Cytoprotective autophagy as a pro-survival strategy in ART-resistant malaria parasites.

Despite several initiatives to subside the global malaria burden, the spread of artemisinin-resistant parasites poses a big threat to malaria elimination. Mutations in PfKelch13 are predictive of ART resistance, whose underpinning molecular mechanism remains obscure. Recently, endocytosis and stress response pathways such as the ubiquitin-proteasome machinery have been linked to artemisinin resistance. With Plasmodium, however, ambiguity persists regarding a role in ART resistance for another cellular stress defence mechanism called autophagy. Therefore, we investigated whether, in the absence of ART treatment, basal autophagy is augmented in PfK13-R539T mutant ART-resistant parasites and analyzed whether PfK13-R539T endowed mutant parasites with an ability to utilize autophagy as a pro-survival strategy. We report that in the absence of any ART treatment, PfK13-R539T mutant parasites exhibit increased basal autophagy compared to PfK13-WT parasites and respond aggressively through changes in autophagic flux. A clear cytoprotective role of autophagy in parasite resistance mechanism is evident by the observation that a suppression of PI3-Kinase (PI3K) activity (a master autophagy regulator) rendered difficulty in the survival of PfK13-R539T ART-resistant parasites. In conclusion, we now show that higher PI3P levels reported for mutant PfKelch13 backgrounds led to increased basal autophagy that acts as a pro-survival response to ART treatment. Our results highlight PfPI3K as a druggable target with the potential to re-sensitize ART-resistant parasites and identify autophagy as a pro-survival function that modulates ART-resistant parasite growth.

A C2 domain containing plasma membrane protein of Plasmodium falciparum merozoites mediates calcium-dependent binding and invasion to host erythrocytes.

BACKGROUND: Invasion of red blood cells by Plasmodium falciparum merozoites is governed by multiple receptor-ligand interactions which are critical for bridging the two cells together. The critical function of these ligands for invasion and their direct exposure to the host immune system makes them lucrative vaccine candidates. This necessitates the discovery of new adhesins with less redundancy that mediates the binding of merozoite to the red cell, and furthermore invasion into it. Here we have identified a novel membrane associated antigen (PfC2DMA) that is conserved throughout the Plasmodium species and has a membrane targeting C2 domain at its extreme N-terminal region. METHODS: Recombinant C2dom was expressed heterologously in bacteria and purified to homogeneity. Mice antisera against C2dom was raised and used to check the expression and intraparasitic localization of the protein. RBC and Ca2+ ion binding activity of C2dom was also checked. RESULTS: C2dom exhibited specific binding to Ca2+ ions and not to Mg2+ ions. PfC2DMA localized to the surface of merozoite and recombinant C2dom bound to the surface of human RBCs. RBC receptor modification by treatment with different enzymes showed that binding of C2dom to RBC surface is neuraminidase sensitive. Mice antisera raised against C2dom of Pf C2DMA showed invasion inhibitory effects. CONCLUSION: Our findings suggest that C2dom of PfC2DMA binds to surface of red cell in a Ca2+-dependent manner, advocating a plausible role in invasion and can serve as a potential novel blood stage vaccine candidate.

N-sulfonylpiperidinedispiro-1,2,4,5-tetraoxanes exhibit potent in vitro antiplasmodial activity and in vivo efficacy in mice infected with P. berghei ANKA.

The artemisinin resistance has posed a serious threat against malaria elimination lately. Past few years have seen important development of several peroxide based medicinal compounds and their derivatives such as trioxanes and tetraoxanes. Here, we report a rapid, one-pot method for synthesizing a new series of N-sulfonylpiperidine dispiro-1,2,4,5-tetraoxane analogs with diverse substitution on the tetraoxane ring i.e., various substituted alkyl and aryl sulfonyl chlorides, as well as cyclic, acyclic and aryl substituted ketones. All the synthesized tetraoxanes were characterized by spectroscopic (1H NMR,13C NMR), and spectrometric (High-resolution mass spectrometry) techniques and quantify by High Performance Liquid Chromatography (HPLC) analysis. The structure of compound 19 was confirmed by single crystal XRD. From the overall preliminary in vitro data, analogs 14, 16, 19, 20, 24, 41, and 44 exhibited potential IC50 values in the nanomolar range between 4.7 ± 0.3 to 12.9 ± 1.1 nM against P. falciparum (Pf3D7) strains of human malaria parasite. Furthermore, these selective analogs were evaluated in vivo for their antimalarial potential against P. berghei and results revealed that analogue 24 rapidly kills the infected cell at asexual erythrocytic stage, with activity comparable to positive control chloroquine.

Chorismate synthase mediates cerebral malaria pathogenesis by eliciting salicylic acid-dependent autophagy response in parasite.

