Sergio Romeo

Antiplasmodial activity of Punica granatum L. fruit rind.

AIM OF THE STUDY Sun-dried rind of the immature fruit of Punica granatum L. (Punicaceae) (Pg) is presently used as a herbal formulation (OMARIA) in Orissa, India, for the therapy and prophylaxis of malaria. The aims of this study were (i) to assess in vitro the antiplasmodial activity of the methanolic extract, of a tannin enriched fraction and of compounds/metabolites of the antimalarial plant, (ii) to estimate the curative efficacy of the Pg extracts and (iii) to explore the mechanism of action of the antiplasmodial compounds. Urolithins, the ellagitannin metabolites, were also investigated for antiplasmodial activity. MATERIALS AND METHODS Chloroquine-susceptible (D10) and -resistant (W2) strains of Pf were used for in vitro studies and the rodent malaria model Plasmodium berghei-BALB/c mice was used for in vivo assessments. Recombinant plasmepsins 2 and 4 were used to investigate the interference of Pg compounds with the metabolism of haemoglobin by malaria parasites. RESULTS The Pg methanolic extract (Pg-MeOH) inhibited parasite growth in vitro with a IC(50) of 4.5 and 2.8 microg/ml, for D10 and W2 strain, respectively. The activity was found to be associated to the fraction enriched with tannins (Pg-FET, IC(50) 2.9 and 1.5 microg/ml) in which punicalagins (29.1%), punicalins, ellagic acid (13.4%) and its glycoside could be identified. Plasmepsin 2 was inhibited by Pg-MeOH extract and by Pg-FET (IC(50) 7.3 and 3.0 microg/ml), which could partly explain the antiparasitic effect. On the contrary, urolithins were inactive. Both Pg-MeOH extract and Pg-FET did not show any in vivo efficacy in the murine model. CONCLUSIONS The in vitro studies support the use of Pg as antimalarial remedy. Possible explanations for the negative in vivo results are discussed.

Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria

BackgroundThe sun-dried rind of the immature fruit of pomegranate (Punica granatum) is presently used as a herbal formulation (OMARIA, Orissa Malaria Research Indigenous Attempt) in Orissa, India, for the therapy and prophylaxis of malaria. The pathogenesis of cerebral malaria, a complication of the infection by Plasmodium falciparum, is an inflammatory cytokine-driven disease associated to an up-regulation and activity of metalloproteinase-9 and to the increase of TNF production. The in vitro anti-plasmodial activity of Punica granatum (Pg) was recently described. The aim of the present study was to explore whether the anti-malarial effect of OMARIA could also be sustained via other mechanisms among those associated to the host immune response.MethodsFrom the methanolic extract of the fruit rind, a fraction enriched in tannins (Pg-FET) was prepared. MMP-9 secretion and expression were evaluated in THP-1 cells stimulated with haemozoin or TNF. The assays were conducted in the presence of the Pg-FET and its chemical constituents ellagic acid and punicalagin. The effect of urolithins, the ellagitannin metabolites formed by human intestinal microflora, was also investigated.ResultsPg-FET and its constituents inhibited the secretion of MMP-9 induced by haemozoin or TNF. The effect occurred at transcriptional level since MMP-9 mRNA levels were lower in the presence of the tested compounds. Urolithins as well inhibited MMP-9 secretion and expression. Pg-FET and pure compounds also inhibited MMP-9 promoter activity and NF-kB-driven transcription.ConclusionsThe beneficial effect of the fruit rind of Punica granatum for the treatment of malarial disease may be attributed to the anti-parasitic activity and the inhibition of the pro-inflammatory mechanisms involved in the onset of cerebral malaria.

4-Aminoquinoline quinolizidinyl- and quinolizidinylalkyl-derivatives with antimalarial activity

A set of quinolizidinyl and quinolizidinylalkyl derivatives of 4-amino-7-chloroquinoline and of 9-amino-6-chloro-2-methoxyacridine were prepared and tested in vitro against CQ-sensitive (D-10) and CQ-resistant (W-2) strains of Plasmodium falciparum. All compounds but one exerted significant antimalarial activity. Some of the quinolizidine derivatives were from 5 to 10 times more active than chloroquine on the CQ-resistant strain. No toxicity against mammalian cells was observed.

Novel amodiaquine congeners as potent antimalarial agents.

To develop new classes of antimalarial agents, the possibility of replacing the phenolic ring of amodiaquine, tebuquine, and isoquine with other aromatic nuclei was investigated. Within a first set of pyrrole analogues, several compounds displayed high activity against both D10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. The isoquine structure was also modified by replacing the diethylamino group with more metabolically stable bicyclic moieties and by replacing the aromatic hydroxyl function with a chlorine atom. Among these compounds, two quinolizidinylmethylamino derivatives (6f and 7f) displayed high activity against both CQ-S and CQ-R strains.

