A Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Acyl Hydrolase Activities

Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX4D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions.

Polymorphs and hydrates of Etoricoxib, a selective COX-2 inhibitor

The crystal structures of two polymorphs and two polymorphic hemihydrates of Etoricoxib are reported. Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) that is a selective inhibitor of COX-2. It is used in the treatment of various types of inflammation, pain and fever. Clas et al. have reported four polymorphs (labeled I through IV) and two solvates (hemi-and sesquihydrate) of the API in US patent 6,441,002 (Clas et al, US patent 6,441,002, 2002). However, no crystal structures have been reported for any of these forms. A comparison was made between the PXRD patterns reported in patent `002 and the powder spectra simulated from single crystal data. The two polymorphs characterized here correspond to form I and form IV of the patent. Form II of the patent could not be obtained by us with a variety of experimental conditions. Form III of the patent corresponds to hemihydrate II of this study. Form III is therefore not a polymorph of form I and form IV. What we have termed hemihydrate I in this study is obtained under a wide variety of conditions and it is also the only hemihydrate reported as such in the patent. Because the Etoricoxib molecule contains no conventional hydrogen bond donors, there cannot be any strong hydrogen bonds in the crystal structures of forms I and IV. The packing is accordingly characterized by weak hydrogen bonds of the C-H center dot center dot center dot O=S and C-H center dot center dot center dot N type. Thermal data were collected for form I, form IV and hemihydrate I to shed some light on relative stabilities. PXRD diffractograms show the transformation of form IV to form I at elevated temperature, indicating that form I is more stable than form IV. However, this transformation occurs only in samples of form IV that contain some form I; it does not occur in pure form IV. The formation of the two hemihydrates could follow from the known tendency of an acceptor-rich molecule to crystallize as a hydrate.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

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.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of thymidylate kinase (TTHA1607) from Thermus thermophilus HB8

Nucleotide biosynthesis plays a key role in cell survival and cell proliferation. Thymidylate kinase is an enzyme that catalyses the conversion of dTMP to dTDP using ATP-Mg2+ as a phosphoryl-donor group. This enzyme is present at the junction of the de novo and salvage pathways; thus, any inhibitor designed against it will result in cell death. This highlights the importance of this enzyme as a drug target. Thymidylate kinase from the extremely thermophilic organism Thermus thermophilus HB8 has been expressed, purified and crystallized using the microbatch method. The crystals diffracted to a resolution of 1.83 angstrom and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 39.50, b = 80.29, c = 122.55 angstrom. Preliminary studies revealed the presence of a dimer in the asymmetric unit with a Matthews coefficient (V-M) of 2.18 angstrom(3) Da(-1).

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and In vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, (3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro2.5]octan-6-yl] N-(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only similar to 10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ((3R, 4S, 5S, 6R)-5-methoxy-4-(2R, 3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro2.5]octan-6 -yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus

Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the beta-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

Dual enzyme responsive microcapsules simulating an ``OR'' logic gate for biologically triggered drug delivery applications

Hollow microcapsules capable of disintegrating in response to dual biological stimuli have been synthesized from two FDA approved drug molecules. The capsules fabricated from protamine and chondroitin sulphate disintegrate in the presence of either trypsin or hyaluronidase enzymes, which are documented to be simultaneously over-expressed under some pathological conditions.

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.

Structural and kinetic studies on adenylosuccinate lyase from Mycobacterium smegmatis and Mycobacterium tuberculosis provide new insights on the catalytic residues of the enzyme

Adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis, has been recognized as a drug target against microbial infections. In the present study, ASL from Mycobacteriumsmegmatis (MsASL) and Mycobacteriumtuberculosis (MtbASL) were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at a resolution of 2.16 angstrom. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. These structures suggest that His149 and either Lys285 or Ser279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. Most of the active site residues were found to be conserved, with the exception of Ser148 and Gly319 of MsASL. Ser148 is structurally equivalent to a threonine in most other ASLs. Gly319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria and their high GC containing genomes, as well as their dependence on other salvage pathways for the supply of purine nucleotides. Structured digital abstract andby()

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.

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA, a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme-DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria. (C) 2014 Elsevier Inc. All rights reserved.

Advances in the molecular design of potential anticancer agents via targeting of human telomeric DNA

Telomerases are an attractive drug target to develop new generation drugs against cancer. A telomere appears from the chromosomal termini and protects it from double-stranded DNA degradation. A short telomere promotes genomic instability, like end-to-end fusion and regulates the over-expression of the telomere repairing enzyme, telomerase. The telomerase maintains the telomere length, which may lead to genetically abnormal situations, leading to cancer. Thus, the design and synthesis of an efficient telomerase inhibitor is a viable strategy toward anticancer drugs development. Accordingly, small molecule induced stabilization of the G-quadruplex structure, formed by the human telomeric DNA, is an area of contemporary scientific art. Several such compounds efficiently stabilize the G-quadruplex forms of nucleic acids, which often leads to telomerase inhibition. This Feature article presents the discovery and development of the telomere structure, function and evolution in telomere targeted anticancer drug design and incorporates the recent advances in this area, in addition to discussing the advantages and disadvantages in the methods, and prospects for the future.

Consequences of Converting Graded to Action Potentials upon Neural Information Coding and Energy Efficiency

Information is encoded in neural circuits using both graded and action potentials, converting between them within single neurons and successive processing layers. This conversion is accompanied by information loss and a drop in energy efficiency. We investigate the biophysical causes of this loss of information and efficiency by comparing spiking neuron models, containing stochastic voltage-gated Na+ and K+ channels, with generator potential and graded potential models lacking voltage-gated Na+ channels. We identify three causes of information loss in the generator potential that are the by-product of action potential generation: (1) the voltage-gated Na+ channels necessary for action potential generation increase intrinsic noise and (2) introduce non-linearities, and (3) the finite duration of the action potential creates a `footprint' in the generator potential that obscures incoming signals. These three processes reduce information rates by similar to 50% in generator potentials, to similar to 3 times that of spike trains. Both generator potentials and graded potentials consume almost an order of magnitude less energy per second than spike trains. Because of the lower information rates of generator potentials they are substantially less energy efficient than graded potentials. However, both are an order of magnitude more efficient than spike trains due to the higher energy costs and low information content of spikes, emphasizing that there is a two-fold cost of converting analogue to digital; information loss and cost inflation.

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery

In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC50 values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced recA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.