Shagun Krishna

Pharmacophore-Based Screening and Identification of Novel Human Ligase I Inhibitors with Potential Anticancer Activity

Human DNA ligases are enzymes that are indispensable for DNA replication and repair processes. Among the three human ligases, ligase I is attributed to the ligation of thousands of Okazaki fragments that are formed during lagging strand synthesis during DNA replication. Blocking ligation therefore can lead to the accumulation of thousands of single strands and subsequently double strand breaks in the DNA, which is lethal for the cells. The reports of the high expression level of ligase I protein in several cancer cells (versus the low ligase expression level and the low rate of division of most normal cells in the adult body) support the belief that ligase I inhibitors can target cancer cells specifically with minimum side effects to normal cells. Recent publications showing exciting data for a ligase IV inhibitor exhibiting antitumor activity in mouse models also strengthens the argument for ligases as valid antitumor targets. Keeping this in view, we performed a pharmacophore-based screening for potential ligase inhibitors in the Maybridge small molecule library and procured some of the top-ranking compounds for enzyme-based and cell-based in vitro screening. We report here the identification of novel ligase I inhibitors with potential anticancer activity against a colon cancer cell line.

Human DNA Ligases: A Comprehensive New Look for Cancer Therapy

Living organisms belonging to all three domains of life, viz., eubacteria, archaeabacteria, and eukaryotes encode one or more DNA ligases. DNA ligases are indispensable in various DNA repair and replication processes and a deficiency or an inhibition of their activity can lead to accumulation of DNA damage and strand breaks. DNA damage, specially strand breaks at unsustainable levels can lead to replication block and/or cell death. DNA ligases as potential anticancer targets have been realized only recently. There is enough rationale to suggest that ligases have a tremendous potential for novel therapeutics including anticancer and antibacterial therapy, specially when the world is facing acute problems of drug resistance and chemotherapy failure, with an immediate need for new therapeutic targets. Here, we review the current state of the art in the development of human ligase inhibitors, their structures, molecular mechanisms, physiological effects, and their potential in future cancer therapy. Citing examples, we focus on strategies for improving the activity and specificity of existing and novel inhibitors by using structure‐based rational approaches. In the end, we describe potential new sites on the ligase I protein that can be targeted for the development of novel inhibitors. This is the first comprehensive review to compile all known human ligase inhibitors and to provide a rationale for the further development of ligase inhibitors for cancer therapy.

Virtual screening strategies: Recent advances in the identification and design of anti-cancer agents

Virtual screening (VS) is a well-established technique, which is now routinely employed in computer aided drug designing process. VS can be broadly classified into two categories, i.e., ligand-based and structure-based approach. In recent years, VS has emerged as a time saving and cost effective technique, capable of screening millions of compounds in a user friendly manner. In the area of cancer drug design, VS methods have been widely used and helped in identifying novel molecules as potential anti-cancer agents. Both ligand-based VS (LBVS) structure-based VS (SBVS) methods have been highly useful in the identification of a number of potential anti-cancer agents exhibiting activities in nanomolar range. In tune with the rapid progress in the enhancement of computational power, VS has witnessed significant change in terms of speed and hit rate and in future it is expected that VS will be a preferential alternative to high throughput screening (HTS). This review, discusses recent trends and contribution of VS in the area of anti-cancer drug discovery.

Novel β-carboline–quinazolinone hybrid as an inhibitor of Leishmania donovani trypanothione reductase: Synthesis, molecular docking and bioevaluation

Trypanothione reductase (TR) is a vital enzyme in the trypanothione based redox metabolism of trypanosomatid parasites. It is one of the few chemically validated targets for Leishmania. Herein, we report the synthesis of novel β-carboline–quinazolinone hybrids that are able to inhibit Leishmania donovani TR (LdTR) and subsequently inhibit cell growth. A molecular modeling approach based on docking studies and subsequent binding free energy estimation was performed in the active site of LdTR to understand their possible binding sites. With the enzymatic assay on LdTR with compounds, we were able to identify six hit compounds (8j–8o) that were all found to be the competitive inhibitors of TR with Ki in the range of 0.8–9.2 μM. The whole-cell screening assay highlighted the analogues 8k, 8l and 8n as the most active compounds with IC50 of 4.4, 6.0 and 4.3 μM, respectively, along with an adequate selectivity index (SI) of >91, 36 and 24, respectively.

