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Exploring Heme and Hemoglobin Binding Regions of Plasmodium Heme Detoxification Protein for New Antimalarial Discovery
Abstract
Hemoglobin degradation/hemozoin formation, essential steps in Plasmodium life cycle, are targets of existing antimalarials. The pathway still offers vast possibilities to be explored for new antimalarial discoveries. Here, we characterize Heme Detoxification Protein; PfHDP, a major protein involved in hemozoin formation, as a novel drug target. Using in silico and biochemical approaches, we identified two heme binding sites and a hemoglobin binding site in PfHDP. Treatment of Plasmodium falciparum 3D7 parasites with peptide corresponding to the hemoglobin binding domain in PfHDP resulted in food vacuole abnormalities similar to that seen with a cysteine protease inhibitor, E-64 (I-1). Screening of compounds that bound the modeled PfHDP structure in the heme/ hemoglobin-binding pockets from Maybridge Screening Collection identified a compound, ML-2 that inhibited parasite growth in a dose dependent manner, thus paving the way for testing its potential as a new drug candidate. These results provide functional insights into the role of PfHDP in Hz formation and further suggest that PfHDP could be an important drug target to combat malaria.
... In transport vesicles, it typically coexists with hemoglobin. At the site of hemozoin (Hz) formation, heme-containing HDP is endocytosed and transported to the food vacuole (Chiabrando et al., 2014;Gupta et al., 2017). Being involved in Hz formation, which is necessary for survival, raises the possibility that HDP could be a good target for antimalarial drugs (Gupta et al., 2017). ...
... At the site of hemozoin (Hz) formation, heme-containing HDP is endocytosed and transported to the food vacuole (Chiabrando et al., 2014;Gupta et al., 2017). Being involved in Hz formation, which is necessary for survival, raises the possibility that HDP could be a good target for antimalarial drugs (Gupta et al., 2017). The quantity of HDP produced by one million parasites is roughly 40 femtomoles (Jani et al., 2008). ...
... The current study focuses on HDP, a protein crucial for the malaria parasite's survival within erythrocytes. HDP's role in converting toxic heme into 'hemozoin,' reducing heme toxicity, and promoting parasite survival is well-documented (Gupta et al., 2017;Jani et al., 2008;Nema, 2022). Targeting HDP with antimalarial drugs could be a promising approach. ...
... [9][10][11] PfHDP (Heme Detoxification Protein) has been shown to be extremely potent in converting heme to hemozoin. 12 PfHDP, a food vacuoleassociated protein, possesses two heme-binding sites and a Hb-binding site 13 and is a part of a ∼ 200 kDa complex with other proteins including falcipain 2/2, Plasmepsin II, Plasmepsin IV, and histo-aspartic protease inside the food vacuole. 14 The interaction of PfHDP and Hb paved the way for a hypothesis suggesting that PfHDP might be playing some role in the uptake of Hb from the erythrocyte cytoplasm. ...
... 14 The interaction of PfHDP and Hb paved the way for a hypothesis suggesting that PfHDP might be playing some role in the uptake of Hb from the erythrocyte cytoplasm. 13 Although the mechanism of Hb degradation has been extensively studied; the mechanisms of uptake of the Hb from infected erythrocyte cytosol to the parasite remain poorly understood. Four distinct pathways have been proposed to assist in the uptake of Hb. 15,16 The uptake begins with the folding of parasites around erythrocyte cytoplasm followed by the development of vesicles and cytostome that continue uptake of Hb inside the parasite. ...
... 23 The predicted 3D model of PfHDP was used as reported previously. 13 The cryo-EM structure of Pfexp-2 has been resolved, 24 hence its corresponding PDB structure (6E10.pdb) was retrieved from the RCSB PDB. ...
Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP); a protein localized in the parasitophorus vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP‐GFP fusion protein from a transgenic line using GFP trap beads showed the association of PfHDP with Hb as well as with the members of PTEX translocon complex. Association of PfHDP with Hb or Pfexp‐2, a component of translocon complex was confirmed by protein‐protein interaction and immunolocalization tools. Based on these associations, we studied the role of PfHDP in Hb uptake using the PfHDP‐HA‐GlmS transgenic parasites line. PfHDP knock‐down significantly reduced the Hb uptake in these transgenic parasites in comparison to the wild type parasites. Morphological analysis of PfHDP‐HA‐GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Transient knockdown of a member of translocon complex, PfHSP101 in HSP101‐DDDHA parasites also showed a decreased uptake of Hb inside the parasite. Together, these results advocate an interaction between PfHDP and the translocon complex at the parasitophorus vacuole membrane and also suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.
... Because heme can catalyze a Fenton type reaction, the parasite mustbecome free of the heme without releasing free iron (Fe 2+ ). The solution evolved by the parasite has been to polymerize released heme into a polymer, termed hemozoin, which is largely inactive in catalyzing reactive oxygen species (ROS) production [21,22]. The parasite also produces glucose-6-phosphate dehydrogenase (G6PDH) to facilitate an extra production of reduced glutathione (GSH) [23] in an effort to control the oxidative stress. ...
... We examined the P. falciparum protein ATP-ase 6 (PfATP6) in detail, due to the fact that ARTs inhibit specifically and selectively the SERCA orthologue (PfATP6) of P. falciparum [32]. More so, the major parasite protein involved in the conversion of heme to hemozoin, the heme detoxification protein (PfHDP), was investigated as a possible target of the newly synthesized ARTs [22]. ...
... The first protein we used was PfATP6 (1U5N), i.e., PDB ID 1U5N [9]. The second protein used for the in silico study was PfHDP, created by ITASSER [44], to solve the 3D structure for PfHDP, as long as PfHDP did not have a significant sequence homology with any of the PDB structures deposited in the protein database [22]. The proteins were prepared with MGlTools-1.5.7. ...
Artemisinin-based Combination Therapies (ACTs) are currently the frontline treatment against Plasmodium falciparum malaria, but parasite resistance to artemisinin (ART) and its derivatives, core components of ACTs, is spreading in the Mekong countries. In this study, we report the synthesis of several novel artemisinin derivatives and evaluate their in vitro and in silico capacity to counteract Plasmodium falciparum artemisinin resistance. Furthermore, recognizing that the malaria parasite devotes considerable resources to minimizing the oxidative stress that it creates during its rapid consumption of hemoglobin and the release of heme, we sought to explore whether further augmentation of this oxidative toxicity might constitute an important addition to artemisinins. The present report demonstrates, in vitro, that FM-AZ, a newly synthesized artemisinin derivative, has a lower IC50 than artemisinin in P. falciparum and a rapid action in killing the parasites. The docking studies for important parasite protein targets, PfATP6 and PfHDP, complemented the in vitro results, explaining the superior IC50 values of FM-AZ in comparison with ART obtained for the ART-resistant strain. However, cross-resistance between FM-AZ and artemisinins was evidenced in vitro.
... The inhibitory activities of the synthesized compounds were analyzed by assessing their ability to block the in vitro protease activity of recombinant falcipain-2, as well as to block the heme polymerization activity of heme-detoxification protein (HDP) protein. To test the inhibitory activities of artemisinin-dipeptidyl vinyl phosphonate hybrid molecules, we expressed and purified recombinant falcipain-2 and HDP proteins by protocols published previously [19,57,58]. Recombinant falcipain-2 activity was analyzed by quantitatively measuring the blue fluorescence generated as a result of the release of free AMC (7-amino-4methylcoumarin) after the hydrolysis of Z-FR-AMC (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin hydrochloride or Cbz-Phe-Arg-AMC) by the enzyme. ...
... Recombinant falcipain-2 activity was analyzed by quantitatively measuring the blue fluorescence generated as a result of the release of free AMC (7-amino-4methylcoumarin) after the hydrolysis of Z-FR-AMC (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin hydrochloride or Cbz-Phe-Arg-AMC) by the enzyme. Functional activity of recombinant HDP was assessed by its ability to convert free Heme to Hz, both recombinant falcipain-2 as well as HDP proteins used in the assays were functionally active as shown earlier [19,57,58]. We next tested the artemisinin-dipeptidyl vinyl phosphonate hybrid molecules for their abilities to block falcipain-2 as well as HDP activities. ...
... Using two recombinant proteins; rPf falcipain-2 and rPfHDP, an in vitro hemozoin formation assay was developed to demonstrate the conversion of hemoglobin to hemozoin. We have recently shown that PfHDP possesses heme as well as Hb binding sites and has multifunctional roles [58]. As shown in Fig. 3, the HDP alone was able to convert heme to hemozoin in this assay; however, all the three compounds significantly inhibited Heme to Hz conversion. ...
Artemisinin-based combination therapies (ACTs) have been able to reduce the clinical and pathological malaria cases in endemic areas around the globe. However, recent reports have shown a progressive decline in malaria parasite clearance in South-east Asia after ACT treatment, thus envisaging a need for new artemisinin (ART) derivatives and combinations. To address the emergence of drug resistance to current antimalarials, here we report the synthesis of artemisinin-peptidyl vinyl phosphonate hybrid molecules that show superior efficacy than artemisinin alone against chloroquine-resistant as well as multidrug-resistant Plasmodium falciparum strains with EC50 in pico-molar ranges. Further, the compounds effectively inhibited the survival of ring-stage parasite for laboratory-adapted artemisinin-resistant parasite lines as compared to artemisinin. These hybrid molecules showed complete parasite clearance in vivo using P. berghei mouse malaria model in comparison to artemisinin alone. Studies on the mode of action of hybrid molecules suggested that these artemisinin-peptidyl vinyl phosphonate hybrid molecules possessed dual activities: inhibited falcipain-2 (FP-2) activity, a P. falciparum cysteine protease involved in hemoglobin degradation, and also blocked the hemozoin formation in the food-vacuole, a step earlier shown to be blocked by artemisinin. Since these hybrid molecules blocked multiple steps of a pathway and showed synergistic efficacies, we believe that these lead compounds can be developed as effective antimalarials to prevent the spread of resistance to current antimalarials.
