An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells, and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

Palladium(II) complexes as biologically potent metallo-drugs: Synthesis, spectral characterization, DNA interaction studies and antibacterial activity

Four novel mononuclear Pd(II) complexes have been synthesized with the biologically active Schiff base ligands (L-1-L-4) derived from 3-amino-2-methyl-4(3H)-quinazolinone. The structure of the complexes has been proposed by elemental analysis, molar conductance, IR, H-1 NMR, mass, UV-Vis spectrometric and thermal studies. The investigation of interaction of the complexes with calf thymus DNA (CT-DNA) has been performed with absorption and fluorescence spectroscopic studies. The nuclease activity was done using pUC19 supercoiled DNA by gel-electrophoresis. All the ligands and their Pd(II) complexes have also been screened for their antibacterial activity by discolor diffusion technique. (C) 2013 Elsevier B.V. All rights reserved.

Design of an Escherichia coli expressed HIV-1 gp120 fragment Immunogen that binds to b12 and induces broad and potent neutralizing antibodies

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals.

Probing p300/CBP associated Factor (PCAF)-dependent pathways with a small molecule inhibitor

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.

Stable and Potent Analogues Derived from the Modification of the Dicarbonyl Moiety of Curcumin

Curcumin has shown promising therapeutic utilities for many diseases, including cancer; however, its clinical application is severely limited because of its poor stability under physiological conditions. Here we find that curcumin also loses its activity instantaneously in a reducing environment. Curcumin can exist in solution as a tautomeric mixture of keto and enol forms, and the enol form was found to be responsible for the rapid degradation of the compound. To increase the stability of curcumin, several analogues were synthesized in which the diketone moiety of curcumin was replaced by isoxazole (compound 2) and pyrazole (compound 3) groups. Isoxazole and pyrazole curcumins were found to be extremely stable at physiological pH, in addition to reducing atmosphere, and they can kill cancer cells under serum-depleted condition. Using molecular modeling, we found that both compounds 2 and 3 could dock to the same site of tubulin as the parent molecule, curcumin. Interestingly, compounds 2 and 3 also show better free radical scavenging activity than curcumin. Altogether, these results strongly suggest that compounds 2 and 3 could be good replacements for curcumin in future drug development.

Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

Endoplasmic Reticulum Stress-Mediated Activation of p38 MAPK, Caspase-2 and Caspase-8 Leads to Abrin-Induced Apoptosis

Abrin from Abrus precatorius plant is a potent protein synthesis inhibitor and induces apoptosis in cells. However, the relationship between inhibition of protein synthesis and apoptosis is not well understood. Inhibition of protein synthesis by abrin can lead to accumulation of unfolded protein in the endoplasmic reticulum causing ER stress. The observation of phosphorylation of eukaryotic initiation factor 2 alpha and upregulation of CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. Moreover, abrin-induced apoptosis was found to be dependent on p38 MAPK but not JNK. We also observed that abrin induced the activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery.

Conserved water-mediated recognition and dynamics of NAD(+) (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP-NAD(+) complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD(+). Three conserved water molecules (W1, W, and W1 `) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD(+)) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP-NAD(+)-enzyme complexes and their recognition to NAD(+), some covalent modification at carboxamide group of di-nucleotide (NAD(+)) has been made by substituting the -CONH(2)group by -CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

The basics and advances of immunomodulators and antigen presentation: a key to development of potent memory response against pathogens

Introduction: Immunomodulators are agents, which can modulate the immune response to specific antigens, while causing least toxicity to the host system. Being part of the modern vaccine formulations, these compounds have contributed remarkably to the field of therapeutics. Despite the successful record maintained by these agents, the requirement of novel immunomodulators keeps increasing due to the increasing severity of diseases. Hence, research regarding the same holds great importance. Areas covered: In this review, we discuss the role of immunomodulators in improving performance of various vaccines used for counteracting most threatening infectious diseases, mechanisms behind their action and criteria for development of novel immunomodulators. Expert opinion: Understanding the molecular mechanisms underlying immune response is a prerequisite for development of effective therapeutics as these are often exploited by pathogens for their own propagation. Keeping this in mind, the present research in the field of immunotherapy focuses on developing immunomodulators that would not only enhance the protection against pathogen, but also generate a long-term memory response. With the introduction of advanced formulations including combination of different kinds of immunomodulators, one can expect tremendous success in near future.

Charge density distribution and electrostatic interactions of ethionamide: an inhibitor of the enoyl acyl carrier protein reductase (inhA) enzyme of Mycobacterium tuberculosis

An experimental charge density analysis of an anti-TB drug ethionamide was carried out from high resolution X-ray diffraction at 100 K to understand its charge density distribution and electrostatic properties. The experimental results were validated from periodic theoretical charge density calculations performed using CRYSTAL09 at the B3LYP/6-31G** level of theory. The electron density rho(bcp)(r) and the Laplacian of electron density del(2)(rho bcp)(r) of the molecule calculated from both the methods display the charge density distribution of the ethionamide molecule in the crystal field. The electrostatic potential map shows a large electropositive region around the pyridine ring and a large electronegative region at the vicinity of the thiol atom. The calculated experimental dipole moment is 10.6D, which is higher than the value calculated from theory (8.2D). The topological properties of C-H center dot center dot center dot S, N-H center dot center dot center dot N and N-H center dot center dot center dot S hydrogen bonds were calculated, revealing their strength. The charge density analysis of the ethionamide molecule determined from both the experiment and theory gives the topological and electrostatic properties of the molecule, which allows to precisely understand the nature of intra and intermolecular interactions.

Chemical specificity and conformational flexibility in proteinase-inhibitor interaction: Scaffolds for promiscuous binding

One of the most important roles of proteins in cellular milieu is recognition of other biomolecules including other proteins. Protein protein complexes are involved in many essential cellular processes. Interfaces of protein protein complexes are traditionally known to be conserved in evolution and less flexible than other solvent interacting tertiary structural surface. But many examples are emerging where these features do not hold good. An understanding of inter-play between flexibility and sequence conservation is emerging, providing a fresh dimension to the paradigm of sequence structure function relationship. The functional manifestation of the inter-relation between sequence conservation and flexibility of interface is exemplified in this review using proteinase inhibitor protein complexes. (C) 2014 Elsevier Ltd. All rights reserved.

A Kunitz-type serine protease inhibitor from Butea monosperma seed and its influence on developmental physiology of Helicoverpa armigera

A protease inhibitor from the seeds of Butea monosperma (BmPI) was purified, characterized and studied for its influence on developmental physiology of Helicover-pa armigera. BmPI on two-dimensional separations indicated the presence of a 14 kDa protein with an isoelectric point in the acidic region (pl 5.6). Multiple Sequence Analysis data suggested that the BmPI contains a sequence motif which is conserved in various trypsin and chymotrypsin inhibitors of Kunitz-type. The inhibitor exhibited trypsin inhibitory activity in a broad range of pH (4-10) and temperature (10-80 degrees C). The enzyme kinetic studies revealed BmPI as a competitive inhibitor with a K-i value of 1.2 x 10(-9) M. In vitro studies with BmPI indicated measurable inhibitory activity on total gut proteolytic enzymes of H. armigera (IC(50)2.0 mu g/ml) and bovine trypsin. BmPI supplemented artificial diet caused dose dependent mortality and reduction in growth and weight. The fertility and fecundity of H. armigera, declined whereas the larval-pupal duration of the insect life cycle extended. These detrimental effects on H. armigera suggest the usefulness of BmPl in insect pest management of food crops. (C) 2014 Elsevier Ltd. All rights reserved.