Screening the Medicines for Malaria Venture "Malaria Box" against the Plasmodium falciparum Aminopeptidases,M1, M17 and M18

Malaria is a parasitic disease that remains a global health burden. The ability of the parasite to rapidly develop resistance to therapeutics drives an urgent need for the delivery of new drugs. The Medicines for Malaria Venture have compounds known for their antimalarial activity, but not necessarily the molecular targets. In this study, we assess the ability of the “MMV 400” compounds to inhibit the activity of three metalloaminopeptidases from Plasmodium falciparum, PfA-M1, PfA-M17 and PfM18 AAP. We have developed a multiplex assay system to allow rapid primary screening of compounds against all three metalloaminopeptidases, followed by detailed analysis of promising compounds. Our results show that there were no PfM18AAP inhibitors, whereas two moderate inhibitors of the neutral aminopeptidases PfA-M1 and PfA-M17 were identified. Further investigation through structure-activity relationship studies and molecular docking suggest that these compounds are competitive inhibitors with novel binding mechanisms, acting through either non-classical zinc coordination or independently of zinc binding altogether. Although it is unlikely that inhibition of PfA-M1 and/or PfA-M17 is the primary mechanism responsible for the antiplasmodial activity reported for these compounds, their detailed characterization, as presented in this work, pave the way for their further optimization as a novel class of dual PfA-M1/PfA-M17 inhibitors utilising non-classical zinc binding groups.     

Development of binding assays to screen ligands for Plasmodium falciparum thioredoxin and glutathione reductases by ultrafiltration and liquid chromatography/mass spectrometry

To identify potential lead compounds for malaria drug discovery, ultrafiltration and liquid chromatography and mass spectrometry (UF and LC/MS) based binding assays were developed for the first time for Plasmodium falciparum thioredoxin (PfTrxR) and glutathione (PfGR) reductases. In the binding assays, curcuminoids (bis-demethoxycurcumin 1, demethoxycurcumin 2, and curcumin 3) were used to study the binding affinity for PfTrxR and PfGR enzymes. The optimum binding was observed when the curcumimoids mixture (1 μM) was incubated with 1 μM PfTrxR and 0.5 μM PfGR enzymes separately for 60 min at 25 °C. The peak areas of the ligands in the chromatogram corresponding to incubation with active PfTrxR and PfGR enzymes increased by 1.6- and 2.0-fold respectively compared to the chromatogram of test compounds incubated with denatured enzymes. Further, binding assay experiments were carried out for compound 2 under non-competitive and competitive incubation conditions with 1 μM PfTrxR and 0.5 μM PfGR enzymes, separately. The binding affinity of compound 2 was higher for both the enzymes under non-competitive incubation conditions. To validate the binding assay developed, we have tested bis-2,4-dinitrophenyl sulfide (4) which is reported as an inhibitor of PfTrxR and PfGR enzymes. Compound 4 showed greater binding affinity for both enzymes under competitive incubation conditions. The relative peak area of compound 4 increased by 3.2- and 6-fold when incubated with active PfTrxR (1 μM) and PfGR (0.5 μM) enzymes respectively compared to the peak areas of the compound in control experiments. The current method developed has a potential for automated high-throughput screening to rapidly determine the binding affinity of ligands for these enzymes.     

Identification and Validation of a Potent Dual Inhibitor of the P. falciparum M1 and M17 Aminopeptidases Using Virtual Screening

The Plasmodium falciparum PfA-M1 and PfA-M17 metalloaminopeptidases are validated drug targets for the discovery of antimalarial agents. In order to identify dual inhibitors of both proteins, we developed a hierarchical virtual screening approach, followed by in vitro evaluation of the highest scoring hits. Starting from the ZINC database of purchasable compounds, sequential 3D-pharmacophore and molecular docking steps were applied to filter the virtual ‘hits’. At the end of virtual screening, 12 compounds were chosen and tested against the in vitro aminopeptidase activity of both PfA-M1 and PfA-M17. Two molecules showed significant inhibitory activity (low micromolar/nanomolar range) against both proteins. Finally, the crystal structure of the most potent compound in complex with both PfA-M1 and PfA-M17 was solved, revealing the binding mode and validating our computational approach.     

Modeling of human M1 aminopeptidases for in silico screening of potential Plasmodium falciparum alanine aminopeptidase (PfA-M1) specific inhibitors

Plasmodium falciparum alanine M1-aminopeptidase (PfA-M1) is a validated target for anti-malarial drug development. Presence of significant similarity between PfA-M1 and human M1-aminopeptidases, particularly within regions of enzyme active site leads to problem of non-specificity and off-target binding for known aminopeptidase inhibitors. Molecular docking based in silico screening approach for off-target binding has high potential but requires 3D-structure of all human M1-aminopeptidaes. Therefore, in the present study 3D structural models of seven human M1-aminopeptidases were developed. The robustness of docking parameters and quality of predicted human M1-aminopeptidases structural models was evaluated by stereochemical analysis and docking of their respective known inhibitors. The docking scores were in agreement with the inhibitory concentrations elucidated in enzyme assays of respective inhibitor enzyme combinations (r2≈0.70). Further docking analysis of fifteen potential PfA-M1 inhibitors (virtual screening identified) showed that three compounds had less docking affinity for human M1-aminopeptidases as compared to PfA-M1. These three identified potential lead compounds can be validated with enzyme assays and used as a scaffold for designing of new compounds with increased specificity towards PfA-M1.     

Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene derivatives

Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a preliminary assessment, these compounds were shown to possess activities in the low nM range with the most active being compound 6 with IC₅₀ values of 2.79?nM against Pf K1 and 3.2?nM against Pf W2. Overall the resistance indices indicate that the compounds have a low potential for cross resistance. Cytotoxicities were determined with Hek293 human embryonic kidney cells and activities against proliferating cells were assessed against A375 human malignant melanoma cells. The selectivity indices of the amino-artemisinin ferrocene derivatives indicate there is overall an appreciably higher selectivity towards the malaria parasite than mammalian cells.     

Synthesis,antimalarial activities and cytotoxicities of amino-artemisinin-1,2-disubstituted ferrocene hybrids

Artemisinin-ferrocene conjugates incorporating a 1,2-disubstituted ferrocene analogous to that embedded in ferroquine but attached via a piperazine linker to C10 of the artemisinin were prepared from the piperazine artemisinin derivative, and activities were evaluated against asexual blood stages of chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf). The most active was the morpholino derivative 5 with IC₅₀ of 0.86?nM against Pf K1 and 1.4?nM against Pf W2. The resistance indices were superior to those of current clinical artemisinins. Notably, the compounds were active against Pf NF54 early and late blood stage gametocytes – these exerted >86% inhibition at 1?µM against both stages; they are thus appreciably more active than methylene blue (～57% inhibition at 1?µM) against late stage gametocytes. The data portends transmission blocking activity. Cytotoxicity was determined against human embryonic kidney cells (Hek293), while human malignant melanoma cells (A375) were used to assess their antitumor activity.     

Celastrol inhibits Plasmodium falciparum enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein reductase (ENR), a critical enzyme in type II fatty acid biosynthesis, is a promising target for drug discovery against hepatocyte-stage Plasmodium falciparum. In order to identify PfENR-specific inhibitors, we docked 70 FDA-approved, bioactive, and/or natural product small molecules known to inhibit the growth of whole-cell blood-stage P. falciparum into several PfENR crystallographic structures. Subsequent in vitro activity assays identified a noncompetitive low-micromolar PfENR inhibitor, celastrol, from this set of compounds.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

Potential Interaction of Plasmodium falciparum Hsp60 and Calpain

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.     

The Structure of Plasmodium falciparum Blood-Stage 6-Cys Protein Pf41 Reveals an Unexpected Intra-Domain Insertion Required for Pf12 Coordination

Plasmodium falciparum is an apicomplexan parasite and the etiological agent of severe human malaria. The complex P. falciparum life cycle is supported by a diverse repertoire of surface proteins including the family of 6-Cys s48/45 antigens. Of these, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with the glycophosphatidylinositol-anchored Pf12. Our recent structural characterization of Pf12 revealed two juxtaposed 6-Cys domains (D1 and D2). Pf41, however, contains an additional segment of 120 residues predicted to form a large spacer separating its two 6-Cys domains. To gain insight into the assembly mechanism and overall architecture of the Pf12-Pf41 complex, we first determined the 2.45 Å resolution crystal structure of Pf41 using zinc single-wavelength anomalous dispersion. Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1. Notably, the ID is largely proteolyzed in the final structure suggesting inherent flexibility. To assess the contribution of the ID to complex formation, we engineered a form of Pf41 where the ID was replaced by a short glycine-serine linker and showed by isothermal titration calorimetry that binding to Pf12 was abrogated. Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12. Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.     

Design,synthesis and biological evaluation of 4-aminoquinoline-guanylthiourea derivatives as antimalarial agents

Guanylthiourea (GTU) has been identified as an important antifolate antimalarial pharmacophore unit, whereas, 4-amino quinolones are already known for antimalarial activity. In the present work molecules carrying 4-aminoquinoline and GTU moiety have been designed using molecular docking analysis with PfDHFR enzyme and heme unit. The docking results indicated that the necessary interactions (Asp54 and Ile14) and docking score (−9.63 to −7.36 kcal/mmol) were comparable to WR99210 (−9.89 kcal/mol). From these results nine molecules were selected for synthesis. In vitro analysis of these synthesized compounds reveal that out of the nine molecules, eight show antimalarial activity in the range of 0.61–7.55 μM for PfD6 strain and 0.43–8.04 μM for PfW2 strain. Further, molecular dynamics simulations were performed on the most active molecule to establish comparative binding interactions of these compounds and reference ligand with Plasmodium falciparum dihydrofolate reductase (PfDHFR).     

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria.     

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole

Background:Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate.Methods:The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined.Results:PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg²⁺ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly.Conclusion:Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum.General significance:This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

Investigation of acyclic uridine amide and 5′-amido nucleoside analogues as potential inhibitors of the Plasmodium falciparum dUTPase

Previously we have shown that trityl and diphenyl deoxyuridine derivatives and their acyclic analogues can inhibit Plasmodium falciparum dUTPase (PfdUTPase). We report the synthesis of conformationally restrained amide derivatives as inhibitors PfdUTPase, including both acyclic and cyclic examples. Activity was dependent on the orientation and location of the amide constraining group. In the case of the acyclic series, we were able to obtain amide-constrained analogues which showed similar or greater potency than the unconstrained analogues. Unfortunately these compounds showed lower selectivity in cellular assays.     

A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R² = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R² = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.     

A metal ion–dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.