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH₂, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was − 2.96 × 10^− 4 a.u μM^− 1 × s^− 1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate] ÷ (final − initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K_m and overestimation of V_max.     

In vitro and in vivo effects of the phytohormone inhibitor fluridone against Neospora caninum infection

Neospora caninum causes abortion and stillbirth in cattle. Identification of effective drugs against this parasite remains a challenge. Previous studies have suggested that disruption of abscisic acid (ABA)-mediated signaling in apicomplexan parasites such as Toxoplasma gondii offers a new drug target. In this study, the ABA inhibitor, fluridone (FLU), was evaluated for its action against N. caninum. Production of endogenous ABA within N. caninum was confirmed by ultra-performance liquid chromatography–tandem quadruple mass spectrometry. Subsequently, FLU treatment efficacy was assessed using in vitro. Results revealed that FLU inhibited the growth of N. caninum and T. gondii in vitro (IC₅₀ 143.1 ± 43.96 μM and 330.6 ± 52.38 μM, respectively). However, FLU did not affect parasite replication at 24 h post-infection, but inhibited egress of N. caninum thereafter. To evaluate the effect of FLU in vivo, N. caninum-infected mice were treated with FLU for 15 days. FLU treatment appeared to ameliorate acute neosporosis induced by lethal parasite challenge. Together, our data shows that ABA might control egress in N. caninum. Therefore, FLU has potential as a candidate drug for the treatment of acute neosporosis.     

A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-β-lactamases

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63 × 10^?10 and 5.38 × 10^?9 s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-β-lactamases (MβLs) L1 and CcrA, exhibiting K_m and k_cat values of 18 μM and 5 s^?1 and 11 μM and 17 s^?1, respectively.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.     

A lactose specific lectin from the sponge Cinachyrella apion: Purification,characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca²⁺, Mg²⁺ and Mn²⁺ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 °C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.     

Antiprotozoal activity of dicationic 3,5-diphenylisoxazoles,their prodrugs and aza-analogues

Fifty novel prodrugs and aza-analogues of 3,5-bis(4-amidinophenyl)isoxazole and its derivatives were prepared. Eighteen of the 24 aza-analogues exhibited IC₅₀ values below 25 nM against Trypanosoma brucei rhodesiense or Plasmodium falciparum. Six compounds had antitrypanosomal IC₅₀ values below 10 nM. Twelve analogues showed similar antiplasmodial activities, including three with sub-nanomolar potencies. Forty-four diamidines (including 16 aza-analogues) and the 26 prodrugs were evaluated for efficacy in mice infected with T. b. rhodesiense STIB900. Six diamidines cured 4/4 mice at daily 5 mg/kg intraperitoneal doses for 4 days, giving results far superior to pentamidine and furamidine. One prodrug attained 3/4 cures at daily 25 mg/kg oral doses for 4 days.