Shailja Singh

Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes

Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin‐like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium‐dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2‐deficient [PPLP2(−)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(−) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(−) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(−) parasites to the Anopheles vector was reduced. Pore‐forming equinatoxin II rescued both PPLP2(−) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild‐type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(−) loss‐of‐function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm.

Spiro[pyrrolidine-3, 3´-oxindole] as potent anti-breast cancer compounds: Their design, synthesis, biological evaluation and cellular target identification

The spiro[pyrrolidine-3, 3´-oxindole] moiety is present as a core in number of alkaloids with substantial biological activities. Here in we report design and synthesis of a library of compounds bearing spiro[pyrrolidine-3, 3´-oxindole] motifs that demonstrated exceptional inhibitory activity against the proliferation of MCF-7 breast cancer cells. The synthesis involved a one pot Pictet Spengler-Oxidative ring contraction of tryptamine to the desired scaffolds and occurred in 1:1 THF and water with catalytic trifluoroacetic acid and stoichiometric N-bromosuccinimide as an oxidant. Phenotypic profiling indicated that these molecules induce apoptotic cell death in MCF-7 cells. Target deconvolution with most potent compound 5l from the library, using chemical proteomics indicated histone deacetylase 2 (HDAC2) and prohibitin 2 as the potential cellular binding partners. Molecular docking of 5l with HDAC2 provided insights pertinent to putative binding interactions.

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP–EPAC1–AKAP9 direction to the development of additional biotherapeutics for breast cancer.

Neurogenic plasticity of mesenchymal stem cell, an alluring cellular replacement for traumatic brain injury

Traumatic brain injury (TBI) imposes horrendous neurophysiological alterations leading to most devastating forms of neuro-disability. Which includes impaired cognition, distorted locomotors activity and psychosomatic disability in both youths and adults. Emerging evidence from recent studies has identified mesenchymal stem cells (MSCs) as one of the promising category of stem cells having excellent neuroregenerative capability in TBI victims. Some of the clinical and animal studies reported that MSCs transplantation could cure neuronal damage as well as improve cognitive and locomotors behaviors in TBI. However, mechanism behind their broad spectrum neuroregenerative potential in TBI has not been reviewed yet. Therefore, in the present article, we present a comprehensive data on the important attributes of MSCs, such as neurotransdifferentiation, neuroprotection, axonal repair and plasticity, maintenance of blood-brain integrity, reduction of reactive oxygen species (ROS) and immunomodulation. We have reviewed in detail the crucial neurogenic capabilities of MSCs in vivo and provided consolidated knowledge regarding their cellular remodeling in TBI for future therapeutic implications.

Diverse synthesis of natural product inspired fused and spiro-heterocyclic scaffolds via ring distortion and ring construction strategies

Several natural product inspired fused and spiro-heterocyclic scaffolds were prepared by ring distortion and ring construction strategies and evaluated for anti-breast cancer activity. A facile domino Pictet–Spengler lactamization (PSL) afforded nine natural product inspired indolo[2,3-a]quinolizidine and indolo[8,7-b]indolizidine scaffolds which are converted to seven other scaffolds by functional group transformation, ring distortion and ring construction strategies. In vitro screenings of this library of sixteen scaffolds with six distinct architectures against MCF7 cell lines afforded two compounds (10 and 21) with modest activity. Principal component analysis of this library against databases of FDA approved drugs, commercial compounds and FDA approved breast cancer compounds indicated an eclectic mix of structures among the molecules.

Cytotoxic effect of carotenoid phytonutrient lycopene on P. falciparum infected erythrocytes.

Resistance to almost all class of antimalarial drugs has emerged as one of the greatest challenges to malaria control. Strategies to limit the advent and spread of drug-resistant malaria include development of new drugs and its combination with existing drugs. In this work we provide a strong evidence for phytonutrient lycopene, a non-provitamin A carotenoid, to be effective against Plasmodium falciparum growth in vitro. Consistent with the previous findings in mammalian cells, lycopenes prooxidant activity promoted the production of reactive oxygen species (ROS) in P. falciparum. Also a significant loss of mitochondrial functionality and thus, the loss of the membrane potential was observed in lycopene treated schizonts. Taken together, our results indicated that the generation of ROS and loss of mitochondrial membrane potential accounted for lycopenes cytotoxicity against P. falciparum growth in vitro. These insights will help in the design of new treatment strategies to combat malaria.

