Manoj Kathuria

Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by orally active clerodane diterpene

ABSTRACT This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes that was previously demonstrated to be safe and orally active against visceral leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and the generation of reactive oxygen species, triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in the release of cytochrome c into the cytosol, impairing ATP production. Oxidative stress caused the depletion of reduced glutathione, while pretreatment with antioxidant N-acetyl cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore the mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment, suggesting that the generation of oxidative stress is a downstream event relative to the other events. Caspase-3/-7-like protease activity and genomic DNA fragmentation were observed. Electron microscopy studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of the nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations, and the displacement of organelles. Moreover, an increased number of lipid droplets was detected after K-09 treatment, which is suggestive of altered lipid metabolism. Our results indicate that K-09 induces mitochondrial dysfunction and oxidative stress-mediated apoptotic cell death in L. donovani promastigotes, sharing many features with metazoan apoptosis. These mechanistic insights provide a basis for further investigation toward the development of K-09 as a potential drug candidate for VL.

New Fluoranthene FLUN-550 as a Fluorescent Probe for Selective Staining and Quantification of Intracellular Lipid Droplets

A new class of live cell permeant, nontoxic fluoranthene-based fluorescent probe (FLUN-550) having a high Stokes shift in aqueous medium has been discovered. It showed selective staining of lipid droplets (LDs, dynamic cytoplasmic organelles) at a low concentration without background noise in in vitro live cell imaging of 3T3-L1 preadipocytes, J774 macrophages, MCF7 breast cancer cells, and single-celled, parasitic protozoa Leishmania donovani promastigotes and in vivo nonparasitic soil nematode C. elegans.

Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study.

Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis.

Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity and In Vitro and In Vivo Antiendotoxin Activity

ABSTRACT Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat.

An unprecedented alteration in mode of action of IsCT resulting its translocation into bacterial cytoplasm and inhibition of macromolecular syntheses.

IsCT, a 13-residue, non-cell-selective antimicrobial peptide is comprised of mostly hydrophobic residues and lesser cationic residues. Assuming that placement of an additional positive charge in the non-polar face of IsCT could reduce its hydrophobic interaction, resulting in its reduction of cytotoxicity, an analog, I9K-IsCT was designed. Two more analogs, namely, E7K-IsCT and E7K,I9K-IsCT, were designed to investigate the impact of positive charges in the polar face as well as polar and non-polar faces at a time. These amino acid substitutions resulted in a significant enhancement of therapeutic potential of IsCT. IsCT and E7K-IsCT seem to target bacterial membrane for their anti-bacterial activity. However, I9K-IsCT and E7K,I9K-IsCT inhibited nucleic acid and protein syntheses in tested E. coli without perturbing its membrane. This was further supported by the observation that NBD-IsCT localized onto bacterial membrane while NBD-labeled I9K-IsCT and E7K,I9K-IsCT translocated into bacterial cytoplasm. Interestingly, IsCT and E7K-IsCT were significantly helical while I9K-IsCT and E7K,I9K-IsCT were mostly unstructured with no helix content in presence of mammalian and bacterial membrane-mimetic lipid vesicles. Altogether, the results identify two novel cell-selective analogs of IsCT with new prototype amino acid sequences that can translocate into bacterial cytoplasm without any helical structure and inhibit macromolecular syntheses.

Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

Autophagy, a regulated nutrient recycling program can affect both cell survival and cell death. Here, we show that Ormeloxifene (ORM), a selective estrogen receptor modulator approved for oral contraceptive use induces autophagic flux in ovarian cancer cells, which is activated by an ER stress response upstream of autophagy. The ER stress response is characterized by activation of IRE1α, PERK and ATF6 and is under regulation of JNK. Pharmacological inhibition of either autophagy or ER stress increased cell survival, as did silencing of autophagy proteins LC3 and Beclin 1, implying that ORM-induced autophagy is pro-death in nature. Ultrastructural observations of treated cells confirmed stages of autophagic maturation. Caspase-dependent apoptosis succeeded these events and was characterized by generation of reactive oxygen species and disruption of mitochondrial membrane potential. A concomitant inhibition of the Akt/mTOR axis was also observed with possible regulation of Akt by ORM. ORM inhibited tumor growth in ovarian xenograft model and displayed autophagic activity. In summary, in vitro and in vivo results reveal that ORM induces autophagy-associated cell death to attenuate proliferation of ovarian cancer cells. Our results demonstrate that using ORM in combination with ER stress and autophagy modulators could offer better therapeutic outcome in ovarian cancer.