... Free heme (Fe 3+ ) might also interfere with hemoglobin degradation pathway 12,13 . Plasmodium uses a system to detoxify heme (Fe 3+ ) called biocrystallization based on the formation of hemozoin pigment which appears as a dark black crystalline spot (a dark brown pigment) in red blood cells of infected patients [14][15][16][17][18] . Hemozoin is chemically and structurally identical to β-hematin, a heme dimer that crystallizes under the acidic conditions of digestive vacuole of Plasmodium (pH values of 4.8-5.0) ...
... Inhibition of proteolysis is a current target for new antimalarial drugs 4,15,72 . A number of protease inhibitors are active against Plasmodium 5,73 . ...
... Once inside the parasite, hemoglobin is shuttled to digestive vacuole where it is digested by successive action of aspartate and cysteine proteases such as papain-like falcipains 5 . This process results in high levels of globin, peptides and amino acids that are transported into cytoplasm and used for cell growth 15 . Antimalarial drugs could interfere with hemoglobin digestion during blood stages of malarial life-cycle 33,68,74 . ...
Malaria caused by Plasmodium affects millions people worldwide. Plasmodium consumes hemoglobin during its intraerythrocytic stage leaving toxic heme. Parasite detoxifies free heme through formation of hemozoin (β-hematin) pigment. Proteolysis of hemoglobin and formation of hemozoin are two main targets for antimalarial drugs. Quinoline antimarial drugs and analogs (β-carbolines or nitroindazoles) were studied as inhibitors of β-hematin formation. The most potent inhibitors were quinacrine, chloroquine, and amodiaquine followed by quinidine, mefloquine and quinine whereas 8-hydroxyquinoline and β-carbolines had no effect. Compounds that inhibited β-hematin increased free hemin that promoted peroxidative reactions as determined with TMB and ABTS substrates. Hemin-catalyzed peroxidative reactions were potentiated in presence of proteins (i.e. globin or BSA) while antioxidants and peroxidase inhibitors decreased peroxidation. Free hemin increased by chloroquine action promoted oxidative reactions resulting in inhibition of proteolysis by three cysteine proteases: papain, ficin and cathepsin B. Glutathione reversed inhibition of proteolysis. These results show that active quinolines inhibit hemozoin and increase free hemin which in presence of H2O2 that abounds in parasite digestive vacuole catalyzes peroxidative reactions and inhibition of cysteine proteases. This work suggests a link between the action of quinoline drugs with biochemical processes of peroxidation and inhibition of proteolysis.
... FQ retains activity against chloroquine-and piperaquine-resistant parasites in vitro and has a long elimination half-life of 16 days. It is only moderately efficacious as monotherapy but when combined with artesunate (daily dose of 4 or 6 mg/kg FQ plus artesunate 4 mg/kg for 3 days) the polymerase chain reaction (PCR)corrected efficacy at 28 days for the treatment of uncomplicated falciparum malaria was 99% (95% CI [93][94][95][96][97][98][99][100] [46]. The potential for cross resistance of artemisinin-resistant parasites to artefenomel has been investigated. ...
... -ATPase 4, the site of action of the potent cipargamin and SJ557733, both in clinical development, and of 21A092, a pyrazoleamide in the discovery phase [98]. Pathways involved in haem detoxification are also under study [99]. Work continues on developing parasite-selective inhibitors of phenylalanyl-tRNA synthetase, histone deacetylase, hypoxanthine-guanine-(xanthine) phosphoribosyltransferase and N-myristoyltransferase [100][101][102][103]. Another avenue of research and development is the creation of hybrid molecules, i.e. combining two synergistic agents with different targets as a way of increasing the barrier to resistance [104,105]. ...
The last two decades have seen a surge in antimalarial drug development with product development partnerships taking a leading role. Resistance of Plasmodium falciparum to the artemisinin derivatives, piperaquine and mefloquine in Southeast Asia means new antimalarials are needed with some urgency. There are at least 13 agents in clinical development. Most of these are blood schizonticides for the treatment of uncomplicated falciparum malaria, under evaluation either singly or as part of two-drug combinations. Leading candidates progressing through the pipeline are artefenomel–ferroquine and lumefantrine-KAF156, both in Phase 2b. Treatment of severe malaria continues to rely on two parenteral drugs with ancient forebears: artesunate and quinine, with sevuparin being evaluated as an adjuvant therapy. Tafenoquine is under review by stringent regulatory authorities for approval as a single-dose treatment for Plasmodium vivax relapse prevention. This represents an advance over standard 14-day primaquine regimens; however, the risk of acute haemolytic anaemia in patients with glucose-6-phosphate dehydrogenase deficiency remains. For disease prevention, several of the newer agents show potential but are unlikely to be recommended for use in the main target groups of pregnant women and young children for some years. Latest predictions are that the malaria burden will continue to be high in the coming decades. This fact, coupled with the repeated loss of antimalarials to resistance, indicates that new antimalarials will be needed for years to come. Failure of the artemisinin-based combinations in Southeast Asia has stimulated a reappraisal of current approaches to combination therapy for malaria with incorporation of three or more drugs in a single treatment under consideration.
... This study considers the HDP as one of the proposed native mediators for heme to Hz transformation, as reported in the previous literature. 19,21,[37][38][39][40][41]61 However, it is possible that other mediators such as free lipids or lipids in the plasma membrane could actually be facilitating this transformation within the parasite's digestive vacuole. 62 Nonetheless, in our in vitro settings, HDP was found to be the most efficient mediator of heme to β-hematin transformation, 19 which allowed us to observe results that other studies may have missed. ...
Malaria parasites generate toxic heme during hemoglobin digestion, which is neutralized by crystallizing into inert hemozoin (β-hematin). Chloroquine blocks this detoxification process, resulting in heme-mediated toxicity in malaria parasites. However, the exact mechanism of chloroquine's action remains unknown. This study investigates the impact of chloroquine on the transformation of heme into β-hematin. The results show that chloroquine does not completely halt the transformation process but rather slows it down. Additionally, chloroquine complexation with free heme does not affect substrate availability or inhibit β-hematin formation. Scanning electron microscopy (SEM) and X-ray powder diffraction (XRD) studies indicate that the size of β-hematin crystal particles and crystallites increases in the presence of chloroquine, suggesting that chloroquine does not impede crystal growth. These findings suggest that chloroquine delays hemozoin production by perturbing the nucleation events of crystals and/or the stability of crystal nuclei. Thus, contrary to prevailing beliefs, this study provides a new perspective on the working mechanism of chloroquine.
... Chloroquine (CQ) exerts its antimalarial function by inhibiting hemozoin formation during the critical ferriheme detoxification process of the Plasmodium parasite [35][36][37][38][39]. The molecule acts either by: (a) directly complexing monomeric PPIX preventing its incorporation into the growing hemozoin crystals [36,[40][41][42], (b) capping the hemozoin crystal [43,44], (c) binding to PPIX via π-π interactions between the its aromatic ring and the porphyrin ring [45,46], (d) shifting the haematin/μ-oxo PPIX dimer equilibrium [40], or (e) docking onto the fastest growing crystal face [47]. ...
A small library of aminoquinoline and imidazolopiperidine (IMP)-based ligands, containing the 1,2,3-triazole moiety, and their corresponding tricarbonyl rhenium complexes were synthesised and their inhibitory activities evaluated against the chloroquine-sensitive (CQS) and multidrug-resistant (MDR) strains (NF54 and K1, respectively) of P. falciparum. The quinoline-based compounds (L1, L2, ReL1, and ReL2) were at least six-fold more potent than their IMP-based counterparts (L3, L4, ReL3, and ReL4) against both strains of P. falciparum, with the most promising compound (L1) displaying activity comparable to chloroquine diphosphate (CQDP) in the MDR strain. Additionally, all of the synthesised compounds have resistance indices less than CQDP. To gain insight into a possible mechanism of action, in silico hemozoin docking simulations were performed. These studies proposed that the tested compounds may act via hemozoin inhibition, as the new aminoquinoline-derivatives, with the exception of complex ReL2 (binding affinity: -12.62 kcal/mol), showed higher binding affinities than the reference drug chloroquine (CQ, -13.56 kcal/mol). Furthermore, the ligands exhibited superior binding affinity relative to their corresponding Re(I) complexes, which is reflected in their antiplasmodial activity.
... Because of the important role that heme plays in ROS generation, immediately after its production, a detoxification process takes place where approximately 95% of the produced heme (Fe 2+ ) is polymerized into hemozoin, a nontoxic crystal [46]. This reaction involves the heme detoxification protein (HDP), whose action is aided by histidine-rich protein-2 (HRP2) (Figure 7) [48,56]. HRP2 is said to have a very high affinity for heme [57,58], and elevated HRP2 reduces the vulnerability of the parasite. ...