Synthetic modified pyrrolo[1,4] benzodiazepine molecules demonstrate selective anticancer activity by targeting the human ligase 1 enzyme: An in silico and in vitro mechanistic study

Human DNA ligase1 (hLig1) is the major replicative enzyme in proliferating mammalian cells that join Okazaki fragments of the lagging strand during DNA replication. Interruptions in the process of ligation cause DNA damage to accumulate, resulting in cytotoxicity and cell death. In the present study we demonstrate that pyrrolo[1,4] benzodiazepine (PBD) derivatives exhibit anticancer properties by targeting the nick sealing activity of hLig1 as opposed to the DNA interaction activity known for such compounds. Our in silico and in vitro assays demonstrate the binding of these molecules with amino acid residues present in the DNA binding domain (DBD) of the hLig1 enzyme. Two of these hLig1 inhibitors S010-015 and S010-018 demonstrated selective cytotoxicity against DLD-1 (colon cancer) and HepG2 (hepatic cancer) cells in a dose dependant manner. The molecules also reduced cell viability and colony formation at concentrations of ⩽20μM in DLD-1 and HepG2 cells and induced apoptotic cell death. In yet another significant finding, the molecules reduced the migration of cancer cells in wound healing experiments, indicating their anti-metastatic property. In summary, we report the anticancer activity of PBD derivatives against DLD-1 and HepG2 cells and propose a new molecular target for their activity.

Dithiocarbamate-thiourea hybrids useful as vaginal microbicides also show reverse transcriptase inhibition: design, synthesis, docking and pharmacokinetic studies.

Prophylactic prevention is considered as the most promising strategy to tackle STI/HIV. Twenty-five dithiocarbamate-thiourea hybrids (14-38) were synthesized as woman controlled topical vaginal microbicides to counter Trichomonas vaginalis and sperm along with RT inhibition potential. The four promising compounds (18, 26, 28 and 33) were tested for safety through cytotoxic assay against human cervical cell line (HeLa) and compatibility with vaginal flora, Lactobacillus. Docking study of most promising vaginal microbicide (33) revealed that it docked in a position and orientation similar to known reverse transcriptase inhibitor Nevirapine. The preliminary in vivo pharmacokinetics of compound 33 was performed in NZ-rabbits to evaluate systemic toxicity in comparison to Nonoxynol-9.

Design, synthesis and anticancer activity of dihydropyrimidinone–semicarbazone hybrids as potential human DNA ligase 1 inhibitors

A series of new dihydropyrimidinone–semicarbazone hybrids were successfully synthesised by integrating regioselective multicomponent reaction with the pharmacophore hybridization approach. All the synthesised compounds were evaluated for their hLig1 inhibition potency and most of them were found to be good to moderately active. Out of the tested derivatives, compound 6f showed selective anti-proliferative activity against HepG2 cells in a dose-dependent manner with an IC50 value of 10.07 ± 1.2. It also reduced cell survival at ≤20 μM concentration. Further, analysis of treated HepG2 cell lysates by western blot assay showed increased γ-H2AX levels and upregulation of p53, leading to apoptosis. In silico docking results explain the binding modes of compound 6f to the DNA-binding domain of hLig1 enzyme thereby preventing its nick sealing activity. In addition, the favourable pharmacokinetic properties suggest that this new class of hLig1 inhibitors could be promising leads for further drug development.

Identification of potent inhibitors of DNA methyltransferase 1 (DNMT1) through a pharmacophore-based virtual screening approach

DNA methylation is an epigenetic change that results in the addition of a methyl group at the carbon-5 position of cytosine residues. DNA methyltransferase (DNMT) inhibitors can suppress tumour growth and have significant therapeutic value. However, the established inhibitors are limited in their application due to their substantial cytotoxicity. Additionally, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects. In the present study, we have designed a workflow by integrating various ligand-based and structure-based approaches to discover new agents active against DNMT1. We have derived a pharmacophore model with the help of available DNMT1 inhibitors. Utilising this model, we performed the virtual screening of Maybridge chemical library and the identified hits were then subsequently filtered based on the Naïve Bayesian classification model. The molecules that have returned from this classification model were subjected to ensemble based docking. We have selected 10 molecules for the biological assay by inspecting the interactions portrayed by these molecules. Three out of the ten tested compounds have shown DNMT1 inhibitory activity. These compounds were also found to demonstrate potential inhibition of cellular proliferation in human breast cancer MDA-MB-231 cells. In the present study, we have utilized a multi-step virtual screening protocol to identify inhibitors of DNMT1, which offers a starting point to develop more potent DNMT1 inhibitors as anti-cancer agents.

Suppression in PHLPP2 induction by morin promotes Nrf2-regulated cellular defenses against oxidative injury to primary rat hepatocytes

Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3β/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas

BackgroundLeptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins.ResultsDuring change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes.ConclusionOur study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.