Cerebral malaria caused by Plasmodium falciparum is the severest form of the disease resulting in the morbidity of a huge number of people worldwide. Development of effective curatives is essential in order to overcome the fatality of cerebral malaria. Earlier studies have shown the presence of salicylic acid (SA) in malaria parasite P. falciparum, which plays a critical role in the manifestation of cerebral malaria. Further, the application of SA for the treatment of acute symptoms in cerebral malaria increases the activity of iNOS leading to severe inflammation-mediated death, also called as Reye's syndrome. Therefore, modulation of the level of SA might be a novel approach to neutralize the symptoms of cerebral malaria. The probable source of parasite SA is the shikimate pathway, which produces chorismate, a precursor to aromatic amino acids and other secondary metabolites like SA in the parasite. In this work, we performed the immunological, pathological and biochemical studies in mice infected with chorismate synthase knocked-out Plasmodium berghei ANKA, which does not produce SA. Fewer cerebral outcomes were observed as compared to the mice infected with wild-type parasite. The possible mechanism behind this protective effect might be the hindrance of SA-mediated induction of autophagy in the parasite, which helps in its survival in the stressed condition of brain microvasculature during cerebral malaria. The absence of SA leading to reduced parasite load along with the reduced pathological symptoms contributes to less fatality outcome by cerebral malaria.

Amplified Activity of Artesunate Mediated by Iron Oxide Nanoparticles Loaded on a Graphene Oxide Carrier for Cancer Therapeutics.

Development of drugs to tackle the ever-increasing cases of cancer and many other diseases including any pandemic is itself challenging. Repurposing existing drugs is an upcoming drug development strategy established for the reuse of existing licensed drugs to ensure accessible, sustainable, and affordable care against cancer. Herein, we presented a nanochemotherapeutic approach based on PEGylated graphene oxide (GO-PEG) loaded with superparamagnetic iron oxide nanoparticles (NPs) and a sustainable natural origin drug, artesunate (ART) to kill cancerous cells. GO-PEG provided a larger surface area to load the dual cargo, iron oxide NPs (∼40%) and ART (∼13%), at a high loading efficiency and simultaneously affected nanotization and crystallinity of the iron oxide NPs. The morphology and internalization of NPs were determined qualitatively and quantitatively by atomic force microscopy (AFM)-Raman imaging and atomic absorption spectroscopy (AAS) analysis, respectively. Furthermore, the loading and unloading of iron reserves were characterized by high-resolution transmission electron microscopy (TEM) images. The loaded iron oxide NPs underwent a pH-triggered release of iron ions, which is higher in acidic pH than in neutral pH. A ∼sevenfold reduction in the IC50 value of ART upon treatment with the designed nanoconjugate is observed. ART is repositioned as a therapeutic drug against cancer cells, and its efficacy is amplified by the Fenton reaction due to iron oxide NPs, as confirmed by a high oxidative stress generated within the cells. The current work suggests that ART and iron oxide NPs loaded on GO-PEG, a biocompatible carrier, are a promising drug-nanoparticle conjugate for cancer treatment.

Pre-clinical study of iron oxide nanoparticles fortified artesunate for efficient targeting of malarial parasite.

BACKGROUND: Artesunate the most potent antimalarial is widely used for the treatment of multidrug-resistant malaria. The antimalarial cytotoxicity of artesunate has been mainly attributed to its selective, irreversible and iron- radical-mediated damage of parasite biomolecules. In the present research, iron oxide nanoparticle fortified artesunate was tested in P. falciparum and in an experimental malaria mouse model for enhancement in the selectivity and toxicity of artesunate towards parasite. Artesunate was fortified with nontoxic biocompatible surface modified iron oxide nanoparticle which is specially designed and synthesized for the sustained pH-dependent release of Fe2+ within the parasitic food vacuole for enhanced ROS spurt. METHODS: Antimalarial efficacy of Iron oxide nanoparticle fortified artesunate was evaluated in wild type and artemisinin-resistant Plasmodium falciparum (R539T) grown in O + ve human blood and in Plasmodium berghei ANKA infected swiss albino mice. Internalization of nanoparticles, the pH-dependent release of Fe2+, production of reactive oxygen species and parasite biomolecule damage by iron oxide nanoparticle fortified artesunate was studied using various biochemical, biophysical, ultra-structural and fluorescence microscopy. For determining the efficacy of ATA-IONP+ART on resistant parasite ring survival assay was performed. RESULTS: The nanoparticle fortified artesunate was highly efficient in the 1/8th concentration of artesunate IC50 and led to retarded growth of P. falciparum with significant damage to macromolecules mediated via enhanced ROS production. Similarly, preclinical In vivo studies also signified a radical reduction in parasitemia with ~8-10-fold reduced dosage of artesunate when fortified with iron oxide nanoparticles. Importantly, the ATA-IONP combination was efficacious against artemisinin-resistant parasites. INTERPRETATION: Surface coated iron-oxide nanoparticle fortified artesunate can be developed into a potent therapeutic agent towards multidrug-resistant and artemisinin-resistant malaria in humans. FUND: This study is supported by the Centre for Study of Complex Malaria in India funded by the National Institute of Health, USA.