Several measures are in place to combat the worldwide spread of malaria, especially in regions of high endemicity. In part, most common antimalarials, such as quinolines and artemisinin and its derivatives, deploy an ROS-mediated approach to kill malaria parasites. Although some antimalarials may share similar targets and mechanisms of action, varying levels of reactive oxygen species (ROS) generation may account for their varying pharmacological activities. Regardless of the numerous approaches employed currently and in development to treat malaria, concerningly, there has been increasing development of resistance by Plasmodium falciparum, which can be connected to the ability of the parasites to manage the oxidative stress from ROS produced under steady or treatment states. ROS generation has remained the mainstay in enforcing the antiparasitic activity of most conventional antimalarials. However, a combination of conventional drugs with ROS-generating ability and newer drugs that exploit vital metabolic pathways, such antioxidant machinery, could be the way forward in effective malaria control.
... To prevent this, the free heme is detoxified by biocrystallization to form inert, insoluble hemozoin in the parasitic digestive vacuole (Schmitt et al., 1993;Becker et al., 2004;Kumar and Bandyopadhyay, 2005;Kumar et al., 2007;Coronado et al., 2014;Roy, 2017;Herraiz et al., 2019). The resulting hemozoin (biocrystallized heme) is a dark pigment that represents the characteristic black crystalline spot observed in red blood cells during diagnosis of patients infected with malaria (Egan, 2002;Combrinck et al., 2013;Coronado et al., 2014;Gupta et al., 2017). Thus, sequestration of heme into hemozoin is essential for the survival of P. falciparum, and the vital pathway of hemozoin pigment formation is one of the main targets of antimalarial drugs (Hempelmann, 2007;Herraiz et al., 2019;Kapishnikov et al., 2019). ...
We investigated the potential antimalarial and toxicological effects of 16 medicinal plants frequently used by traditional healers to treat malaria, fever, and related disorders in the Greater Mpigi region in Uganda. Species studied were Albizia coriaria, Cassine buchananii, Combretum molle, Erythrina abyssinica, Ficus saussureana, Harungana madagascariensis, Leucas calostachys, Microgramma lycopodioides, Morella kandtiana, Plectranthus hadiensis, Securidaca longipedunculata, Sesamum calycinum subsp. angustifolium, Solanum aculeastrum, Toddalia asiatica, Warburgia ugandensis, and Zanthoxylum chalybeum. In addition, the traditional healers indicated that P. hadiensis is used as a ritual plant to boost fertility and prepare young women and teenagers for motherhood in some Ugandan communities where a high incidence of rapidly growing large breast masses in young female patients was observed (not necessarily breast cancer). We present results from various in vitro experiments performed with 56 different plant extracts, namely, 1) an initial assessment of the 16 species regarding their traditional use in the treatment of malaria by identifying promising plant extract candidates using a heme biocrystallization inhibition library screen; 2) follow-up investigations of antiprotozoal effects of the most bioactive crude extracts against chloroquine-resistant P. falciparum K1; 3) a cytotoxicity counterscreen against human MRC-5SV2 lung fibroblasts; 4) a genotoxicity evaluation of the extract library without and with metabolic bioactivation with human S9 liver fraction; and 5) an assessment of the mutagenicity of the ritual plant P. hadiensis. A total of seven extracts from five plant species were selected for antiplasmodial follow-up investigations based on their hemozoin formation inhibition activity in the heme biocrystallization assay. Among other extracts, an ethyl acetate extract of L. calostachys leaves exhibited antiplasmodial activity against P. falciparum K1 (IC50 value: 5.7 µg/ml), which was further characterized with a selectivity index of 2.6 (CC50 value: 14.7 µg/ml). The experiments for assessment of potential procarcinogenic properties of plant extracts via evaluation of in vitro mutagenicity and genotoxicity indicated that few extracts cause mutations. The species T. asiatica showed the most significant genotoxic effects on both bacterial test strains (without metabolic bioactivation at a concentration of 500 µg/plate). However, none of the mutagenic extracts from the experiments without metabolic bioactivation retained their genotoxic activity after metabolic bioactivation of the plant extract library through pre-incubation with human S9 liver fraction. While this study did not show that P. hadiensis has genotoxic properties, it did provide early stage support for the therapeutic use of the medicinal plants from the Greater Mpigi region.
... The reaction was initiated by the addition of 4 µM of Histagged-PfPRL, and the release of 3-O-methylfluorescein (OMF) was monitored at 485/535 nm in Victor 1420 multilabel counter (Perkin Elmer, USA). His-HDP (heme detoxification protein, a protein used in process of hemozoin formation [24,25] and does not have phosphatase domain or show phosphatase activity) was used as a negative control. The compound Novartis_003209, identified from in silico study, was analyzed in an inhibition assay. ...
Post‐translational modifications, especially reversible phosphorylation, are among the most common mechanisms that regulate protein function and biological processes in Plasmodium species. Of the Plasmodium phosphatases, phosphatase of regenerating liver (PfPRL) is secreted and is an essential phosphatase. Here, we expressed PfPRL in a heterologous expression system, and then purified and characterized its phosphatase activity. We found that Novartis_003209, a previously identified inhibitor, inhibited the PfPRL phosphatase activity of recombinant PfPRL and blocked in vitro parasite growth in a dose‐dependent manner. Further, in silico docking analysis of Novartis_003209 with all four P. falciparum tyrosine phosphatases (PTP) demonstrated that Novartis_003209 is a Plasmodium PTP inhibitor. Overall, our results identify a scaffold as a potential starting point to design a PTP specific inhibitor.
... Among the probable targets for drugs leads falcipain-2 (PfFP-2), known cysteine proteases hydrolyze Hb to provide amino acids for parasite protein synthesis. It is important to find inhibitors of falcipain-2 with suitable properties for novel antimalarial drugs [57][58][59][60]. ...
In an effort to develop a potent anti-malarial agent against Plasmodium falciparum, a structure-guided virtual screening using an in-house library comprising 652 compounds was performed. By docking studies, we identified two compounds (JMI-105 and JMI-346) which formed significant non-covalent interactions and fit well in the binding pocket of PfFP-2. We affirmed this observation by MD simulation studies. As evident by the biochemical analysis, such as enzyme inhibition assay, Surface Plasmon Resonance (SPR), live-cell imaging and hemozoin inhibition, JMI-105 and JMI-346 at 25µM concentration showed an inhibitory effect on purified PfFP-2. JMI-105 and JMI-346 inhibited the growth of CQS (3D7; IC50 = 8.8 and 13µM) and CQR (RKL-9; IC50 = 14.3 and 33µM) strains of P. falciparum. Treatment with compounds resulted in defect in parasite growth and development. No significant hemolysis or cytotoxicity towards human cells was observed suggesting that these molecules are non-toxic. We pursued, structural optimization on JMI-105 and in the process, SAR oriented derivatives (5a-5l) were synthesized and evaluated for growth inhibition potential. JMI-105 significantly decreased parasitemia and prolonged host survival in a murine model with P. berghei ANKA infection. The compounds (JMI-105 and JMI-346) against PfFP-2 have the potential to be used as an anti-malarial agent.
... PfFP-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. PfFP-2 that was inhibited with trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) formed a complex with Hb [60,61]. A peptide encoding the motif blocked hemoglobin hydrolysis, indicat- ing that PfFP-2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor [62,63]. ...
Background:
Novel drug development against malaria parasite over old conventional antimalarial drugs is essential due to rapid and indiscriminate use of drugs, which led to the emergence of resistant strains.
Methods:
In this study, previously reported triazole-amino acid hybrids (13-18) are explored against Plasmodium falciparum as antimalarial agents. Among six compounds, 15 and 18 exhibited antimalarial activity against P. falciparum with insignificant hemolytic activity and cytotoxicity towards HepG2 mammalian cells. In molecular docking studies, both compounds bind into the active site of PfFP-2 and block its accessibility to the substrate that leads to the inhibition of target protein further supported by in vitro analysis.
Results:
Antimalarial half-maximal inhibitory concentration (IC50) of 15 and 18 compounds were found to be 9.26 μM and 20.62 μM, respectively. Blood stage specific studies showed that compounds, 15 and 18 are effective at late trophozoite stage and block egress pathway of parasites. Decreased level of free monomeric heme was found in a dose dependent manner after the treatment with compounds 15 and 18, which was further evidenced by the reduction in percent of hemoglobin hydrolysis. Compounds 15 and 18 hindered hemoglobin degradation via intra- and extracellular cysteine protease falcipain-2 (PfFP-2) inhibitory activity both in in vitro and in vivo in P. falciparum.
Conclusion:
We report antimalarial potential of triazole-amino acid hybrids and their role in the inhibition of cysteine protease PfFP-2 as its mechanistic aspect.
... A better understanding of this mode would allow us to understand the mechanism of antiplasmodial resistance associated with their use on the one hand, and to produce new molecules more effective or improve the activity of antimalarial molecules available on the other hand. Several experimental evidence highlighted the crucial role played by hemin in the therapeutic activity of antimalarials, both quinoline and non-quinoline [2][3][4][5][6][7][8][9]. In this mechanism, the hemin or ferriprotoporphyrin IX [Fe (IX) PP] acts as receptor of antimalarials with quinoline core (APQ) with which it forms complexes [10]. ...
