Mohammad Shameem

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.

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.

Glutamatergic and GABAergic metabolism in mouse brain under chronic nicotine exposure: implications for addiction.

Background and Purpose The effects of nicotine on cerebral metabolism and its influence on smoking behavior is poorly understood. An understanding of the chronic effects of nicotine on excitatory and inhibitory metabolic demand, and corresponding neurotransmission may provide clues for designing strategies for the optimal smoking cessation intervention. The objective of the current study was to investigate neuronal and astroglial metabolism in mice exposed to nicotine (0.5 and 2.0 mg/kg, sc) three times in a day for 4 weeks. Experimental Approach/Principal Findings Metabolic measurements were carried out by co-infusing [U-13C6]glucose and [2-13C]acetate, and monitoring 13C labeling of amino acids in brain tissue extract using 1H-[13C] and 13C-[1H]-NMR spectroscopy. Concentration of 13C-labeled glutamate-C4 was increased significantly from glucose and acetate with chronic nicotine treatment indicating an increase in glucose oxidation by glutamatergic neurons in all brain regions and glutamate-glutamine neurotransmitter cycle in cortical and subcortical regions. However, chronic nicotine treatment led to increased labeling of GABA-C2 from glucose only in the cortical region. Further, increased labeling of glutamine-C4 from [2-13C]acetate is suggestive of increased astroglial activity in subcortical and cerebellum regions of brain with chronic nicotine treatment. Conclusions and Significance Chronic nicotine exposure enhanced excitatory activity in the majority of brain regions while inhibitory and astroglial functions were enhanced only in selected brain regions.

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.

Synthesis and characterization of iron-rich FexPt1−x ferrofluid for magnetic resonance imaging

Iron-rich FexPt1?x ultrafine nanodots were prepared by a simple and versatile polyol process using a combinatorial strategy of introducing a strong reducing agent and decreasing the synthesis temperature. The native hydrophobic nanodots were converted into a wettable dispersion by ligand exchange-mediated phase transformation using tetramethyl ammonium hydroxide. The microstructural study confirmed the formation of Fe-rich FePt nanodots having an average particle size of ~3.5?nm with a narrow size distribution. An MTT (methylthiazolyldiphenyl-tetrazolium bromide [3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) study on mammalian leukocyte cultures confirmed the high degree of biocompatibility for the ferrofluid. The ferrofluid, when studied for its concentration-dependent transverse relaxation time and contrast properties, was found to exhibit promising properties as a magnetic resonance imaging T2 contrast agent.

Identification and characterization of novel ligase I inhibitors

The terminal step of ligation of single and/or double‐strand breaks during physiological processes such as DNA replication, repair and recombination requires participation of DNA ligases in all mammals. DNA Ligase I has been well characterised to play vital roles during these processes. Considering the indispensable role of DNA Ligase I, a therapeutic strategy to impede proliferation of cancer cells is by using specific small molecule inhibitors against it. In the present study, we have designed and chemically synthesised putative DNA Ligase I inhibitors. Based on various biochemical and biophysical screening approaches, we identify two prospective DNA Ligase I inhibitors, SCR17 and SCR21. Both the inhibitors blocked ligation of nicks on DNA in a concentration‐dependent manner, when catalysed by cell‐free extracts or purified Ligase I. Docking studies in conjunction with biolayer interferometry and gel shift assays revealed that both SCR17 and SCR21 can bind to Ligase I, particularly to the DNA Binding Domain of Ligase I with KD values in nanomolar range. The inhibitors did not show significant affinity towards DNA Ligase III and DNA Ligase IV. Further, addition of Ligase I could restore the joining, when the inhibitors were treated with testicular cell‐free extracts. Ex vivo studies using multiple assays showed that even though cell death was limited in the presence of inhibitors in cancer cells, their proliferation was compromised. Hence, we identify two promising DNA Ligase I inhibitors, which can be used in biochemical and cellular assays, and could be further modified and optimised to target cancer cells.

Label-free dopamine imaging in live rat brain slices.

Dopaminergic neurotransmission has been investigated extensively, yet direct optical probing of dopamine has not been possible in live cells. Here we image intracellular dopamine with sub-micrometer three-dimensional resolution by harnessing its intrinsic mid-ultraviolet (UV) autofluorescence. Two-photon excitation with visible light (540 nm) in conjunction with a non-epifluorescent detection scheme is used to circumvent the UV toxicity and the UV transmission problems. The method is established by imaging dopamine in a dopaminergic cell line and in control cells (glia), and is validated by mass spectrometry. We further show that individual dopamine vesicles/vesicular clusters can be imaged in cultured rat brain slices, thereby providing a direct visualization of the intracellular events preceding dopamine release induced by depolarization or amphetamine exposure. Our technique opens up a previously inaccessible mid-ultraviolet spectral regime (excitation ~270 nm, emission < 320 nm) for label-free imaging of native molecules in live tissue.

