Identification of new oxospiro chromane quinoline-carboxylate antimalarials that arrest parasite growth at ring stage.

Malaria still threatens half the globe population despite successful Artemisinin-based combination therapy. One of the reasons for our inability to eradicate malaria is the emergence of resistance to current antimalarials. Thus, there is a need to develop new antimalarials targeting Plasmodium proteins. The present study reported the design and synthesis of 4, 6 and 7-substituted quinoline-3-carboxylates 9(a-o) and carboxylic acids 10(a-b) for the inhibition of Plasmodium N-Myristoyltransferases (NMTs) using computational biology tools followed by chemical synthesis and functional analysis. The designed compounds exhibited a glide score of -9.241 to -6.960 kcal/mol for PvNMT and -7.538 kcal/mol for PfNMT model proteins. Development of the synthesized compounds was established via NMR, HRMS and single crystal X-ray diffraction study. The synthesized compounds were evaluated for their in vitro antimalarial efficacy against CQ-sensitive Pf3D7 and CQ-resistant PfINDO lines followed by cell toxicity evaluation. In silico results highlighted the compound ethyl 6-methyl-4-(naphthalen-2-yloxy)quinoline-3-carboxylate (9a) as a promising inhibitor with a glide score of -9.084 kcal/mol for PvNMT and -6.975 kcal/mol for PfNMT with IC50 values of 6.58 µM for Pf3D7 line. Furthermore, compounds 9n and 9o exhibited excellent anti-plasmodial activity (Pf3D7 IC50 = 3.96, 6.71 µM, and PfINDO IC50 = 6.38, 2.8 µM, respectively). The conformational stability of 9a with the active site of the target protein was analyzed through MD simulation and was found concordance with in vitro results. Thus, our study provides scaffolds for the development of potent antimalarials targeting both Plasmodium vivax and Plasmodium falciparum.Communicated by Ramaswamy H. Sarma.

Combating multi-drug resistant malaria parasite by inhibiting falcipain-2 and heme-polymerization: Artemisinin-peptidyl vinyl phosphonate hybrid molecules as new antimalarials.

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.

Quinoline carboxamide core moiety-based compounds inhibit P. falciparumfalcipain-2: Design, synthesis and antimalarial efficacy studies.

Targeting Falcipain-2 (FP2) for the development of antimalarials is a promising and established concept in antimalarial drug discovery and development. FP2, a member of papain-family cysteine protease of the malaria parasite Plasmodium falciparum holds an important role in hemoglobin degradation pathway. A new series of quinoline carboxamide-based compounds was designed, synthesized and evaluated for antimalarial activity. We integrated molecular hybridization strategy with in-silico drug design to develop FP2 inhibitors. In-vitro results of FP2 inhibition by Qs17, Qs18, Qs20 and Qs21 were found to be in low micromolar range with IC50 4.78, 7.37, 2.14 and 2.64 µM, respectively. Among the 25 synthesized compounds, four compounds showed significant antimalarial activities. These compounds also depicted morphological and food-vacuole abnormalities much better than that of E-64, an established FP2 inhibitor. Overall these aromatic substituted quinoline carboxamides can serve as promising leads for the development of novel antimalarial agents.

Enantioselective Synthesis of γ-Phenyl-γ-amino Vinyl Phosphonates and Sulfones and Their Application to the Synthesis of Novel Highly Potent Antimalarials.

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.

Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity.

Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study.

Quinoline-triazole hybrids inhibit falcipain-2 and arrest the development of Plasmodium falciparum at the trophozoite stage.

Falcipain-2 (FP2) is a papain family cysteine protease and a key member of the hemoglobin degradation pathway, a process that is required at erythrocytic stages of Plasmodium falciparum to obtain amino acids. In this study, we report a set of 10 quinoline-triazole-based compounds (T1-T10) which exhibit a good binding affinity for FP2, inhibit its catalytic activity at micromolar concentrations and thereby arrest the parasite growth. Compounds T4 and T7 inhibited FP2 with IC50 values of 16.16 µM and 25.64 µM respectively. Both the compounds T4 and T7 arrested the development of P. falciparum at the trophozoite stage with an EC50 value 21.89 µM and 49.88 µM. These compounds also showed morphological and food-vacuole abnormalities like E-64, a known inhibitor of FP2. Our results thus identify the quinoline-triazole-based compounds as a probable starting point for the design of FP2 inhibitors and they should be further investigated as potential antimalarial agents.

Biochemical characterization of Plasmodium complement factors binding protein for its role in immune modulation.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite Plasmodium employs a series of intricate mechanisms that enables it to evade different arms of immune system, including the complement system. Here, we show the expression of a multi-domain Plasmodium Complement Control Protein 1, PfCCp1 at asexual blood stages and its binding affinity with C3b as well as C4b proteins of human complement cascade. Using a biochemical assay, we demonstrate that PfCCp1 binds with complement factors and inhibits complement activation. Active immunization of mice with PfCCp1 followed by challenge with Plasmodium berghei resulted in the loss of biphasic growth of parasites and early death in comparison to the control group. The study also showed a role of PfCCp1 in modulating Toll-like receptor (TLR)-mediated signalling and effector responses on antigen-presenting cells. PfCCp1 binds with dendritic cells that down-regulates the expression of signalling molecules and pro-inflammatory cytokines, thereby dampening the TLR2-mediated signalling; hence acting as a potent immuno-modulator. In summary, PfCCp1 appears to be an important component of malaria parasite directed immuno-modulating strategies that promote the adaptive fitness of pathogens in the host.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Protein-protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation that binds to human erythrocytes.