Malaria is a serious public health problem in developing countries and affects about 500 million people per year. However, resistance of some plasmodial strains to the chemotherapy effect of quinoline based-antimalarial drugs has caused the resurgence of malaria in endemic areas. The aim of this study was to evaluate the antimalarial activity of complexes of hemin with chloroquine, amodiaquine, quinine, quinidine and mefloquine. The complexes of hemin with chloroquine, amodiaquine, quinine, quinidine and mefloquine were prepared in vitro, using water-propylène glycol 30% mixture as solvent. The in vitro antimalarial assays were carried out according to WHO protocol and expressed as IC 50 i.e concentration of drug which kills 50% of the parasite. The in vivo antiplasmodial activity was evaluated by thick smear while the average weights of mice before and after the exposure to the hemin-amodiaquin complex were used to evaluate the complex toxicity. The IC 50 shows a relatively high activity for all complexes tested, in comparison with the corresponding quinoline antimalarial based drug alone (p<005). The in vivo antimalarial assay with hemin-amodiaquine complex (H-AQ) shows a total disappearance of parasitaemia at day 7 after infection of Balb/c mice with P. berghei. The preliminary toxicity test on mice has revealed that H-AQ is not toxic. Besides, any correlation was found between the antimalarial activity and the thermodynamic stability considering all complexes. This could be explained by the great discrepancy observed in susceptibility of used parasite strains. It is therefore desirable that the antiplasmodial test be carried on standardized strains using the isotopic test. It would be interesting to include other quinoline derivatives and corresponding complexes in order to confirm the thermodynamic-biological activity correlation of complexes by determining an area of temperature, pH, ionic strength, etc. to which the complexation constant would respect the order of parasitological activity.
... The number of proteins in oxygen binding (GO:0019825) in water buffalo is far higher than in beef cattle and dairy cattle, which increases the significance of deep investigation of these two proteins. Hemoglobin is vital to hemoprotozoan survival inside the RBC and, to date, it is regarded as the main energy source for most of the hemoprotozoan (35). Moreover, hemoglobin is covalently modified in order to inhibit the intake of amino acids by plasmodium but does not affect the normal functions (36). ...
A number of studies have demonstrated that Babesia orientalis (B. orientalis) can only infect water buffalo (Bubalus bubalis) and not dairy cattle (Bos taurus) or beef cattle (Bos taurus), even though all three belong to the tribe Bovini and have close evolutionary relationships. In addition, Babesia species are intracellular protozoans that obligately parasitize in erythrocytes. This may indicate that the infection specificity is due to differences in erythrocyte proteins. Totals of 491, 1,143, and 1,145 proteins were identified from water buffalo, beef cattle, and dairy cattle, respectively, by searching the Uniprot and NCBI databases. The number of proteins identified for water buffalo was far lower than for beef cattle and dairy cattle, particularly in the range from 15 to 25 kDa. Remarkably, 290 identified proteins were unique to water buffalo, of which putative gamma-globin and putative epsilon-globin had a significant possibility of being relevant to the survival of B. orientalis only in water buffalo. A total of 2,222 proteins were annotated in terms of molecular function, biological process, and cellular component according to GO annotation. The number of proteins of water buffalo in oxygen binding was far higher than for beef cattle and dairy cattle. This is the first time that the protein profiles of water buffalo, beef cattle, and dairy cattle have been comparatively analyzed. The uniquely expressed proteins in water buffalo obtained in this study may provide new insights into the mechanism of B. orientalis infection exclusivity in water buffalo and may be a benefit for the development of strategies against B. orientalis.
... On the basis of the screening of compounds that bound the modeled Pf HDP structure in the heme/hemoglobin-binding pockets in silicon, compound 130 was identified with the Pf HDP inhibitory activity against the Hz formation. 229 In addition, the discovery of pigment HZ inhibitor from natural products library and drug repurposing also becomes the hot topic. Inokuchi group synthesized a series of indoloquinolines analogues originated from Cryptolepis sanguinolenta. ...
The fascinating and dangerous colored pathogens contain unique chemically pigmented molecules, which give varied and efficient assistance as virulence factors to the crucial reproduction and growth of microbes. Therefore, multiple novel strategies and inhibitors have been developed in recent years that target virulence factor pigments. However, despite the importance and significance of this topic, it has not yet been comprehensively reviewed. Moreover, research groups around the world have made successful progress against antibacterial infections by targeting pigment production, including our serial works on the discovery of CrtN inhibitors against staphyloxanthin production in Staphylococcus aureus. On the basis of the previous achievements and recent progress of our group in this field, this article will be the first comprehensive review of pigment inhibitors against colored pathogens, especially S. aureus infections, and this article includes design strategies, representative case studies, advantages, limitations, and perspectives to guide future research.
... Chloroquine (CQ) exerts its antimalarial function by inhibiting hemozoin formation during the critical ferriheme detoxification process of the Plasmodium parasite [35][36][37][38][39]. The molecule acts either by: (a) directly complexing monomeric PPIX preventing its incorporation into the growing hemozoin crystals [36,[40][41][42], (b) capping the hemozoin crystal [43,44], (c) binding to PPIX via π-π interactions between the its aromatic ring and the porphyrin ring [45,46], (d) shifting the haematin/µ-oxo PPIX dimer equilibrium [40], or (e) docking onto the fastest growing crystal face [47]. ...
We have explored the possibility of using organometallic derivatives of cobalamin as a scaffold for the delivery of the same antimalarial drug to both erythro- and hepatocytes. This hybrid molecule approach, intended as a possible tool for the development of multi-stage antimalarial agents, pivots on the preparation of azide-functionalized drugs which, after coupling to the vitamin, are released with a 4-(4-ethynylphenyl)-triazole functionality. Three chloroquine and one imidazolopiperazine derivative (based on the KAF156 structure) were selected as model drugs. One hybrid chloroquine conjugate was extensively studied via fluorescent labelling for in vitro and in vivo bio-distribution studies and gave proof-of-concept for the design. It showed no toxicity in vivo (zebrafish model) as well as no hepatotoxicity, no cardiotoxicity or developmental toxicity of the embryos. All 4-(4-ethynylphenyl)-triazole derivatives of chloroquine were equally active against chloroquine-resistant (CQR) and chloroquine-sensitive (CQS) Plasmodium falciparum strains.
... A routinely used scoring function is Verify3D. [41,42] Verify3D assesses segments of the model based on how well the environment of the residues in that segments correlate with their detected propensities for being in that environment. [43] Statistics-based scoring functions, such as ProsaII, [44,45] measure the stability of a polypeptide from the frequency that the interactions (atom-atom or residue-residue) identified in that conformation becomes clear in the database of known structures. ...
Proteins are composed of linear chains of amino acids that form a unique three-dimensional structure in their native environment. Such native structure favors the proteins to perform their biochemical activity. Protein is formed of some levels of structure. The primary structure of a protein is specified by the particular amino acid sequence. In an amino acid sequence, patterns of local bonding can be identified as secondary structure. The final level that forms a tertiary protein structure is composed of the mentioned elements and form after the protein folds into its native state. To find the native structure of proteins, the physicochemical principles as well as identifying the lowest free-energy states are considered as the best properties and to predict target proteins with unknown structures, the bioinformatics-based methods have earned considerable success. Protein structure prediction methods have been mainly classified into three types: ab Initio folding, comparative (homology) modeling and threading. Each mentioned method may be applied for a protein structure, depending on the existence of related experimental structures that are deposited in the PDB. Once an initial model is generated, refinement simulations are conducted to reassemble the global topology and the local structures of the protein chains. Since significant features of a model may be in regions that are structurally distinct from the template, refining of a primary model is influential. A trustful strategy is included a stereo-chemical check and discovering how the model deviates from the basic disciplines of known experimental structures.
... Inside the erythrocyte, the parasite digests the host hemoglobin to supply itself with amino acids essential for its biosynthetic processes. The toxic free heme is detoxified by crystallization to an insoluble polymer called hemozoin (Gupta et al., 2017). The schizont forms 16-32 merozoites that are released into the bloodstream under rupture of the erythrocyte (Figure 1.2C) Mitchell, 2003). ...
Peroxiredoxins (Prx) are ubiquitous thiol-dependent hydroperoxidases and belong to the most abundant enzymes in a variety of organisms from all kingdoms of life. Furthermore, Prx act as redox sensors in hydrogen peroxide signaling. The Antioxidant Protein (PfAOP), a Prx5-type enzyme from the malaria parasite Plasmodium falciparum, was recently shown to belong to a special subclass of Prx using glutaredoxin (Grx) and reduced glutathione (GSH) as electron donors. In this work, two modulatory residues of PfAOP were characterized that influence the enzyme parameters and the inactivation susceptibility. Gain- and loss-of-function mutants were generated in which the size of residue 109 was altered and the catalytically relevant but nonessential cysteine residue 143 was present or absent. Using steady-state kinetic measurements with recombinant PfAOP, the overall reactivity of these mutant enzymes was analyzed and the mechanism of the reduction of the oxidized enzyme was unraveled. Rate constants for the oxidation and over-oxidation of the peroxidatic cysteine residues were determined by stopped-flow kinetic measurements. A mutant enzyme was identified that is catalytically more active and more durable regarding hydrogen peroxide dependent inactivation compared to the wild-type enzyme. This suggests that PfAOP not only acts as a hydroperoxidase but might also have an additional function as part of a redox relay in vivo. Fusion constructs between redox-sensitive green fluorescent protein 2 (roGFP2) and Prx are valuable tools for redox measurements in living cells. Using the in vitro well characterized gain- and loss-of-function mutants of PfAOP, different fusion constructs with roGFP2 were generated and the in vivo roGFP2 readouts were analyzed in yeast. We showed that the ratiometrically measured degree of oxidation of the roGFP2 fusion proteins correlates with the corresponding in vitro enzyme properties of the attached Prx. Additionally, the roGFP2 signal can be used to map the over-oxidation based inactivation of the Prx. This will allow the assessment of protein structure-function relationships, like post-translational protein modifications, in vivo. Further future applications are the estimation of absolute intracellular peroxide concentrations and the improvement of redox sensors. To gain more insight into the physiological relevance of PfAOP, knockout parasites were generated in the P. falciparum strain 3D7 using the CRSIPR/Cas9 system. The 3D7Δpfaop knockout lines were viable and showed no significant growth phenotype under standard cell culture conditions. Furthermore, the IC50 values for external oxidants remained unchanged. In a previous conducted quantitative trait locus analysis a locus on chromosome 7, encoding 49 genes including PFAOP, was associated with altered artemisinin susceptibility in malaria parasites. Here, we showed that the deletion of the gene encoding PfAOP does neither affect IC50 values nor ring stage survival rates for artemisinin. Thus, the correlation between chromosome 7 and the artemisinin susceptibility is probably based on one of the other genes within the identified locus. Western blot analyses of PFAOP over-expressing P. falciparum strains in combination with peroxide challenges revealed a band corresponding to a probable interaction partner of PfAOP in the cytosol. However, this protein could not be identified so far and it remains to be shown if it is part of a peroxide dependent redox relay involved in redox signaling. In summary, the findings of this thesis lead to a better understanding of the kinetic mechanism of peroxiredoxins. Furthermore, it could be shown that in vitro kinetic properties of peroxidases correlate with the roGFP2 readout of corresponding fusion constructs inside living cells. So far, the physiological relevance of PfAOP remains unknown, but our results suggest that PfAOP might exert an additional function as a redox sensor in vivo.