A Novel Benzocoumarin-Stilbene Hybrid as a DNA ligase i inhibitor with in vitro and in vivo anti-tumor activity in breast cancer models

Existing cancer therapies are often associated with drug resistance and toxicity, which results in poor prognosis and recurrence of cancer. This necessitates the identification and development of novel therapeutics against existing as well as novel cellular targets. In this study, a novel class of Benzocoumarin-Stilbene hybrid molecules were synthesized and evaluated for their antiproliferative activity against various cancer cell lines followed by in vivo antitumor activity in a mouse model of cancer. The most promising molecule among the series, i.e. compound (E)-4-(3,5-dimethoxystyryl)-2H-benzo[h]chromen-2-one (19) showed maximum antiproliferative activity in breast cancer cell lines (MDA-MB-231 and 4T1) and decreased the tumor size in the in-vivo 4T1 cell-induced orthotopic syngeneic mouse breast cancer model. The mechanistic studies of compound 19 by various biochemical, cell biology and biophysical approaches suggest that the compound binds to and inhibits the human DNA ligase I enzyme activity that might be the cause for significant reduction in tumor growth and may constitute a promising next-generation therapy against breast cancers.

Identification of a novel human DNA ligase I inhibitor that promotes cellular apoptosis in DLD-1 cells: an in silico and in vitro mechanistic study

The processes of DNA replication and repair are accomplished by the concerted action of several proteins. Among them human DNA ligases play an important role during the last step of almost all DNA replication and repair processes, where they seal the nicks between DNA strands. In humans, three kinds of DNA ligases (human DNA ligase I, III, IV) are found. DNA ligase I (hLigI) is involved in both DNA replication as well as in DNA repair pathways and is reported to be over-expressed in rapidly dividing cells, including cancer cells. For this reason, in this study we have targeted hLigI for studying its response as a novel anticancer target. We have screened for ligase I inhibitors from our in-house small molecule library by a previously validated pharmacophore based virtual screening method and found a novel hLigI inhibitor. This compound (S-097/98) demonstrated antiproliferative activities specifically in DLD-1 (colon), MDA-MB-231 (triple negative breast) and HepG2 (liver) cancer cell lines at low micromolar concentrations of 6–7 μM. Mechanistic studies show that the compound can directly interacts with the hLigI protein and inhibits ligation of both the purified protein in vitro, as well as in cell lysate of DLD-1 cells treated with the inhibitor. The compound also arrests cell cycle progression at the G2/M phase and increases the nuclear size of DLD-1 cancer cells, thereby demonstrating its antiproliferative activity. Finally, the compound promotes cellular apoptosis in DLD-1 cells.

Altered neurotransmitter levels in different regions of rat brain following acute stress

Lakshmi Subramanian 1, Anindita Sarkar 1, Ashwin S. Shetty 1, Bhavana Muralidharan 1, Hari Padmanabhan 1, Michael Piper 2, Edwin S. Monuki 3, Ingolf Bach 4, Richard M. Gronostajski 5,6, Linda J. Richards 2, Shubha Tole 2 1 Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India 2 Queensland Brain Institute and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia 3 Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Irvine, CA 92697, United States 4 Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States 5 Department of Biochemistry, State University of New York, Buffalo, NY 14203, United States 6 Developmental Genomics Group, New York State Centre of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203, United States The sequential production of neurons and astrocytes from neuroepithelial precursors is a fundamental feature of central nervous system development. We report that LIM-homeodomain (LIM-HD) transcription factor Lhx2 regulates this transition in the developing hippocampus. Disrupting Lhx2 function in the embryonic hippocampus by in utero electroporation and in organotypic slice culture caused the premature production of astrocytes at stages when neurons are normally generated. Lhx2 function is therefore necessary to suppress astrogliogenesis during the neurogenic period. Furthermore, Lhx2 overexpression was sufficient to suppress astrogliogenesis and prolong the neurogenic period. We provide evidence that Lhx2 overexpression can counteract the instructive astrogliogenic effect of Notch activation. Lhx2 overexpression was also able to override and suppress the activation of the GFAP promoter by Nfia, a Notch-regulated transcription factor that is required for gliogenesis. Thus, Lhx2 appears to act as a “brake” on Notch/Nfia-mediated astrogliogenesis. This critical role for Lhx2 is spatially restricted to the hippocampus, because loss of Lhx2 function in the neocortex did not result in premature astrogliogenesis at the expense of neurogenesis. Our results therefore place Lhx2 as a central regulator of the neuron-glia cell fate decision in the hippocampus and reveal a striking regional specificity of this fundamental function within the dorsal telencephalon.