Plasmodium falciparum merozoite surface protein (PfMSP) 1 has been studied extensively as a vaccine candidate antigen. PfMSP-1 undergoes proteolytic processing into four major products, such as p83, p30, p38, and p42, that are associated in the form of non-covalent complex(s) with other MSPs. To delineate MSP1 regions involved in the interaction with other MSPs, here we expressed recombinant proteins (PfMSP-165) encompassing part of p38 and p42 regions and PfMSP-119 PfMSP-165 interacted strongly with PfMSP-3, PfMSP-6, PfMSP-7, and PfMSP-9, whereas PfMSP-119 did not interact with any of these proteins. Since MSP-1 complex binds human erythrocytes, we examined the ability of these proteins to bind human erythrocyte. Among the proteins of MSP-1 complex, PfMSP-6 and PfMSP-9 bound to human erythrocytes. Serological studies showed that PfMSP-165 was frequently recognized by sera from malaria endemic regions, whereas this was not the case for PfMSP-119 In contrast, antibodies against PfMSP-119 showed much higher inhibition of merozoite invasion compared with antibodies against the larger PfMSP-165 fragment. Importantly, anti-PfMSP-119 antibodies recognized both recombinant proteins, PfMSP-119 and PfMSP-165; however, anti-PfMSP-165 antibody failed to recognize the PfMSP-119 protein. Taken together, these results demonstrate that PfMSP-1 sequences upstream of the 19 kDa C-terminal region are involved in molecular interactions with other MSPs, and these sequences may probably serve as a smoke screen to evade antibody response to the membrane-bound C-terminal 19 kDa region.

Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum.

Invasion of human erythrocytes by Plasmodium falciparum merozoites involves multiple interactions between host receptors and their merozoite ligands. Here we report human Cyclophilin B as a receptor for PfRhopH3 during merozoite invasion. Localization and binding studies show that Cyclophilin B is present on the erythrocytes and binds strongly to merozoites. We demonstrate that PfRhopH3 binds to the RBCs and their treatment with Cyclosporin A prevents merozoite invasion. We also show a multi-protein complex involving Cyclophilin B and Basigin, as well as PfRhopH3 and PfRh5 that aids the invasion. Furthermore, we report identification of a de novo peptide CDP3 that binds Cyclophilin B and blocks invasion by up to 80%. Collectively, our data provide evidence of compounded interactions between host receptors and merozoite surface proteins and paves the way for developing peptide and small-molecules that inhibit the protein-protein interactions, individually or in toto, leading to abrogation of the invasion process.

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells.

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (∼0.8 mg/g dry weight) at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments (chloroplast, nucleus, and mitochondria). The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of dihydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.

Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP.

Signal recognition particle (SRP) is a ubiquitous ribonucleoprotein complex that targets proteins to endoplasmic reticulum (ER) in eukaryotes. Here we report that Plasmodium falciparum SRP is composed of six polypeptides; SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72 and a 303nt long SRP RNA. We generated four transgenic parasite lines expressing SRP-GFP chimeric proteins and co-localization studies showed the nucleo-cytoplasmic localization for these proteins. The evaluation of the effect of known SRP and nuclear import/export inhibitors on P. falciparum revealed that ivermectin, an inhibitor of importin α/ß mediated nuclear import inhibited the nuclear import of PfSRP polypeptides at submicromolar concentration, thereby killing the parasites. These findings provide insights into dynamic structure of P. falciparum SRP and also raise the possibility that ivermectin could be used in combination with other antimalarial agents to control the disease.

Assessment of Her-2/neu status using immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology smears: experience from a tertiary care centre in India.

Breast carcinoma shows amplification/overexpression of Her-2/neu in ∼20-30% of cases. The determination of Her-2/neu expression accurately is vital in clinical practice as it has significant predictive value and eligibility for anti Her-2/neu therapy. Amplification and overexpression of Her-2/neu gene is traditionally identified by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) on tissue sections; only a few studies have evaluated feasibility of these techniques on cytological smears. One hundred cases of breast cancer with fine-needle aspiration cytology (FNAC) samples and corresponding surgically resected specimen were selected. Immunocytochemistry (ICC) and FISH for Her-2/neu was done on FNA smears, whereas IHC was performed on corresponding tissue sections. Diagnostic accuracy of ICC was 99% when compared with IHC. Comparison of FISH results with IHC showed 100% concordance. Unlike many centers in West, FNAC is still routinely performed in developing countries like India where vast majority of breast cancer cases present as palpable lumps. The high rates of accuracy of ICC and FISH for Her-2/neu detection can make FNAC a relevant first line of investigation as a cost effective model with a rapid turn-around time, providing complete information necessary for initial management of breast cancer patients.