Malaria caused by Plasmodium protozoa, transmitted by the Anopheles mosquitoes' is one ofthe most important diseases. Current antimalarial drugs target vital parasite progressions ormetabolic pathways essential for parasitic development, thus aiding the host in overcomingthe infection by inhibiting protozoa growth. However, at the same time, it also produces adisruptive consequence on the host cells. These cause severe adverse effects on the host andlead to drug resistance. The urgent need for novel, non‐toxic anti‐protozoal compounds isevident due to the resistance developed against drugs like chloroquine andhydroxychloroquine. Metal complexes of various elements like iron, gold, ruthenium, and othershave shown their anti‐malarial potential. We have reviewed the research ongoing globally on the developments of new molecules of chloroquine coupled with different transition elements and describe the structure–activity relationship of chloroquine, which also provides insights into the chemistry of these compounds.
Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.
Malaria parasites generate toxic heme during hemoglobin digestion, which is neutralized by crystallizing into inert hemozoin (β-hematin). Chloroquine blocks this detoxification process, resulting in heme-mediated toxicity in malaria parasites. However, the exact mechanism of chloroquine’s action remains unknown. This study investigates the impact of chloroquine on the transformation of heme into β-hematin. The results show that chloroquine does not completely halt the transformation process but rather slows it down. Additionally, chloroquine complexation with free heme does not affect substrate availability or inhibit β-hematin formation. SEM and XRD studies indicate that the size of β-hematin crystal particles and crystallite increases in the presence of chloroquine, suggesting that chloroquine does not impede crystal growth. These findings suggest that chloroquine delays hemozoin production by perturbing the nucleation events of crystals and/or the stability of crystal nuclei. Thus, contrary to prevailing beliefs, this study provides a new perspective on the working mechanism of chloroquine.
The challenges concerning the control of malaria remain due to the continuous emergence of drug resistant strains. Over the years, the use, misuse, and abuse of antimalarials have created a conducive environment for the development of resistant Plasmodium falciparum strains. We herein report on the synthesis, characterisation and antimalarial activity of a library of seven novel 1,2,3-triazoles as part of the drug discovery campaign against drug-resistant Plasmodium falciparum. The interactions of the triazoles with plasmepsin II, plasmepsin IV, falcipain-2 and the heme detoxifying protein-all key proteins of Plasmodium falciparum degradosequesterome (Dsq) were also investigated by molecular docking. The compounds 3a-d, 4-6 were synthesized by CuAAC click reaction in good to excellent yields of 73-98% and characterized by melting point, UV-visible, infrared and nuclear magnetic resonance (¹H and ¹³C) and MS techniques. Compounds 3a-d displayed high in vitro potency (IC50s: 0.62-22.11 ug/ml) against the chloroquine-resistant Dd2 lab strain of Plasmodium falciparum and low toxicity (SI > 1 except compound 4) to human erythrocytes. Computational studies indicated that the compounds 3a-3d had an absorption of 76-91%, and they were category III acute oral toxins (LD50 from 500 to 5000 mg/kg). The molecular docking study suggests that compounds 3a-d interacted with plasmepsin IV and the heme detoxifying protein with high affinity and a moderate affinity for falcipain-2.
As India sets its sight on malaria elimination, using tools for vector control, protection, diagnosis, and treatment, it is important to look at the progress made in efforts for new drug discovery in the country. This chapter focuses on antimalarial drug discovery and development efforts for new chemical entities, natural products, and formulations, and the current research scenario for exploring new biological targets in the parasite. It also identifies certain gaps and recommends future measures for strengthening research on antimalarial drugs in India.
Racemic and enantioselective syntheses of γ-phenyl-γ-amino vinyl phosphonates and sulfones have been achieved using Horner−Wadsworth−Emmons olefination of trityl protected α-phenyl-α-amino aldehydes with tetraethyl methylenedi-phosphonate and diethyl ((phenylsulfonyl)methyl)phosphonate, respectively, without any racemization. The present strategy has also been successfully applied to the synthesis of peptidyl vinyl phosphonate and peptidyl vinyl sulfone derivatives as potential cysteine protease inhibitors of Chagas disease, K11002, with 100% de. The developed synthetic protocol was further utilized to synthesize hybrid molecules consisting of artemisinin as an inhibitor of major cysteine protease falcipain-2 present in the food vacuole of the malarial parasite. The synthesized artemisinin−dipeptidyl vinyl sulfone hybrid compounds showed effective in vitro inhibition of falcipain-2 and potent parasiticidal efficacies against Plasmodium falciparum in nanomolar ranges. Overall, the developed synthetic protocol could be effectively utilized to design cysteine protease inhibitors not only as novel antimalarial compounds but also to be involved in other life-threatening diseases.
Racemic and enantioselective syntheses of γ-phenyl-γ-amino vinyl phosphonates and sulfones have been achieved using Horner–Wadsworth–Emmons olefination of trityl protected α-phenyl-α-amino aldehydes with tetraethyl methylenediphosphonate and diethyl ((phenylsulfonyl)methyl)phosphonate, respectively, without any racemization. The present strategy has also been successfully applied to the synthesis of peptidyl vinyl phosphonate and peptidyl vinyl sulfone derivatives as potential cysteine protease inhibitors of Chagas disease, K11002, with 100% de. The developed synthetic protocol was further utilized to synthesize hybrid molecules consisting of artemisinin as an inhibitor of major cysteine protease falcipain-2 present in the food vacuole of the malarial parasite. The synthesized artemisinin–dipeptidyl vinyl sulfone hybrid compounds showed effective in vitro inhibition of falcipain-2 and potent parasiticidal efficacies against Plasmodium falciparum in nanomolar ranges. Overall, the developed synthetic protocol could be effectively utilized to design cysteine protease inhibitors not only as novel antimalarial compounds but also to be involved in other life-threatening diseases.
Artemisinins are the most rapidly acting of currently available antimalarial drugs. Artesunate has become the treatment of choice for severe malaria, and artemisinin-based combination therapies (ACTs) are the foundation of modern falciparum malaria treatment globally. Their safety and tolerability profile is excellent. Unfortunately, Plasmodium falciparum infections with mutations in the 'K13' gene, with reduced ring-stage susceptibility to artemisinins, and slow parasite clearance in patients treated with ACTs, are now widespread in Southeast Asia. We review clinical efficacy data from the region (2000-2015) that provides strong evidence that the loss of first-line ACTs in western Cambodia, first artesunate-mefloquine and then DHA-piperaquine, can be attributed primarily to K13 mutated parasites. The ring-stage activity of artemisinins is therefore critical for the sustained efficacy of ACTs; once it is lost, rapid selection of partner drug resistance and ACT failure are inevitable consequences. Consensus methods for monitoring artemisinin resistance are now available. Despite increased investment in regional control activities, ACTs are failing across an expanding area of the Greater Mekong subregion. Although multiple K13 mutations have arisen independently, successful multidrug-resistant parasite genotypes are taking over and threaten to spread to India and Africa. Stronger containment efforts and new approaches to sustaining long-term efficacy of antimalarial regimens are needed to prevent a global malaria emergency.
Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes. However, the reaction mechanisms of HDP are poorly understood. We identified the active site residues in HDP using a combination of Hz formation assay and spectroscopic characterization of mutant proteins. Replacement of the critical histidine residues His122, His172, His175, and His197 resulted in a reduction in the Hz formation activity to approximately 50% of the wild-type protein. Spectroscopic characterization of histidine-substituted mutants revealed that His122 binds heme and that His172 and His175 form a part of another heme-binding site. Our results show that the histidine residues could be present in the individual active sites and could be ligated to each heme. The interaction between heme and the histidine residues would serve as a molecular tether, allowing the proper positioning of two hemes to enable heme dimer formation. The heme dimer would act as a seed for the crystal growth of Hz in P. falciparum.
Protein structure homology modelling has become a routine technique to generate 3D models for proteins when experimental structures
are not available. Fully automated servers such as SWISS-MODEL with user-friendly web interfaces generate reliable models
without the need for complex software packages or downloading large databases. Here, we describe the latest version of the
SWISS-MODEL expert system for protein structure modelling. The SWISS-MODEL template library provides annotation of quaternary
structure and essential ligands and co-factors to allow for building of complete structural models, including their oligomeric
structure. The improved SWISS-MODEL pipeline makes extensive use of model quality estimation for selection of the most suitable
templates and provides estimates of the expected accuracy of the resulting models. The accuracy of the models generated by
SWISS-MODEL is continuously evaluated by the CAMEO system. The new web site allows users to interactively search for templates,
cluster them by sequence similarity, structurally compare alternative templates and select the ones to be used for model building.
