Richa Verma

Glycyrrhetinic acid and its analogs: a new class of antifilarial agents.

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5 μM; IC50: 1.20 μM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a-7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50 μM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC50 values were found to be 8.8 and 2.2 μM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100mg/kg, s.c. ×5 days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A-D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold. In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines.

Inhibition of lytic activity of Escherichia coli toxin hemolysin E against human red blood cells by a leucine zipper peptide and understanding the underlying mechanism.

To investigate as to whether a peptide derived from hemolysin E (HlyE) can inhibit the cytotoxic activity of this protein or not, several peptides were examined for their efficacy to inhibit the lytic activity of the protein against human red blood cells (hRBCs). It was found that a wild-type peptide, H-205, derived from an amphipathic leucine zipper motif, located in the amino acid region 205-234, inhibited the lytic activity of hemolysin E against hRBCs. To understand the basis of this inhibition, several functional and structural studies were performed. Western blotting analysis indicated that the preincubation of HlyE with H-205 did not inhibit its binding to hRBC. The results indicated that H-205 but not its mutant inhibited the hemolysin E-induced depolarization of hRBCs. Flow cytometric studies with annexin V-FITC staining of hRBCs after incubation with either protein or protein/peptide complex suggested that H-205 prevented the hemolysin E-induced damage of the membrane organization of hRBCs. Tryptophan fluorescence and circular dichroism studies showed that H-205 induced a conformational change in HlyE, which was accompanied by the enhancement of an appreciable helical structure. Fluorescence studies with rhodamine-labeled peptides showed that H-205 reversibly self-assembled in aqueous environment, which raised a possibility that the H-205 peptide could interact with its counterpart in the protein and thus disturb the proper conformation of HlyE, resulting in the inhibition of its cytotoxic activity. The peptides derived from the homologous segments of other members of this toxin family may also act as inhibitors of the corresponding toxin.

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity.

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63μg/ml, IC50: 6.00μg/ml) than macrofilaricidal (LC100: >250; IC50: 88μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250-500μg/ml, IC50: 44.16μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100-200mg/kg; fractions: 100mg/kg, i.p.×5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38-40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63μg/ml, IC50: 6.57-10.31μg/ml) and microfilaricidal (LC100: 31.25-62.5μg/ml, IC50: 11.05-22.10μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).

In Vitro, In Silico and In Vivo Studies of Ursolic Acid as an Anti-Filarial Agent

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM) and female adult worms (LC100: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuters ‘Metadrug’ tool screening predictions.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K+ channel with six transmembrane (S1–S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218–239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: plausible implications in the toxic activity of the protein.

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89th position was replaced by aspartic acid and in the other both glycine and valine at the 88th and 89th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.

Cross reactive molecules of human lymphatic filaria Brugia malayi inhibit Leishmania donovani infection in hamsters

Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.

Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human

Mutations in various voltage gated cardiac ion channels are the cause of different forms of long QT syndrome (LQTS), which is an inherited arrhythmic disorder marked as a prolonged QT interval on electrocardiogram. Of these LQTS1 is associated with mutations in the gene encoding KCNQ1 (KvLQT1) channel. One responsible mutation, G269S, in the S5 segment of KvLQT1, that affects the proper expression and function of channel protein leads to LQTS1. Our objective was to study how G269S mutation interferes with the structure and function of a synthetic S5 segment of KvLQT1 channel. One wild type 22-residue peptide and another mutant peptide of the same length with G269S mutation, derived from the S5 segment were synthesized and labeled with fluorescent probes. The mutant peptide exhibited lower affinity towards phospholipid vesicles as compared to the wild type peptide and showed impaired assembly and localization onto the lipid vesicles as evidenced by membrane-binding, energy transfer and proteolytic cleavage experiments. Loss in the helical content of S5 mutant peptide in membrane-mimetic environments was observed. Furthermore, it was observed that G269S mutation significantly inhibited the ability of S5 peptide to permeabilize the lipid vesicles. The present studies show the basis of change in function of the selected S5 segment as a result of G269S mutation which is associated with LQT1 syndrome. We speculate that the structural and functional changes related to the glycine to serine amino acid substitution in the S5 segment may also influence the activity of the whole KvLQT1 channel.

Phospholipid membrane-interaction of a peptide from S4 segment of KvAP K(+) channel and the influence of the positive charges and an identified heptad repeat in its interaction with a S3 peptide.

In order to examine the ability of S3 and S4 segments of a Kv channel to interact with each other, two wild type short peptides derived from the S3 and S4 segments of KvAP channel were synthesized. Additionally, to evaluate the role of positive charges and an identified heptad repeat in the S4 segment, two S4 mutants of the same size as the S4 peptide, one with substitution of two leucine residues in the heptad repeat sequence by two alanine residues and in the other two arginine residues replaced by two glutamines residues were synthesized. Our results show that only the wild type S4 peptide, but not its mutants, self-assembled and permeabilized negatively charged phospholipid vesicles. The S3 peptide showed lesser affinity toward the same kind of lipid vesicles and localized onto its surface. However, the S3 peptide interacted only with S4 wild type peptide, but not with S4 mutants, and altered its localization onto the phospholipid membrane with increased resistance against the proteolytic enzyme, proteinase-k, in the presence of the S4 peptide. The results demonstrate that the selected, synthetic S3 and S4 segments possess the required amino acid sequences to interact with each other and show that the positive charges and the identified heptad repeat in S4 contribute to its assembly and interaction with S3 segment.