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The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC 50 ) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

Synthesis of new 1-aryl-3-substituted propanol derivatives followed by structure-activity relationship, in silico drug-likeness, cytotoxicity, genotoxicity, in silico metabolism, in silico pharmacophore modeling, and in vivo studies led to the identification of compounds 22 and 23 with significant in vitro antiplasmodial activity against drug sensitive (D6 IC 50  ≤ 0.19 μM) and multidrug resistant (FCR-3 IC 50  ≤ 0.40 μM and C235 IC 50  ≤ 0.28 μM) strains of Plasmodium falciparum. Adequate selectivity index and absence of genotoxicity was also observed. Notably, compound 22 displays excellent parasitemia reduction (98 ± 1%), and complete cure with all treated mice surviving through the entire period with no signs of toxicity. One important factor is the agreement between in vitro potency and in vivo studies. Target exploration was performed; this chemotype series exhibits an alternative antimalarial mechanism., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We report the discovery of new potent inhibitors of the growth of Plasmodium falciparum chloroquine (CQ)-resistant W2 strain. These compounds were designed using the double drug approach by introducing a residue able to enhance the accumulation of plasmepsins inhibitors into the food vacuole. Some of the molecules were more active than CQ against CQ-resistant strain and showed good selectivity against cathepsin D., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Catestatin, an endogenous peptide derived from bovine chromogranin A, and its active domain cateslytin display powerful antimicrobial activities. We have tested the activities of catestatin and other related peptides on the growth of Plasmodium falciparum in vitro. Catestatin inhibits growth of the chloroquine-sensitive strain of P. falciparum 3D7, exhibiting 88% inhibition at 20 microM. A similar partial inhibition of parasite growth was observed for the chloroquine-resistant strain, 7G8 (64%,) and the multidrug-resistant strain, W2 (62%). In the presence of parasite-specific lactate dehydrogenase, a specific protein-protein interaction between catestatin and plasmepsin II precursor was demonstrated. In addition, catestatin partially inhibited the parasite-specific proteases plasmepsin in vitro. A specific interaction between catestatin and plasmepsins II and IV from P. falciparum and plasmepsin IV from the three remaining species of Plasmodium known to infect man was observed, suggesting a catestatin-induced reduction in availability of nutrients for protein synthesis in the parasite.

A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in Escherichia coli. The automaturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally occurring form isolated from parasites. The S3-S3' subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. On the basis of the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 A structure of the PfPM2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 1-6, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4.

The carboxylate atoms of the two catalytic aspartic acid residues in aspartic proteases are nearly coplanar and in the uncomplexed form share an in-plane nucleophilic water molecule that is central to the mechanism of these enzymes. This note reports that while reviewing the electron-density maps derived from the deposited data for uncomplexed plasmepsin II from Plasmodium falciparum [Asojo et al. (2003), J. Mol. Biol. 327, 173-181; PDB code 1lf4], it was discovered that the aspartic acid residues in this structure should in fact be distinctly noncoplanar. The crystallographic model from the deposited coordinates has been re-refined against the 1.9 A resolution published diffraction data to an R(cryst) of 21.2% and an R(free) of 22.2%. The catalytic water molecule is present, but the plane of the carboxylate group of Asp214 is rotated by 66 degrees from its original position.

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the "double-drug" approach: primaquine, which has been linked to statine-based inhibitors of plasmepsins (PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC(50)) as low as 0.1 microM was obtained, an excellent improvement in comparison with inhibitors previously reported (IC(50) = 2-20 microM). The killing activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar range (K(i) = 1-700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human fibroblasts at 100 microM and were highly selective for PLMs vs human cathepsin D.

The first macrocyclic inhibitor of the Plasmodium falciparum aspartic proteases plasmepsin I, II, and IV with considerable selectivity over the human aspartic protease cathepsin D has been identified. A series of macrocyclic compounds were designed and synthesized. Cyclizations were accomplished using ring-closing metathesis with the second generation Grubbs catalyst. These compounds contain either a 13-membered or a 16-membered macrocycle and incorporate a 1,2-dihydroxyethylene as transition state mimicking unit. The binding mode of this new class of compounds was predicted with automated docking and molecular dynamics simulations, with an estimation of the binding affinities through the linear interaction energy (LIE) method.