In cases where multiple alternative template structures are available for a protein of interest, a user-guided template selection
step allows building models in different functional states. SWISS-MODEL is available at http://swissmodel.expasy.org/.
Malaria parasites use hemoglobin (Hb) as a major nutrient source in the intraerythrocytic stage, during which heme is converted to hemozoin (Hz). The formation of Hz is essential for parasite survival, but to date, the underlying mechanisms of Hb degradation and Hz formation are poorly understood. We report the presence of a ∼200-kDa protein complex in the food vacuole that is required for Hb degradation and Hz formation. This complex contains several parasite proteins, including falcipain 2/2', plasmepsin II, plasmepsin IV, histo aspartic protease, and heme detoxification protein. The association of these proteins is evident from coimmunoprecipitation followed by mass spectrometry, coelution from a gel filtration column, cosedimentation on a glycerol gradient, and in vitro protein interaction analyses. To functionally characterize this complex, we developed an in vitro assay using two of the proteins present in the complex. Our results show that falcipain 2 and heme detoxification protein associate with each other to efficiently convert Hb to Hz. We also used this in vitro assay to elucidate the modes of action of chloroquine and artemisinin. Our results reveal that both chloroquine and artemisinin act during the heme polymerization step, and chloroquine also acts at the Hb degradation step. These results may have important implications in the development of previously undefined antimalarials.
Motivation:
Molecular simulation has historically been a low-throughput technique, but faster computers and increasing amounts of genomic and structural data are changing this by enabling large-scale automated simulation of, for instance, many conformers or mutants of biomolecules with or without a range of ligands. At the same time, advances in performance and scaling now make it possible to model complex biomolecular interaction and function in a manner directly testable by experiment. These applications share a need for fast and efficient software that can be deployed on massive scale in clusters, web servers, distributed computing or cloud resources.
Results:
Here, we present a range of new simulation algorithms and features developed during the past 4 years, leading up to the GROMACS 4.5 software package. The software now automatically handles wide classes of biomolecules, such as proteins, nucleic acids and lipids, and comes with all commonly used force fields for these molecules built-in. GROMACS supports several implicit solvent models, as well as new free-energy algorithms, and the software now uses multithreading for efficient parallelization even on low-end systems, including windows-based workstations. Together with hand-tuned assembly kernels and state-of-the-art parallelization, this provides extremely high performance and cost efficiency for high-throughput as well as massively parallel simulations.
Availability:
GROMACS is an open source and free software available from http://www.gromacs.org.
Supplementary information:
Supplementary data are available at Bioinformatics online.
Drug-likeness is a key consideration when selecting compounds during the early stages of drug discovery. However, evaluation of drug-likeness in absolute terms does not reflect adequately the whole spectrum of compound quality. More worryingly, widely used rules may inadvertently foster undesirable molecular property inflation as they permit the encroachment of rule-compliant compounds towards their boundaries. We propose a measure of drug-likeness based on the concept of desirability called the quantitative estimate of drug-likeness (QED). The empirical rationale of QED reflects the underlying distribution of molecular properties. QED is intuitive, transparent, straightforward to implement in many practical settings and allows compounds to be ranked by their relative merit. We extended the utility of QED by applying it to the problem of molecular target druggability assessment by prioritizing a large set of published bioactive compounds. The measure may also capture the abstract notion of aesthetics in medicinal chemistry.
NCBI’s Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved
domain footprints, and functional sites inferred from these footprints. CDD includes manually curated domain models that make
use of protein 3D structure to refine domain models and provide insights into sequence/structure/function relationships. Manually
curated models are organized hierarchically if they describe domain families that are clearly related by common descent. As
CDD also imports domain family models from a variety of external sources, it is a partially redundant collection. To simplify
protein annotation, redundant models and models describing homologous families are clustered into superfamilies. By default,
domain footprints are annotated with the corresponding superfamily designation, on top of which specific annotation may indicate
high-confidence assignment of family membership. Pre-computed domain annotation is available for proteins in the Entrez/Protein
dataset, and a novel interface, Batch CD-Search, allows the computation and download of annotation for large sets of protein
queries. CDD can be accessed via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.
Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.
Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.
Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.
Virtual screening methods start to be well established as effective approaches to identify hits, candidates and leads for drug discovery research. Among those, structure based virtual screening (SBVS) approaches aim at docking collections of small compounds in the target structure to identify potent compounds. For SBVS, the identification of candidate pockets in protein structures is a key feature, and the recent years have seen increasing interest in developing methods for pocket and cavity detection on protein surfaces.
Fpocket is an open source pocket detection package based on Voronoi tessellation and alpha spheres built on top of the publicly available package Qhull. The modular source code is organised around a central library of functions, a basis for three main programs: (i) Fpocket, to perform pocket identification, (ii) Tpocket, to organise pocket detection benchmarking on a set of known protein-ligand complexes, and (iii) Dpocket, to collect pocket descriptor values on a set of proteins. Fpocket is written in the C programming language, which makes it a platform well suited for the scientific community willing to develop new scoring functions and extract various pocket descriptors on a large scale level. Fpocket 1.0, relying on a simple scoring function, is able to detect 94% and 92% of the pockets within the best three ranked pockets from the holo and apo proteins respectively, outperforming the standards of the field, while being faster.
Fpocket provides a rapid, open source and stable basis for further developments related to protein pocket detection, efficient pocket descriptor extraction, or drugablity prediction purposes. Fpocket is freely available under the GNU GPL license at http://fpocket.sourceforge.net.
Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in folate biosynthesis and a major malarial drug target. This bifunctional enzyme thus presents different design approaches for developing novel inhibitors against drug-resistant mutants. We performed a high-throughput in silico screen of a database of diverse, drug-like molecules against a non-active-site pocket of TS-DHFR. The top compounds from this virtual screen were evaluated by in vitro enzymatic and cellular culture studies. Three compounds active to 20 microM IC(50)'s in both wildtype and antifolate-resistant P. falciparum parasites were identified; moreover, no inhibition of human DHFR enzyme was observed, indicating that the inhibitory effects appeared to be parasite-specific. Notably, all three compounds had a biguanide scaffold. However, relative free energy of binding calculations suggested that the compounds might preferentially interact with the active site over the screened non-active-site region. To resolve the two possible modes of binding, co-crystallization studies of the compounds complexed with TS-DHFR enzyme were performed. Surprisingly, the structural analysis revealed that these novel, biguanide compounds do indeed bind at the active site of DHFR and additionally revealed the molecular basis by which they overcome drug resistance. To our knowledge, these are the first co-crystal structures of novel, biguanide, non-WR99210 compounds that are active against folate-resistant malaria parasites in cell culture.
We previously identified a Plasmodium falciparum trophozoite cysteine proteinase (TCP) and hypothesized that it is required for the degradation of host hemoglobin by intraerythrocytic malaria parasites. To test this hypothesis and to evaluate TCP as a chemotherapeutic target, we examined the antimalarial effects of a panel of peptide fluoromethyl ketone proteinase inhibitors. For each inhibitor, effectiveness at inhibiting the activity of TCP correlated with effectiveness at both blocking hemoglobin degradation and killing cultured parasites. Benzyloxycarbonyl (Z)-Phe-Arg-CH2F, the most potent inhibitor, inhibited TCP at picomolar concentrations and blocked hemoglobin degradation and killed parasites at nanomolar concentrations. Micromolar concentrations of the inhibitor were nontoxic to cultured mammalian cells. These results support the hypothesis that TCP is a necessary hemoglobinase and suggest that it is a promising chemotherapeutic target.
The intraerythrocytic malaria parasite uses hemoglobin as a major nutrient source. Digestion of hemoglobin releases heme, which the parasite converts into an insoluble microcrystalline material called hemozoin or malaria pigment. We have purified hemozoin from the human malaria organism Plasmodium falciparum and have used infrared spectroscopy, x-ray absorption spectroscopy, and chemical synthesis to determine its structure. The molecule consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme.
A novel method for differentiating between correctly and incorrectly determined regions of protein structures based on characteristic atomic interactions is described. Different types of atoms are distributed nonrandomly with respect to each other in proteins. Errors in model building lead to more randomized distributions of the different atom types, which can be distinguished from correct distributions by statistical methods.
Atoms are classified in one of three categories: carbon (C), nitrogen (N), and oxygen (O). This leads to six different combinations of pairwise noncovalently bonded interactions (CC, CN, CO, NN, NO, and OO). A quadratic error function is used to characterize the set of pairwise interactions from nine-residue sliding windows in a database of 96 reliable protein structures. Regions of candidate protein structures that are mistraced or misregistered can then be identified by analysis of the pattern of nonbonded interactions from each window.
Hemoglobin degradation in intraerythrocytic malaria parasites is a vast process that occurs in an acidic digestive vacuole. Proteases that participate in this catabolic pathway have been defined. Studies of protease biosynthesis have revealed unusual targeting and activation mechanisms. Oxygen radicals and heme are released during proteolysis and must be detoxified by dismutation and polymerization, respectively. The quinoline antimalarials appear to act by preventing sequestration of this toxic heme. Understanding the disposition of hemoglobin has allowed identification of essential processes and metabolic weakpoints that can be exploited to combat this scourge of mankind.
