Virander S. Chauhan

Artemisinin, an Endoperoxide Antimalarial, Disrupts the Hemoglobin Catabolism and Heme Detoxification Systems in Malarial Parasite

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well asPlasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.

Novel dipeptide nanoparticles for effective curcumin delivery.

Background: Curcumin, the principal curcuminoid of the popular Indian spice turmeric, has a wide spectrum of pharmaceutical properties such as antitumor, antioxidant, antiamyloid, and anti-inflammatory activity. However, poor aqueous solubility and low bioavailability of curcumin is a major challenge in its development as a useful drug. To enhance the aqueous solubility and bioavailability of curcumin, attempts have been made to encapsulate it in liposomes, polymeric nanoparticles (NPs), lipid-based NPs, biodegradable microspheres, cyclodextrin, and hydrogels. Methods: In this work, we attempted to entrap curcumin in novel self-assembled dipeptide NPs containing a nonprotein amino acid, α, β-dehydrophenylalanine, and investigated the biological activity of dipeptide-curcumin NPs in cancer models both in vitro and in vivo. Results: Of the several dehydrodipeptides tested, methionine-dehydrophenylalanine was the most suitable one for loading and release of curcumin. Loading of curcumin in the dipeptide NPs increased its solubility, improved cellular availability, enhanced its toxicity towards different cancerous cell lines, and enhanced curcumin’s efficacy towards inhibiting tumor growth in Balb/c mice bearing a B6F10 melanoma tumor. Conclusion: These novel, highly biocompatible, and easy to construct dipeptide NPs with a capacity to load and release curcumin in a sustained manner significantly improved curcumin’s cellular uptake without altering its anticancer or other therapeutic properties. Curcumin-dipeptide NPs also showed improved in vitro and in vivo chemotherapeutic efficacy compared to curcumin alone. Such dipeptide-NPs may also improve the delivery of other potent hydrophobic drug molecules that show poor cellular uptake, bioavailability, and efficacy.

Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum

Summary Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double‐stranded RNA of cysteine protease genes (falcipain‐1 and ‐2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double‐stranded (ds)RNAs are reminiscent of the effects observed upon administering E‐64, a cysteine protease inhibitor. The parasites treated with falcipains dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs ≈ 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence‐specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.

Stimuli responsive self-assembled hydrogel of a low molecular weight free dipeptide with potential for tunable drug delivery.

Bottom-up fabrication by molecular self-assembly is now widely recognized as a potent method for generating interesting and functional nano- and mesoscale structures. Hydrogels from biocompatible molecules are an interesting class of mesoscale assemblies with potential biomedical applications. The self-assembly of a proteolysis resistant aromatic dipeptide containing a conformational constraining residue (DeltaPhe) into a stable hydrogel has been studied in this work. The reported dipeptide has free -N and -C termini. The hydrogel was self-supportive, was fractaline in nature, and possessed high mechanical strength. It was responsive to environmental conditions like pH, temperature, and ionic strength. The gel matrix could encapsulate and release bioactive molecules in a sustained manner. The described hydrogel showed no observable cytotoxicity to the HeLa and L929 cell lines in culture.

Protective hinge in insulin opens to enable its receptor engagement

Significance Insulin provides a model for analysis of protein structure and evolution. Here we describe in detail a conformational switch that enables otherwise hidden nonpolar surfaces in the hormone to engage its receptor. Whereas the classical closed conformation of insulin enables its stable storage in pancreatic β cells, its active conformation is open and susceptible to nonnative aggregation. Our findings illuminate biophysical constraints underlying the evolution of an essential signaling system and provide a structural foundation for design of therapeutic insulin analogs. Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated “microreceptors” that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of PheB24 to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptors L1–β2 sheet. Opening of this hinge enables conserved nonpolar side chains (IleA2, ValA3, ValB12, PheB24, and PheB25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.

Use of urine volatile organic compounds to discriminate tuberculosis patients from healthy subjects.

