Shahper N. Khan

Interaction of mitoxantrone with human serum albumin : Spectroscopic and molecular modeling studies

Mitoxantrone (MTX) is a clinically used antitumor anthracycline, which is made available to the target tissues by transport protein human serum albumin (HSA). Being less toxic unlike other member of this family, its binding characteristics are therefore of immense interest. The circular dichroism (CD), fluorescence and Fourier transform infrared (FTIR) spectroscopies were employed to elucidate the mode and the mechanism for this interaction. MTX binding is characterized by one high affinity binding site with the association constants of the order of 10(5). Correlation between stability of N-MTX (drug bound N form of HSA) and B-MTX (drug bound B form of HSA) complexes with drug distribution has been discussed. The molecular distance, r, between donor (HSA) and acceptor (MTX) was estimated according to Forsters theory of non-radiation energy transfer. The features of MTX induced structural perturbation of human serum albumin (HSA) has been studied in detail by CD and FTIR analysis. Domain I was assigned to possess high affinity binding site for MTX. Molecular docking showed that the MTX binds HSA to a non-classical drug binding site. The binding dynamics was expounded by synchronous fluorescence, thermodynamic parameters and molecular modeling, which entails that hydrophobic interactions, hydrogen bonding and electrostatic forces, stabilizes the interaction.

Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (−7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of −7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

Novel anti-adherence activity of mulberry leaves: inhibition of Streptococcus mutans biofilm by 1-deoxynojirimycin isolated from Morus alba

OBJECTIVES The present study focused on isolation, characterization and evaluation of purified compounds from Morus alba against Streptococcus mutans biofilm formation. METHODS The effect of crude extract from M. alba leaves was evaluated against oral pathogens, chiefly S. mutans. MICs were determined by the microdilution method. The compound was purified by employing silica gel chromatography and critically analysed with GC-MS, NMR and IR spectroscopy. The S. mutans traits of adherence and biofilm formation were assessed at sub-MIC concentrations of the crude extract and purified compound. Both water-soluble and alkali-soluble polysaccharide were estimated to determine the effect of the purified compound on the extracellular polysaccharide secretion of S. mutans. Its effect on biofilm architecture was also investigated with the help of confocal microscopy. RESULTS The purified compound of M. alba showed an 8-fold greater reduction of MIC against S. mutans than the crude extract (MICs, 15.6 and 125 mg/L, respectively). The extract strongly inhibited biofilm formation of S. mutans at its active accumulation and plateau phases. The purified compound led to a 22% greater reduction in alkali-soluble polysaccharide than in water-soluble polysaccharide. The purified compound was found to be 1-deoxynojirimycin (DNJ). Confocal microscopy revealed that DNJ distorts the biofilm architecture of S. mutans. CONCLUSIONS The whole study reflects a prospective role of DNJ as a therapeutic agent by controlling the overgrowth and biofilm formation of S. mutans.

Role of histone acetylation in cell physiology and diseases: An update

Although the role of histone acetylation in gene regulation has been the subject of many reviews, their impact on cell physiology and pathological states of proliferation, differentiation and genome stability in eukaryotic cells remain to be elucidated. Therefore, this review will discuss the molecular, physiological and biochemical aspects of histone acetylation and focus on the interplay of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in different disease states. Current treatment strategies are mostly limited to enzyme inhibitors, though potential lies in targeting other imperative chromatin remodeling factors involved in gene regulation.

Myeloma Is Characterized by Stage-Specific Alterations in DNA Methylation That Occur Early during Myelomagenesis

Epigenetic changes play important roles in carcinogenesis and influence initial steps in neoplastic transformation by altering genome stability and regulating gene expression. To characterize epigenomic changes during the transformation of normal plasma cells to myeloma, we modified the HpaII tiny fragment enrichment by ligation–mediated PCR assay to work with small numbers of purified primary marrow plasma cells. The nano-HpaII tiny fragment enrichment by ligation–mediated PCR assay was used to analyze the methylome of CD138+ cells from 56 subjects representing premalignant (monoclonal gammopathy of uncertain significance), early, and advanced stages of myeloma, as well as healthy controls. Plasma cells from premalignant and early stages of myeloma were characterized by striking, widespread hypomethylation. Gene-specific hypermethylation was seen to occur in the advanced stages, and cell lines representative of relapsed cases were found to be sensitive to decitabine. Aberrant demethylation in monoclonal gammopathy of uncertain significance occurred primarily in CpG islands, whereas differentially methylated loci in cases of myeloma occurred predominantly outside of CpG islands and affected distinct sets of gene pathways, demonstrating qualitative epigenetic differences between premalignant and malignant stages. Examination of the methylation machinery revealed that the methyltransferase, DNMT3A, was aberrantly hypermethylated and underexpressed, but not mutated in myeloma. DNMT3A underexpression was also associated with adverse overall survival in a large cohort of patients, providing insights into genesis of hypomethylation in myeloma. These results demonstrate widespread, stage-specific epigenetic changes during myelomagenesis and suggest that early demethylation can be a potential contributor to genome instability seen in myeloma. We also identify DNMT3A expression as a novel prognostic biomarker and suggest that relapsed cases can be therapeutically targeted by hypomethylating agents.