The three-dimensional (3D) profile of a protein structure is a table computed from the atomic coordinates of the structure that can be used to score the compatibility of the 3D structure model with any amino acid sequence. Three-dimensional profiles computed from correct protein structures match their own sequences with high scores. An incorrectly modeled segment in an otherwise correct structure can be identified by examining the profile score in a moving-window scan. Thus, the correctness of a protein model can be verified by its 3D profile, regardless of whether the model has been derived by X-ray, nuclear magnetic resonance (NMR), or computational procedures. For this reason, 3D profiles are useful in the evaluation of undetermined protein models, based on low-resolution electron-density maps, on NMR spectra with inadequate distance constraints, or on computational procedures. An advantage of using 3D profiles for testing models is that profiles have not themselves been used in the determination of the structure. Traditional R-factor tests in X-ray analysis depend on the comparison of observed properties—that is, the X-ray structure factor magnitudes with the same property calculated from the final protein model.
The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.
Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61+/-2% of the iron within parasitized erythrocytes, of which 92+/-6% is located within the food vacuole. Of this, 88+/-9% is in the form of haemozoin. (57)Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion.
Falcipain-2 (FP2) is a papain family cysteine protease and important hemoglobinase of erythrocytic Plasmodium falciparum parasites. Inhibitors of FP2 block hemoglobin hydrolysis and parasite development, suggesting that this enzyme is a promising target for antimalarial chemotherapy. FP2 and related plasmodial cysteine proteases have an unusual 14-aa motif near the C terminus of the catalytic domain. Recent solution of the structure of FP2 showed this motif to form a β-hairpin that is distant from the enzyme active site and protrudes out from the protein. To evaluate the function of this motif, we compared the activity of the wild-type enzyme with that of a mutant lacking 10 aa of the motif (Δ10FP2). Δ10FP2 had nearly identical activity to that of the wild-type enzyme against peptide substrates and the protein substrates casein and gelatin. However, Δ10FP2 demonstrated negligible activity against hemoglobin or globin. FP2 that was inhibited with trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (FP2E-64) formed a complex with hemoglobin, but Δ10FP2E-64 did not, indicating that the motif mediates binding to hemoglobin independent of the active site. A peptide encoding the motif blocked hemoglobin hydrolysis, but not the hydrolysis of casein. Kinetics for the inhibition of Δ10FP2 were very similar to those for FP2 with peptidyl and protein inhibitors, but Δ10FP2 was poorly inhibited by the inhibitory prodomain of FP2. Our results indicate that FP2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor.
• macromolecular interaction
• malaria
• drug discovery
• antimalarial chemotherapy
Here, we describe two freely available web servers for molecular docking. The PatchDock method performs structure prediction
of protein–protein and protein–small molecule complexes. The SymmDock method predicts the structure of a homomultimer with
cyclic symmetry given the structure of the monomeric unit. The inputs to the servers are either protein PDB codes or uploaded
protein structures. The services are available at http://bioinfo3d.cs.tau.ac.il. The methods behind the servers are very efficient, allowing large-scale docking experiments.
Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. The community-wide Critical Assessment of Structure Prediction (CASP) experiments have been designed to obtain an objective assessment of the state-of-the-art of the field, where I-TASSER was ranked as the best method in the server section of the recent 7th CASP experiment. Our laboratory has since then received numerous requests about the public availability of the I-TASSER algorithm and the usage of the I-TASSER predictions.
An on-line version of I-TASSER is developed at the KU Center for Bioinformatics which has generated protein structure predictions for thousands of modeling requests from more than 35 countries. A scoring function (C-score) based on the relative clustering structural density and the consensus significance score of multiple threading templates is introduced to estimate the accuracy of the I-TASSER predictions. A large-scale benchmark test demonstrates a strong correlation between the C-score and the TM-score (a structural similarity measurement with values in [0, 1]) of the first models with a correlation coefficient of 0.91. Using a C-score cutoff > -1.5 for the models of correct topology, both false positive and false negative rates are below 0.1. Combining C-score and protein length, the accuracy of the I-TASSER models can be predicted with an average error of 0.08 for TM-score and 2 A for RMSD.
The I-TASSER server has been developed to generate automated full-length 3D protein structural predictions where the benchmarked scoring system helps users to obtain quantitative assessments of the I-TASSER models. The output of the I-TASSER server for each query includes up to five full-length models, the confidence score, the estimated TM-score and RMSD, and the standard deviation of the estimations. The I-TASSER server is freely available to the academic community at http://zhang.bioinformatics.ku.edu/I-TASSER.
Structural details of protein–protein interactions are invaluable for understanding and deciphering biological mechanisms.
Computational docking methods aim to predict the structure of a protein–protein complex given the structures of its single
components. Protein flexibility and the absence of robust scoring functions pose a great challenge in the docking field. Due
to these difficulties most of the docking methods involve a two-tier approach: coarse global search for feasible orientations that treats proteins as rigid bodies, followed by an accurate refinement stage that aims to introduce flexibility into the process. The FireDock web server, presented here, is the first web server
for flexible refinement and scoring of protein–protein docking solutions. It includes optimization of side-chain conformations
and rigid-body orientation and allows a high-throughput refinement. The server provides a user-friendly interface and a 3D
visualization of the results. A docking protocol consisting of a global search by PatchDock and a refinement by FireDock was
extensively tested. The protocol was successful in refining and scoring docking solution candidates for cases taken from docking
benchmarks. We provide an option for using this protocol by automatic redirection of PatchDock candidate solutions to the
FireDock web server for refinement. The FireDock web server is available at http://bioinfo3d.cs.tau.ac.il/FireDock/.
Author Summary
Each year, more than one million people, most of them children under the age of 5, succumb to malaria, a devastating disease caused by Plasmodium parasites. The parasite resides inside the red blood cells of the host, where, during its development, it proteolyzes vast amounts of host hemoglobin. This degradation also releases heme, which is extremely toxic to the parasite. To protect itself (from the toxic effects of heme), the parasite converts free heme into hemozoin. This parasite-specific mechanism is widely accepted as the weakest link in its lifecycle and is targeted by several of the currently available antimalarial drugs, which prevent hemozoin formation by binding to heme. However, due to an incomplete understanding of the parasite processes that lead to hemozoin formation, a drug that specifically targets the parasite factors responsible for hemozoin production has never been developed. Here we identify and characterize Heme Detoxification Protein, a unique Plasmodium protein, which we show as the potent producer of hemozoin. We demonstrate that this protein is highly conserved across the Plasmodium genus, is extremely efficient in producing hemozoin, and is delivered to the food vacuole, the site of hemozoin formation, via a unique trafficking route. We also demonstrate the critical nature of this protein and suggest that it could be targeted to develop novel antimalarial drugs.
Falcipain-2 (FP-2), a papain family cysteine protease of Plasmodium falciparum, is a promising target for antimalarial chemotherapy. Designing inhibitors that are highly selective for falcipain-2 has been difficult because of broad specificity of different cysteine proteinases. Because propeptide regions of cysteine proteases have been shown to inhibit their cognate enzymes specifically and selectively, in the present study, we evaluated the inhibitory potential of few falcipain-2 proregion peptides. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme activity in vitro. Studies on the uptake of PP1 into the parasitized erythrocytes showed access of peptide into the infected RBCs. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation. Together, our results identify a peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite.
Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described. In the discovery setting 'the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5 (or MlogP>4.15). Computational methodology for the rule-based Moriguchi Log P (MLogP) calculation is described. Turbidimetric solubility measurement is described and applied to known drugs. High throughput screening (FITS) leads tend to have higher MWT and Log P and lower turbidimetric solubility than leads in the pre-HTS era. In the development setting, solubility calculations focus on exact value prediction and are difficult because of polymorphism. Recent work on linear free energy relationships and Log P approaches are critically reviewed. Useful predictions are possible in closely related analog series when coupled with experimental thermodynamic solubility measurements. (C) 2012 Published by Elsevier B.V.
In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical (1)H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material.
AutoDock Vina, a new program for molecular docking and virtual screening, is presented. AutoDock Vina achieves an approximately two orders of magnitude speed-up compared with the molecular docking software previously developed in our lab (AutoDock 4), while also significantly improving the accuracy of the binding mode predictions, judging by our tests on the training set used in AutoDock 4 development. Further speed-up is achieved from parallelism, by using multithreading on multicore machines. AutoDock Vina automatically calculates the grid maps and clusters the results in a way transparent to the user.
Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described. In the discovery setting `the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5 (or MlogP>4.15). Computational methodology for the rule-based Moriguchi Log P (MLogP) calculation is described. Turbidimetric solubility measurement is described and applied to known drugs. High throughput screening (HTS) leads tend to have higher MWT and Log P and lower turbidimetric solubility than leads in the pre-HTS era. In the development setting, solubility calculations focus on exact value prediction and are difficult because of polymorphism. Recent work on linear free energy relationships and Log P approaches are critically reviewed. Useful predictions are possible in closely related analog series when coupled with experimental thermodynamic solubility measurements.
Geometrical validation around the Cα is described, with a new Cβ measure and updated Ramachandran plot. Deviation of the observed Cβ atom from ideal position provides a single measure encapsulating the major structure-validation information contained in bond angle distortions. Cβ deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new ϕ,ψ plot using density-dependent smoothing for 81,234 non-Gly, non-Pro, and non-prePro residues with B < 30 from 500 high-resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the γ-turn conformation near +75°,−60°, counted as forbidden by common structure-validation programs; however, it occurs in well-ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the γ-turn H-bond. Favored and allowed ϕ,ψ regions are also defined for Pro, pre-Pro, and Gly (important because Gly ϕ,ψ angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of α-helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two-peptide NHs permits donating only a single H-bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user-uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www-cryst.bioc.cam.ac.uk/rampage). Proteins 2003;50:437–450. © 2003 Wiley-Liss, Inc.
