Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.     

The complete sequence of the mitochondrial genome of the crustacean Penaeus monodon: are malacostracan crustaceans more closely related to insects than to branchiopods?

The complete sequence of the mitochondrial genome of the giant tiger prawn, Penaeus monodon (Arthropoda, Crustacea, Malacostraca), is presented. The gene content and gene order are identical to those observed in Drosophila yakuba. The overall AT composition is lower than that observed in the known insect mitochondrial genomes, but higher than that observed in the other two crustaceans for which complete mitochondrial sequence is available. Analysis of the effect of nucleotide bias on codon composition across the Arthropoda reveals a trend with the crustaceans represented showing the lowest proportion of AT-rich codons in mitochondrial protein genes. Phylogenetic analysis among arthropods using concatenated protein-coding sequences provides further support for the possibility that Crustacea are paraphyletic. Furthermore, in contrast to data from the nuclear gene EF1alpha, the first complete sequence of a malacostracan mitochondrial genome supports the possibility that Malacostraca are more closely related to Insecta than to Branchiopoda.     

Assessment of the humoral immune response to cancer

One of the deadly hallmarks of cancer is its ability to prosper within the constraints of the host immune system. Recent advances in immunoproteomics and high-throughput technologies have lead to profiling of the antibody repertoire in cancer patients. This in turn has lead to the identification of tumour associated antigens/autoantibodies. Autoantibodies are extremely attractive and promising biomarker entities, however there has been relatively little discussion on how to interpret the humoral immune response. It may be that autoantibody profiles hold the key to ultimately uncovering neoplastic associated pathways and through the process of immunosculpting the tumour may have yielded an immune response in the early stages of malignant tumour development. The aim of this review is to discuss the utility of the autoantibody response that is elicited as a result of malignancy and discuss the advantages and limitations of autoantibody profiling. This article is part of a Special Issue entitled: Translational Proteomics.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.     

A Ca2+-induced mitochondrial permeability transition causes complete release of rat liver endonuclease G activity from its exclusive location within the mitochondrial intermembrane space. Identification of a novel endo-exonuclease activity residing within the mitochondrial matrix

Endonuclease G, a protein historically thought to be involved in mitochondrial DNA (mtDNA) replication, repair, recombination and degradation, has recently been reported to be involved in nuclear DNA degradation during the apoptotic process. As a result, its involvement in mtDNA homeostasis has been called into question and has necessitated detailed analyses of its precise location within the mitochondrion. Data is presented localizing rat liver endonuclease G activity exclusively to the mitochondrial intermembrane space with no activity associated with either the interior face of the inner mitochondrial membrane or with the mitochondrial matrix. Additionally, it is shown that endonuclease G can be selectively released from the mitochondrion via induction of a Ca²⁺-induced mitochondrial permeability transition and that, upon its release, a further nuclease activity loosely associated with the interior face of the inner mitochondrial membrane and distinct in its properties from that of endonuclease G can be detected.     

Structure-function analysis of the bestrophin family of anion channels

The bestrophins are a newly described family of anion channels unrelated in primary sequence to any previously characterized channel proteins. The human genome codes for four bestrophins, each of which confers a distinctive plasma membrane conductance on transfected 293 cells. Extracellular treatment with methanethiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophin1 demonstrates that cysteine 69 is the single endogenous cysteine responsible for MTSET inhibition of whole-cell current. Cysteines introduced between positions 78-99 and 223-226 are also accessible to external MTSET, with MTSET modification at positions 79, 80, 83, and 90 producing a 2-6-fold increase in whole-cell current. The latter set of four cysteine-substitution mutants define a region that appears to mediate allosteric control of channel activity. Mapping of transmembrane topography by insertion of N-linked glycosylation sites and tobacco etch virus protease cleavage sites provides evidence for cytosolic N and C termini and an unexpected transmembrane topography with at least three extracellular loops that include positions 60-63, 212-227, and 261-267. These experiments provide the first structural analysis of the bestrophin channel family.     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity

An unconventional use for the polarization optics, associated with a variety of commercially available fluorescence microplate readers, is reported. This novel application has allowed the discrimination of green fluorescent protein (GFP) fluorescence in genetically modified yeast cells from interfering autofluorescent species. The method exploits the unusually high fluorescence anisotropy of GFP compared to smaller fluorophores and autofluorescent species. The principle was successfully applied to resolve the induced GFP signal from that of autofluorescent test compounds, in an assay for genotoxic species. The use of fluorescence polarization enabled both proflavin and methapyrilene to be identified as genotoxic agents in the yeast assay. This would not have been possible using conventional fluorescence alone since these compounds were found to be intensely autofluorescent at the same wavelength as GFP and thus effectively mask the GFP signal.