Plasmepsin 4 from Plasmodium falciparum and orthologs from Plasmodium malariae, Plasmodium ovale and Plasmodium vivax have been expressed in recombinant form, and properties of the active site of each enzyme characterized by kinetic analysis. A panel of chromogenic peptide substrates systematically substituted at the P3, P2, P2' and P3' positions was used to estimate enzyme/ligand interactions in the corresponding enzyme subsites based upon kinetic data. The kinetic parameters kcat, Km and kcat/Km were measured to identify optimal substrates for each enzyme and also sequences that were readily cleaved by the plasmepsins but poorly by host aspartic peptidases. Computer generated models were utilized to compare enzyme structures and interpret kinetic results. The orthologous plasmepsins share highly similar subsite specificities. In the S3 and S2 subsites, the plasmepsin 4 orthologs all preferred hydrophobic amino acid residues, Phe or Ile, but rejected charged residues such as Lys or Asp. In S2' and S3' subsites, these plasmepsins tolerated both hydrophobic and hydrophilic residues. Subsite specificities of the plasmepsin 4 family of orthologs are similar to those of human cathepsins D and E, except in S3' where the plasmepsins accept substrates containing Ser significantly better than either of these human aspartic proteases. Peptidomimetic methyleneamino reduced-peptide inhibitors, which have inhibition constants in the picomolar range, were prepared for each plasmepsin 4 ortholog based upon substrate preferences. A peptidomimetic inhibitor designed for plasmepsin 4 from P. falciparum having Ser in P3' had the lowest Ki of the series of inhibitors prepared, but did not significantly improve the selectivity of the inhibitor for plasmepsin 4 versus human cathepsin D.

Aspartic endopeptidases (plasmepsins) have been implicated in the degradation of hemoglobin in the erythrocytic stage of infection by Plasmodium falciparum. To develop new targets for drug development, these enzymes have been isolated and cloned, expressed, and studied structurally and enzymatically. This study expands this approach to two other species of the malarial parasite, P. vivax and P. malariae. Expression of cloned genes from these species, utilizing methodology similar to that employed in the original reports on the enzymes from P. falciparum, has provided active enzymes for analysis by kinetic methods. We describe here studies of three enzymes, plasmepsin II from P. falciparum, and one plasmepsin from both P. vivax and P. malariae, utilizing oligopeptide substrates and low-molecular-weight inhibitors. These analyses provide new information on the properties of distinct regions of the active site cleft; such data can suggest strategies for drug design to inhibit these critical enzymes of the parasite.

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k(cat)/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

DNA encoding the last 48 residues of the propart and the whole mature sequence of Plasmepsin II was inserted into the T7 dependent vector pET 3a for expression in E. coli. The resultant product was insoluble but accumulated at approximately 20 mg/l of cell culture. Following solubilisation with urea, the zymogen was refolded and, after purification by ion-exchange chromatography, was autoactivated to generate mature Plasmepsin II. The ability of this enzyme to hydrolyse several chromogenic peptide substrates was examined; despite an overall identity of approximately 35% to human renin, Plasmepsin II was not inhibited significantly by renin inhibitors.

A clone encoding the aspartic proteinase (PFAPD) from Plasmodium falciparum strain HB3 was obtained during the course of a project designed to sequence and identify the protein coding regions of the parasite's genome. The protein encoded by the clone contains a sequence identical to the N-terminal sequence determined for an aspartic proteinase isolated from the digestive vacuole of P. falciparum and demonstrated to participate in the hemoglobin digestive pathway (D. Goldberg, personal communication). The translated polypeptide sequence encompasses a number of features characteristic of aspartic proteinases, having > 30% identity and > 50% similarity overall to human cathepsin D, cathepsin E and renin. A model of the three-dimensional structure of PFAPD was constructed using rule-based procedures. This confirms that the primary sequence may be folded as a single chain into a three dimensional structure closely resembling those of other known aspartic proteinases. It includes a lengthy prosegment, two typical-hydrophobic-hydrophobic-Asp-Thr/Ser-Gly motifs and a tyrosine residue positioned in a beta-hairpin loop. The distribution of hydrophobic residues throughout the active site cleft is indicative of a likely preference for hydrophobic polypeptide substrates. The recombinant form of this enzyme expressed using the pGEX2T vector in Escherichia coli is active in digesting hemoglobin at acidic pH and in hydrolyzing a synthetic peptide corresponding to the putative initial cleavage site in hemoglobin. Activity is inhibited completely by pepstatin, confirming the identity of PFAPD as a member of the aspartic proteinase family. Specific mRNA for PFAPD is expressed in the erythrocytic stages of the life cycle.