The AQUA and PROCHECK-NMR programs provide a means of validating the geometry and restraint violations of an ensemble of protein structures solved by solution NMR. The outputs include a detailed breakdown of the restraint violations, a number of plots in PostScript format and summary statistics. These various analyses indicate both the degree of agreement of the model structures with the experimental data, and the quality of their geometrical properties. They are intended to be of use both to support ongoing NMR structure determination and in the validation of the final results.
Computer simulations offer a valuable way to study membrane systems, from simple lipid bilayers to large transmembrane protein complexes and lipid-nucleic acid complexes for drug delivery. Their accuracy depends on the quality of the force field parameters used to describe the components of a particular system. We have implemented the widely used CHARMM22 and CHARMM27 force fields in the GROMACS simulation package to (i) combine the CHARMM22 protein force field with two sets of united-atom lipids parameters; (ii) allow comparisons of the lipid CHARMM27 force field with other lipid force fields or lipid-protein force field combinations. Our tests do not show any particular issue with the combination of the all-atom CHARMM22 force field with united-atoms lipid parameters, although pertinent experimental data are lacking to assess the quality of the lipid-protein interactions. The conversion utilities allow automatic generation of GROMACS simulation files with CHARMM nucleic acids and protein parameters and topologies, starting from pdb files using the standard GROMACS pdb2gmx method. CMAP is currently not implemented.
This report describes a number of substructural features which can help to identify compounds that appear as frequent hitters (promiscuous compounds) in many biochemical high throughput screens. The compounds identified by such substructural features are not recognized by filters commonly used to identify reactive compounds. Even though these substructural features were identified using only one assay detection technology, such compounds have been reported to be active from many different assays. In fact, these compounds are increasingly prevalent in the literature as potential starting points for further exploration, whereas they may not be.
We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using flexible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique.
The malaria parasite Plasmodium falciparum uses host erythrocyte hemoglobin as a major nutrient source. We report the purification of P. falciparum digestive vacuoles and characterization of the degradative process therein. Vacuoles were isolated by a combination of differential centrifugation and density gradient separation. The pure vacuoles were capable of degrading hemoglobin to small fragments with a pH optimum of 5-5.5. Proteolysis in the vacuoles appears to be an ordered process, requiring an aspartic protease to clip intact hemoglobin before other proteolytic activities can function efficiently. The vacuoles do not contain other hydrolases commonly found in lysosomes and therefore appear to be unique proteolytic organelles designed specifically to degrade hemoglobin.
To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucyl-amido-(4-guanidino)-butane (E-64), two peptide inhibitors of cysteine proteinases, inhibited the proteolysis of globin and caused the accumulation of undegraded erythrocyte cytoplasm in parasite food vacuoles, suggesting that a food vacuole cysteine proteinase is necessary for hemoglobin degradation. Proteinase assays of trophozoites demonstrated cysteine proteinase activity with a pH optimum similar to that of the food vacuole and the substrate specificity of lysosomal cathepsin L. We also identified an Mr 28,000 proteinase that was trophozoite stage-specific and was inhibited by leupeptin and E-64. We conclude that the Mr 28,000 cysteine proteinase has a critical, perhaps rate-limiting, role in hemoglobin degradation within the food vacuole of Plasmodium falciparum. Specific inhibitors of this enzyme might provide new means of antimalarial chemotherapy.
The digestive vacuole of Plasmodium falciparum is the site of hemoglobin degradation, heme polymerization into crystalline hemozoin, and antimalarial drug accumulation. Antibodies identified histidine-rich protein II (HRP II) in purified digestive vacuoles. Recombinant or native HRP II promoted the formation of hemozoin, and chloroquine inhibited the reaction. The related HRP III also polymerized heme, and an additional HRP was identified in vacuoles. It is proposed that after secretion by the parasite into the host erythrocyte cytosol, HRPs are brought into the acidic digestive vacuole along with hemoglobin. After hemoglobin proteolysis, HRPs bind the liberated heme and mediate hemozoin formation.
The erythrocytic growth stage of Plasmodium falciparum involves hemoglobin proteolysis as the primary nutrient source with the concomitant release of free heme. The liberated heme is processed by the parasite into hemozoin, a polymeric porphyrin dimer. Histidine-rich protein binds heme and mediates the formation of hemozoin, which is inhibited by the antimalarial drug chloroquine. Interference with heme binding was determined using a microtiterplate assay. Combinatorial libraries were screened and tested against parasite growth, revealing a good correlation between heme binding interference and the inhibition of parasite growth. Several of these compounds retain their potency against a chloroquine-resistant strain of Plasmodium falciparum. The most potent compounds have IC(50) values less than or equal to 50 nM against chloroquine-resistant and chloroquine-sensitive parasites.
The histidine-rich protein II (HRP II) from Plasmodium falciparum has been implicated in the formation of hemozoin, a detoxified, crystalline form of ferric protoporphyrin IX (Fe(3+)-PPIX) produced by the parasite. Fe(3+)-PPIX titrations coupled with quantitative amino acid analysis showed that HRP II binds 15 Fe(3+)-PPIX molecules per 30 kDa monomer. Circular dichroism spectroscopy was used to probe the secondary structure of HRP II with and without bound Fe(3+)-PPIX. These studies have revealed large changes in the secondary structure with Fe(3+)-PPIX binding, changing from a random coil in the absence of Fe(3+)-PPIX to a more ordered helical structure in the presence of Fe(3+)-PPIX. The Fe(3+)-PPIX-bound HRP II structure most closely resembles a 3(10)-helix. Coincident with this structural change caused by Fe(3+)-PPIX binding, the formation of an intermolecular disulfide bond occurs between HRP II monomers. In vitro pull-down assays show an interaction between monomers that is dependent on the presence of Fe(3+)-PPIX. One model that best fits with the data reported here requires formation of 15 intermolecular bishistidyl ligated Fe(3+)-PPIX molecules arranged in a head to head fashion, which would then allow for the formation of an intermolecular disulfide bond. The structure best able to accommodate these requirements is a 3(10)-helix.
Malaria continues to be a major threat in the developing world, with > 1 million clinical episodes and 3000 deaths every day. In the last century, malaria claimed between 150 and 300 million lives, accounting for 2 - 5% of all deaths. Currently approximately 40% of the world population resides in areas of active malaria transmission. The disease symptoms are most severe in young children and pregnant women. A total of 90% of the disease-associated mortality occurs in Subsaharan Africa, despite the fact that malaria is indigenous to most tropical regions. A licensed vaccine for malaria has not become a reality and antimalarial drugs are the only available method of treatment. Although chloroquine, the first synthetically developed antimalarial, proved to be an almost magical cure for > 30 years, the emergence and spread of chloroquine-resistant parasites has made it virtually ineffective in most parts of the world. Currently, artemisinin, a plant-derived antimalarial, is the only available drug that is globally effective against the parasite. Although several new drugs have been introduced in the past 30 years, widespread or isolated cases of resistance indicate that their window of effectiveness will be limited. Thus, there is an urgent need to develop new therapeutics and regimens for malaria control. This article presents an overview of the currently available antimalarial chemotherapy options and the efforts being undertaken to develop new drugs based on both the recent technological advances and modifications to the old remedies, and on combination therapies.
The microsomal heme oxygenase system consists of heme oxygenase (HO) and NADPH-cytochrome P450 reductase, and plays a key role in the physiological catabolism of heme which yields biliverdin, carbon monoxide, and iron as the final products. Heme degradation proceeds essentially as a series of autocatalytic oxidation reactions involving heme bound to HO. Large amounts of HO proteins from human and rat can now be prepared in truncated soluble form, and the crystal structures of some HO proteins have been determined. These advances have greatly facilitated the understanding of the mechanisms of individual steps of the HO reaction. HO can be induced in animals by the administration of heme or several other substances; the induction is shown to involve Bach1, a translational repressor. The induced HO is assumed to have cytoprotective effects. An uninducible HO isozyme, HO-2, has been identified, so the authentic HO is now called HO-1. HOs are also widely distributed in invertebrates, higher plants, algae, and bacteria, and function in various ways according to the needs of individual species.
The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.
Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.
Formation of malaria pigment or haemozoin is the major route of haem detoxification in the malaria parasite Plasmodium falciparum as well as several other species of haematophagous organisms, including other Plasmodium species, helminth worms such as Schistosoma mansoni and blood-sucking insects such as Rhodnius prolixus. Recent advances in our understanding of the formation of haemozoin, both from new observations that it is formed within lipid bodies in P. falciparum and S. mansoni and biomimetic studies on the formation of its synthetic counterpart beta-haematin are reviewed. The review also covers methods available for screening compounds for their ability to inhibit beta-haematin formation.
Because the quinolines inhibit heme crystallization within the malaria parasite much work has focused on mechanism of formation and inhibition of hemozoin. Here we review the recent evidence for heme crystallization within lipids in diverse parasites and the new implications of a lipid site of crystallization for drug targeting. Within leukocytes hemozoin can generate toxic radical lipid metabolites, which may alter immune function or reduce deformability of uninfected erythrocytes.
Geneva: World Health Organization
World Malaria Report 2016. Geneva: World Health Organization. 2016.
AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading
- O Trott
- A J Olson
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5: a high-throughput and highly parallel open source molecular simulation toolkit
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- N Sapay
- D P Tieleman
- A Marchler-Bauer
- S Lu
- J B Anderson
- F Chitsaz
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