Identification of GXXXXG motif in Chrysophsin-1 and its implication in the design of analogs with cell-selective antimicrobial and anti-endotoxin activities

Marine fish antimicrobial peptide, chrysophsin-1 possesses versatile biological activities but its non-selective nature restricts its therapeutic possibilities. Often small alterations in structural motifs result in significant changes in the properties of concerned proteins/peptides. We have identified GXXXXG motif in chrysophsin-1. Glycine residue(s) of this motif in Chrysophsin-1 was/were replaced with alanine, valine and proline residue(s). Of these, proline-substituted Chrysophsin-1 analogs exhibited significantly reduced cytotoxicity towards mammalian cells. Further, these analogs showed broad-spectrum activity against Gram-positive, Gram-negative bacteria, Methicillin-resistant Staphylococcus aureus strains and fungi and also retained antibacterial activity in presence of physiological salts, serum and at elevated temperatures indicative of their therapeutic potential. These Chrysophsin-1 analogs also inhibited lipopolysaccharide (LPS) induced pro-inflammatory responses in THP-1 cells and in murine primary macrophages. One of these single proline-substituted Chrysophsin-1 analogs inhibited LPS-stimulated pro-inflammatory cytokine production in BALB/c mice and elicited appreciable survival of mice administered with a lethal dose of LPS in a model of severe sepsis. The data for the first time showed the implication of GXXXXG motifs in functional and biological properties of an antimicrobial peptide and could be useful to design novel anti-microbial and anti-endotoxin peptides by employing this motif.

Ammonium trichloro [1,2-ethanediolato- O , O ′]-tellurate cures experimental visceral leishmaniasis by redox modulation of Leishmania donovani trypanothione reductase and inhibiting host integrin linked PI3K/Akt pathway

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O′]-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4β7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101’s affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

Imaging and Quantitative Detection of Lipid Droplets by Yellow Fluorescent Probes in Liver Sections of Plasmodium Infected Mice and Third Stage Human Cervical Cancer Tissues

The diagnosis and prognosis of the disease associated with lipid irregularity are areas of extreme significance. In this direction, fluoranthene based yellow fluorescent probes (FLUN-550, FLUN-552, FLUN-547) were designed and synthesized by conjugating the ethanolamine headgroup of the phospholipid phosphatidyl-ethanolamine present in biological membranes. Owing to unique photophysical properties and aqueous compatibility, these probes were successfully employed for staining lipid droplets (LDs) in preadipocytes and Leishmania donovani promastigotes. Furthermore, using the fluorescent probes FLUN-550 and FLUN-552 we successfully imaged and quantitatively detected the excess accumulation of lipids in a liver section of Plasmodium yoelii MDR infected mice (3- to 4-fold) and the tissue sections of third stage human cervical cancer patients (1.5- to 2-fold) compared to normal tissues. To the best of our knowledge, this is the first report of yellow fluorescent probes for imaging and quantitative detection of LDs in human cervical cancer tissues. These new yellow fluorescent lipid probes (FLUN-550 and FLUN-552) showed great potential for diagnosis of cervical cancer patients.

New visible light excitable donor–acceptor 7-hydroxy-coumarins as blue fluorescent probes for selective staining of vacuoles in yeasts and L. donovani

In order to address the existing limitations of the commercially available fluorescent probe CMAC (7-amino-4-chloromethylcoumarin), a new series of highly fluorescent donor-acceptor 7-hydroxy-coumarin derivatives was prepared and these derivatives were used as vacuole specific fluorescent probes for live cell imaging of unicellular parasitic protozoa L. donovani promastigotes and yeast cells S. pombe and S. cerevisiae. The synthesized 7-hydroxy-coumarins exhibited interesting photophysical properties and have advantages such as excitation in the visible region, good water solubility, photo-stability, good quantum yield and low cytotoxicity.