Novel β-carboline-quinazolinone hybrids disrupt Leishmania donovani redox homeostasis and show promising antileishmanial activity

Graphical abstract Figure. No Caption available. ABSTRACT Visceral Leishmaniasis is a deadly parasitic disease caused by Leishmania donovani. Paucity exists in the discovery of novel chemotherapeutics against Leishmaniasis. In this study, we synthesized a natural product inspired Diversity Oriented Synthesis library of L. donovani Trypanothione reductase (LdTR) inhibitor &bgr;‐carboline‐quinazolinone hybrids, which are different in stereochemical architecture and diverse in the bioactive chemical space. It is noteworthy that chirality affects drug‐to‐protein binding affinity since proteins in any living system are present only in one of the chiral forms. Upon evaluation of the hybrids, one of the chiral forms i.e. Compound 1 showed profound cytotoxic effect in micromolar range as compared to its other chiral form i.e. Compound 2. In‐silico docking studies confirmed high binding efficiency of Compound 1 with the catalytic pocket of LdTR. Treatment of L. donovani parasites with Compound 1 inhibits LdTR activity, induces imbalance in redox homeostasis by enhancing ROS, disrupts the mitochondrial membrane potential, modifies actin polymerization and alters the surface topology and architecture. All these cellular modifications eventually led to apoptosis‐like death of promastigotes. Furthermore, we synthesized the analogues of Compound 1 and found that these compounds show profound antileishmanial activity in the nanomolar range both in promastigotes and intracellular amastigotes. The enhanced inhibitory potential of these compounds was further supported by in‐silico analysis of protein‐ligand interactions which revealed high binding efficiency towards the catalytic pocket of LdTR. Taken together, this study reports the serendipitous discovery of &bgr;‐carboline‐quinazolinone hybrids with enhanced antileishmanial activity along with the in‐depth structure‐activity relationships and mechanism of action of these analogues.

In silico analysis of calcium binding pocket of perforin like protein 1: insights into the regulation of pore formation

Plasmodium falciparum perforin like proteins (PfPLPs) are an important arsenal for the entry and exit of malaria parasites. These proteins bind and oligomerize on the membrane in calcium dependent manner and form an open pore. The calcium dependent pore forming activity of PLPs is usually conferred by their C2 like C-terminal domain. Here, we have tried to elucidate the calcium binding residues in the C-terminal domain of PfPLP1, a member of P. falciparum PLPs, playing a crucial role in calcium dependent egress of blood stage parasites. Through our in silico study, we have found that the C-terminal domain of all PfPLPs is rich in β-pleated sheets and is structurally similar to C2 domain of human perforin. Furthermore, homology search based on 3-D structure of PfPLP1 confirmed that it is structurally homologous to the calcium binding C2 domain of many proteins. On further elucidation of the calcium-binding pocket of the C2 like domain of PfPLP1 showed that it binds to two calcium molecules. The calcium-binding pocket could be a target of novel chemotherapeutics for studying functional role of PfPLPs in parasite biology as well as for limiting blood stage growth of malaria parasite.

In silico study of peptide inhibitors against BACE 1

Peptides are increasingly used as inhibitors of various disease specific targets. Several naturally occurring and synthetically developed peptides are undergoing clinical trials. Our work explores the possibility of reusing the non-expressing DNA sequences to predict potential drug-target specific peptides. Recently, we experimentally demonstrated the artificial synthesis of novel proteins from non-coding regions of Escherichia coli genome. In this study, a library of synthetic peptides (Synpeps) was constructed from 2500 intergenic E. coli sequences and screened against Beta-secretase 1 protein, a known drug target for Alzheimer’s disease (AD). Secondary and tertiary protein structure predictions followed by protein–protein docking studies were performed to identify the most promising enzyme inhibitors. Interacting residues and favorable binding poses of lead peptide inhibitors were studied. Though initial results are encouraging, experimental validation is required in future to develop efficient target specific inhibitors against AD.

Diversity oriented synthesis for novel anti-malarials

Malaria a global pandemic has engulfed nearly 0.63 million people globally. It is high time that a cure for malaria is required to stop its ever increasing menace. Our commentary discusses the advent and contribution of diversity oriented synthesis (DOS) in the drug discovery efforts towards developing cure for malaria. DOS based on chemical genetics focusses on design and synthesis of molecular libraries which covers large tracts of biologically relevant chemical space. Herein we will discuss the applications, advantages, disadvantages and future directions of DOS with respect to malaria.