A structure-activity study for the inhibition of metalloproteinase-9 activity and gene expression by analogues of gallocatechin-3-gallate

Abstract.Catechins are able to modulate the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promoter activity and mRNA levels. The effect appears to be dependent on some structural and stereochemical requirements. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (±)-gallocatechin-3-gallate (±)-GCG, selectively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (±)-GCG and (±)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (±)-trans-3-flavanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lowering MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-κB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.

Synthetic peptides derived from the C-terminal 6kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development.

BACKGROUND Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site. METHODS In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential. RESULTS The final proteolytic step of PfSERA5 involves removal of a C-terminal ~6kDa fragment that results in the generation of a catalytically active ~50kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~6kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5. CONCLUSIONS Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress. GENERAL SIGNIFICANCE These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~6kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.

Design and synthesis of protein-protein interaction mimics as Plasmodium falciparum cysteine protease, falcipain-2 inhibitors

Small peptides that mimic the protein-protein interactions between falcipain-2 and egg white cystatin, an endogenous inhibitor of cysteine proteases, were designed and synthesized and their effects on falcipain-2 activity were analyzed. The mimics are characterized by the presence of different linkers: γ-aminobutyric acid, cis-4-aminocyclohexane carboxylic acid and a macrocycle formed by GABA and two cysteines joined by a disulfide linkage. Some of these compounds showed falcipain-2 inhibition in the micromolar range and produced morphological abnormalities in the Plasmodium food vacuole. Although these peptides are less potent than cystatin, considering the reduction of amino acid residues and the capacity to cross membranes, this approach could be an interesting starting point for the development of a new class of anti-malarial drugs.

Parallel synthesis and antileishmanial activity of ether-linked phospholipids

The synthesis and antileishmanial activity of 18 edelfosine analogues are described. Compounds were obtained in parallel combining solid phase and solution phase synthesis. The most active analogue is characterized by the octadecyl group in position 2 of the glycerol chain. Considering that this substitution determines the loss of antitumor activity, a different mechanism of antileishmanial action can be hypothesized.

Atovaquone-statine "double-drugs" with high antiplasmodial activity.

Malaria is one of the most widespread parasitic infections in the world; the unavailability of a vaccine and the spread and intensification of drug resistance over the past 15–20 years have led to a dramatic decline in the efficacy of the most affordable antimalarial drugs. The reported presence of sporadic cases of artemether resistance indicates the need for increased vigilance and a coordinated and rapid deployment of drug combinations. Plasmepsins are a family of proteases comprising aspartic proteases Plasmepsins I, II, and IV (PLM I, PLM II, and PLM IV) and an histoaspartic protease (HAP) localized in the digestive vacuole of Plasmodium falciparum (Pf) together with other proteases (that is, falcipains). These proteases are involved in the degradation of haemoglobin during the intraerythrocytic cycle of Pf. Haemoglobin digestion is a crucial process for the survival of the parasite in the red blood cell (RBC) both for providing amino acids for protein biosynthesis and for reducing the osmotic pressure in the RBC. The inhibition of these enzymes has been proposed as a promising target for the development of a new antimalarial therapeutic approach. The protease machinery developed by the parasite to accomplish this task is quite redundant and the role of each enzyme needs further investigation. The developed PLM inhibitors, including molecules with a statinebased core and peptidomimetic structures, showed a limited effectiveness in inhibiting the intraerythrocytic parasite growth (IC50 range 1–20 mm), although their Ki values against PLMs were in the nanomolar range. Their low potency against cultured parasites may be explained by the redundancy of proteases which implies that the blockade of one or two PLMs may not be lethal. The “double-drug” approach combines two different inhibitors into a single chemical entity by a linker, with the aim of improving the physicochemical characteristics of the individual compounds. We have previously reported the high antiplasmodial activity of double-drug 1, designed by linking a statinebased inhibitor of PLMs, with primaquine, a known antimalarial drug active against the hepatic stage of Plasmodium. 10] We describe herein the synthesis and biological evaluation of a series of ATQ–statine double-drugs. ATQ is a very potent anti-

Bovine hemoglobin cross-linked through the beta chains: functional and structural aspects.

2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled to bovine Hb according to the procedure developed by Benesch and Benesch(1). The reaction of bovine Hb with NFPLP leads to a cross-linkage between the β subunits, which greatly stabilizes the low affinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into αβ dimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confronted with two different structural alternatives; the cross-link of β chains may be formed between lysine 82 of β and the N terminus of methionine 2 of β or between the two lysine 82 residues of both β chains. Digestion of modified β globin chains and isolation of the cross-linked peptide have showed that NFPLP cross-links Met-β2 and Lys-β82. This allowed discussion in some detail of the molecular basis of the Bohr effect of the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb could be considered as a first step toward the synthesis of a potential blood substitute.