Breaking the Spell: Combating Multidrug Resistant 'Superbugs'

Multidrug-resistant (MDR) bacteria have become a severe threat to community wellbeing. Conventional antibiotics are getting progressively more ineffective as a consequence of resistance, making it imperative to realize improved antimicrobial options. In this review we emphasized the microorganisms primarily reported of being resistance, referred as ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacteriaceae) accentuating their capacity to “escape” from routine antimicrobial regimes. The upcoming antimicrobial agents showing great potential and can serve as alternative therapeutic options are discussed. We also provided succinct overview of two evolving technologies; specifically network pharmacology and functional genomics profiling. Furthermore, In vivo imaging techniques can provide novel targets and a real time tool for potential lead molecule assessment. The employment of such approaches at prelude of a drug development process, will enables more informed decisions on candidate drug selection and will maximize or predict therapeutic potential before clinical testing.

Molecular interactions between mitochondrial membrane proteins and the C-terminal domain of PB1-F2: an in silico approach

PB1-F2 is a recently described influenza A viral protein that induces apoptosis by binding with two mitochondrial membrane proteins, i.e. VDAC1 (outer membrane) and ANT3 (inner membrane). Knowledge of this binding mechanism could provide insights that would aid in the design of novel inhibitors against this protein. Therefore, to better understand these interactions, we have undertaken this study to model the PB1-F2 protein of the highly pathogenic influenza A virus subtype H5N1. Moreover, a model of human ANT3 was also established. The dynamics of the molecular interactions between the C-terminal region of PB1-F2 protein and VDAC1 and ANT3 were expounded by employing an in silico approach. Our results suggest the involvement of 12 amino acids of PB1-F2 protein, which form hydrophobic contacts with 22 amino acids of VDAC1. Of these, Leu64, Arg75 and Val76 were found to be crucial for mitochondrial targetting. In the case of the PB1-F2-ANT3 complex, 14 amino acids of ANT3 were found to make hydrophobic contacts with 9 amino acids of PB1-F2. Furthermore, two hydrogen bonds were predicted in both complexes PB1-F2/VDAC1 and PB1-F2/ANT3. This study reveals the molecular interactions required for PB1-F2-induced apoptosis and suggests a hypothetical model for future study.

Template synthesis and physico-chemical characterization of 14-membered tetraimine macrocyclic complexes, [MLX2] [M=Co(II), Ni(II), Cu(II) and Zn(II)]. DNA binding study on [CoLCl2] complex

The template condensation reaction between glyoxal and 1,8-diaminonaphthalene resulted a few novel mononuclear 14-membered tetraimine macrocyclic complexes of the type, [MLX(2)] [M=Co(II), Ni(II), Cu(II) and Zn(II), for X=Cl or NO(3)]. The stoichiometry and the nature of the complexes have been deduced from the results of elemental analyses and conductance data. The formation of macrocyclic framework has been inferred from the appearance of imine upsilon(CN) and upsilon(MN) band in IR spectra and the resonance signals observed in (1)H and (13)C-NMR spectra. However, the overall geometry of the complexes has been assigned on the positions of bands in electronic spectra and magnetic moment data. The distortion in Cu(II) complexes has been deduced on EPR data. The thermal behavior of these complexes has been studied by TGA analysis. Absorption and circular dichroism studies on the complex proved a significant binding to calf thymus DNA.

Characterization of doxorubicin binding site and drug induced alteration in the functionally important structural state of oxyhemoglobin.

Doxorubicin (DOX) binding to hemoglobin (Hb) was studied to investigate the drug induced protein dysfunction. The features of anti-tumor drug doxorubicin infused structural perturbation of human Hb were studied by circular dichroism (CD). The mechanism of DOX-Hb binding was elucidated by steady-state and synchronous fluorescence spectroscopy. The Stern-Volmer analysis indicated that the binding of Hb to DOX is characterized by more than one high affinity binding site with the association constants of the order of 10(5). Hydrophobic probe ANS was employed to elucidate the drug binding site. Binding mode expounded by thermodynamic parameters implied the role of hydrogen bonding, electrostatic and hydrophobic interaction in stabilizing the complex. The molecular distance between donor (Hb) and acceptor (DOX) was calculated according to Försters theory of energy transfer. Fourier transform infrared (FT-IR) spectroscopy provides an insight to the changes occurring in protein on DOX binding. Treatment of Hb with DOX resulted in a dose dependent fragmentation of protein. The quantitative analysis revealed the release of acid soluble amino groups from the photoexcited Hb-DOX mixture. The free radical mediated degradation was suggested by its rescue on mannitol and superoxide dismutase (SOD) appliance. The loss of protein band further corroborates the concentration dependent Hb fragmentation. The molecular modeling complies with the thermodynamic data of forces involved in DOX binding and depicts its interaction in the proximity of oxygen binding pocket of Hb. Thus, this study enriches our understanding of the interaction dynamics of anticancer drugs to the physiologically important protein Hb.

A proteomic approach for exploring biofilm in Streptococcus mutans.

Biofilm formation by Streptococcus mutans is considered as its principal virulence factor, causing dental caries. Mutants of S. mutans defective in biofilm formation were generated and analyzed to study the collective role of proteins in its formation. Mutants were characterized on the basis of adherence to saliva-coated surface, and biofilm formation. The confocal laser microscopy and scanning electron microscopy images showed that the control biofilms had cluster of cells covered by layer of exo-polysaccharide while the biofilms of mutants were thin and spaced. Two-dimensional protein electrophoresis data analysis identified 57 proteins that are either up (44 proteins) or down (13 proteins) regulated. These data points to the importance of up and down regulated proteins in the formation of biofilm in Streptococcus mutans.