A novel benzimidazole derivative binds to the DNA minor groove and induces apoptosis in leukemic cells

DNA minor groove binders are an important class of chemotherapeutic agents. These small molecule inhibitors interfere with various cellular processes like DNA replication and transcription. Several benzimidazole derivatives showed affinity towards the DNA minor groove. In this study we show the synthesis and biological studies of a novel benzimidazole derivative (MH1), that inhibits topoisomerase II activity and in vitro transcription. UV-visible and fluorescence spectroscopic methods in conjunction with Hoechst displacement assay demonstrate that MH1 binds to DNA at the minor groove. Cytotoxic studies showed that leukemic cells are more sensitive to MH1 compared to cancer cells of epithelial origin. Further, we find that MH1 treatment leads to cell cycle arrest at G2/M, at early time points in Molt4 cells. Finally multiple cellular assays demonstrate that MH1 treatment leads to reduction in MMP, induction of apoptosis by activating CASPASE 9 and CASPASE 3. Thus our study shows MH1, a novel DNA minor groove binder, induces cytotoxicity efficiently in leukemic cells by activating the intrinsic pathway of apoptosis.

Phytomedicinal importance of Saraca asoca (Ashoka): an exciting past, an emerging present and a promising future

Since the dawn of civilization, natural resources have remained the mainstay of various remedial approaches of humans vis-a-vis a large number of illnesses. Saraca asoca (Roxb.) de Wilde (Saraca indica L.) belonging to the family Caesalpiniaceae has been regarded as a universal panacea in old Indian Ayurvedic texts and has especially been used to manage gynaecological complications and infections besides treating haemmorhagic dysentery, uterine pain, bacterial infections, skin problems, tumours, worm infestations, cardiac and circulatory problems. Almost all parts of the plant are considered pharmacologically valuable. Extensive folkloric practices and ethnobotanical applications of this plant have even lead to the availability of several commercial S. asoca formulations recommended for different indications though adulteration of these remains a pressing concern. Though a wealth of knowledge on this plant is available in both the classical and modern literature, extensive research on its phytomedicinal worth using state-of-the-art tools and methodologies is lacking. Recent reports on bioprospecting of S. asoca endophytic fungi for industrial bioproducts and useful pharmacologically relevant metabolites provide a silver lining to uncover single molecular bio-effectors from its endophytes. Here, we describe socio-ethnobotanical usage, present the current pharmacological status and discuss potential bottlenecks in harnessing the proclaimed phytomedicinal worth of this prescribed Ayurvedic medicinal plant. Finally, we also look into the possible future of the drug discovery and pharmaceutical R&D efforts directed at exploring its pharma legacy.

Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum

Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) C-13-H-1] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a ``uniqueness score'' and a ``coverage score''. Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of deduttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMIR-based metabolomics data by reducing existing impediments.

Sonic hedgehog-responsive lipoxygenases and cyclooxygenase-2 modulate Dectin-1-induced inflammatory cytokines

Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)-or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-l-responsive pro-inflammatory signature cytokines like TNE-alpha, IL-1 beta and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention. (C) 2015 Elsevier Ltd. All rights reserved.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp-CrP14, obtained from stem tissues, and Talaromyces radicus-CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 mu g/ml and 20 mu g/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus-CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus-CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 mu g/l) in modified M2 medium and of vinblastine (70 mu g/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches

Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygenrich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.