Development of noninvasive methods for tuberculosis (TB) diagnosis, with the potential to be administered in field situations, remains as an unmet challenge. A wide array of molecules are present in urine and reflect the pathophysiological condition of a subject. With infection, an alteration in the molecular constituents is anticipated, characterization of which may form a basis for TB diagnosis. In the present study volatile organic compounds (VOCs) in human urine derived from TB patients and healthy controls were identified and quantified using headspace gas chromatography/mass spectrometry (GC/MS). We found significant (p < 0.05) increase in the abundance of o-xylene (6.37) and isopropyl acetate (2.07) and decreased level of 3-pentanol (0.59), dimethylstyrene (0.37), and cymol (0.42) in TB patients compared to controls. These markers could discriminate TB from healthy controls and related diseases like lung cancer and chronic obstructive pulmonary disorder. This study suggests a possibility of using urinary VOCs for the diagnosis of human TB.

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion

Significance Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is a leading blood-stage malaria vaccine candidate that elicits potent strain-transcending invasion inhibitory antibodies. However, it lacks both transmembrane domains and a GPI-anchor and is thus anchored to the merozoite surface through an unknown mechanism. We have demonstrated that PfRH5 and its known partner, PfRH5-interacting protein (PfRipr), associates with a conserved GPI-anchored protein, Cysteine-rich protective antigen (CyRPA), to form a complex on the merozoite surface. CyRPA was shown to be GPI-linked, refractory to knockout, and like PfRH5, elicited potent strain-transcending invasion inhibitory antibodies. This discovery elucidates the formation of a previously unidentified PfRH5/PfRipr/CyRPA protein complex on the merozoite surface, which facilitates the PfRH5–Basigin interaction and offers another highly conserved, potent target (CyRPA) for novel antimalarial strategies that could abrogate formation of this crucial complex. Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5–Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.

Identification and Characterization of a Novel Plasmodium falciparum Merozoite Apical Protein Involved in Erythrocyte Binding and Invasion

Proteins that coat Plasmodium falciparum merozoite surface and those secreted from its apical secretory organelles are considered promising candidates for the vaccine against malaria. In the present study, we have identified an asparagine rich parasite protein (PfAARP; Gene ID PFD1105w), that harbors a predicted signal sequence, a C-terminal transmembrane region and whose transcription and translation patterns are similar to some well characterized merozoite surface/apical proteins. PfAARP was localized to the apical end of the merozoites by GFP-targeting approach using an inducible, schizont-stage expression system, by immunofluorescence assays using anti-PfAARP antibodies. Immuno-electron microsopic studies showed that PfAARP is localized in the apical ends of the rhoptries in the merozoites. RBC binding assays with PfAARP expressed on COS cells surface showed that it binds to RBCs through its N-terminal region with a receptor on the RBC surface that is sensitive to trypsin and neuraminidase treatments. Sequencing of PfAARP from different P. falciparum strains as well as field isolates showed that the N-terminal region is highly conserved. Recombinant protein corresponding to the N-terminal region of PfAARP (PfAARP-N) was produced in its functional form in E. coli. PfAARP-N showed reactivity with immune sera from individuals residing in P. falciparum endemic area. The anti-PfAARP-N rabbit antibodies significantly inhibited parasite invasion in vitro. Our data on localization, functional assays and invasion inhibition, suggest a role of PfAARP in erythrocyte binding and invasion by the merozoite.

Nanovesicles based on self-assembly of conformationally constrained aromatic residue containing amphiphilic dipeptides.

Peptide-based vesicular structures have been the focus of research in the past decade for their potential application as drug delivery agents. We here report the self-assembly of amphiphilic dipeptides containing conformation-constraining alpha,beta-dehydrophenylalanine into nanovesicles. The vesicles can encapsulate small drug molecules such as riboflavin and vitamin B(12), bioactive peptides, and small protein molecules. The nanovesicles are resistant to treatment of a nonspecific protease, proteinase K, and are stable at low concentrations of monovalent and divalent cations. The vesicles are effectively taken up by actively growing cells in culture and show no observable cytopathic effects. These peptide-based nanostructures can be considered as models for further development as delivery agents for different biomolecules.

Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC(80) values ranging from 15.62 μM to 250 μM. Synergy experiments showed that MIC(80) of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.