Synthetic Toll-like receptor agonists for the development of powerful malaria vaccines: a patent review.

INTRODUCTION: Currently, there is no efficient vaccine available against clinical malaria. However, continuous efforts have been committed to develop powerful antimalarial vaccine by discovery of novel antigens with in-depth understanding of its nature, immunogenicity, and presentation (delivery adjuvants). Moreover, another important part of vaccine development includes discovery of better immunostimulatory formulation components (immunostimulants). A protective vaccine against malaria requires antigen-specific B and T helper cell responses as well as cytotoxic T lymphocyte (CTL) responses. A long-lasting B and T memory cell production is also required for effective malaria vaccine. Since activation of Toll-like receptors (TLRs) promotes both innate inflammatory responses as well as the induction of adaptive immunity, several initiatives have been mounted during the last few years for the use of TLR agonists as malaria vaccine adjuvants. AREAS COVERED: The review summarizes reports related to the use and development of TLR agonists as malaria vaccine adjuvants and describes various strategies involved for the selection of specific antigens and TLR agonists. EXPERT OPINION: TLR agonists are promising adjuvants for the development of effective malaria vaccine, allowing for both innate inflammatory responses as well as the induction of adaptive immunity.

Benzoxazine derivatives of phytophenols show anti-plasmodial activity via sodium homeostasis disruption.

Development of new class of anti-malarial drugs is an essential requirement for the elimination of malaria. Bioactive components present in medicinal plants and their chemically modified derivatives could be a way forward towards the discovery of effective anti-malarial drugs. Herein, we describe a new class of compounds, 1,3-benzoxazine derivatives of pharmacologically active phytophenols eugenol (compound 3) and isoeugenol (compound 4) synthesised on the principles of green chemistry, as anti-malarials. Compound 4, showed highest anti-malarial activity with no cytotoxicity towards mammalian cells. Compound 4 induced alterations in the intracellular Na+ levels and mitochondrial depolarisation in intraerythrocytic Plasmodium falciparum leading to cell death. Knowing P-type cation ATPase PfATP4 is a regulator for sodium homeostasis, binding of compound 3, compound 4 and eugenol to PfATP4 was analysed by molecular docking studies. Compounds showed binding to the catalytic pocket of PfATP4, however compound 4 showed stronger binding due to the presence of propylene functionality, which corroborates its higher anti-malarial activity. Furthermore, anti-malarial half maximal effective concentration of compound 4 was reduced to 490 nM from 17.54 µM with nanomaterial graphene oxide. Altogether, this study presents anti-plasmodial potential of benzoxazine derivatives of phytophenols and establishes disruption of parasite sodium homeostasis as their mechanism of action.

Dually crosslinked injectable hydrogels of poly(ethylene glycol) and poly[(2-dimethylamino)ethyl methacrylate]-b-poly(N-isopropyl acrylamide) as a wound healing promoter.

Rapid gelation, low heat generation, biocompatibility, biodegradability, avoiding the use of a small molecular weight gelator and high gel fraction are the essential criteria for the successful biomedical application of an injectable hydrogel. We have developed a series of dually crosslinked injectable hydrogels of PEG and poly[2-(dimethylamino)ethyl methacrylate]-b-poly(N-isopropyl acrylamide) through extremely simple chemistry. The sequential nucleophilic substitution reaction between PEG containing reactive termini and the copolymer provided chemically crosslinked hydrogels with a gel fraction as high as 96-99% with a gelation time of 1-4 min under physiological conditions. The gelation occurred with ca. 1 °C rise in temperature per gram of the injectable solution, avoids formation of by-products and can be performed in the temperature range of 20-37 °C. The hydrogels undergo hardening at a physiological temperature as confirmed by rheological experiments. The gelation time, water swelling, mechanical properties and degradability of the hydrogels depend on the PEG to copolymer ratio in the injectable solution. The rheological behaviour of the fully hydrated hydrogels showed desirable mechanical properties for soft tissue regeneration. The hydrogels exhibited blood compatibility and retained the viability of HepG2 cells with time. Platelet adhesion and aggregation followed by fibrinogen adsorption ability makes these hydrogels suitable for wound healing applications.