“To Cross-Seed or Not To Cross-Seed”: A Pilot Study Using Metallo-β-lactamases

Knowledge of protein structures is of central importance in modern drug discovery and molecular biology, but to be useful the structures, including those obtained in the crystalline state, must be biologically relevant. Small variations in crystallization conditions can lead to alternative crystal forms, conformations, and oligomerization states, causing changes which can lead to altered fold and active site architectures. In the determination of protein structures by X-ray crystallography, crystallization is an essential prerequisite and remains a major bottleneck in drug discovery. Although many methods have been tried in an attempt to improve the production of protein crystals, it is still largely a “trial and error” process. To our knowledge, crystallization by cross-seeding using homologous proteins has previously only been successful for proteins with greater than 61–74% sequence identity. In the study presented here, we explore the effect of low sequence similarity on cross-seeding using metallo-β-lactamases with sequence identities and sequence similarities as low as 24% and 36%, respectively, but with homologous core folds. Despite the low sequence identities, the results show that micro-cross-seeding matrix screening can increase the number of hits obtained and can shorten crystallization time. It can also help in the identification of new crystallization conditions and different crystal forms

“To Cross-Seed or Not To Cross-Seed”: A Pilot Study Using Metallo-β-lactamases

Knowledge of protein structures is of central importance in modern drug discovery and molecular biology, but to be useful the structures, including those obtained in the crystalline state, must be biologically relevant. Small variations in crystallization conditions can lead to alternative crystal forms, conformations, and oligomerization states, causing changes which can lead to altered fold and active site architectures. In the determination of protein structures by X-ray crystallography, crystallization is an essential prerequisite and remains a major bottleneck in drug discovery. Although many methods have been tried in an attempt to improve the production of protein crystals, it is still largely a “trial and error” process. To our knowledge, crystallization by cross-seeding using homologous proteins has previously only been successful for proteins with greater than 61–74% sequence identity. In the study presented here, we explore the effect of low sequence similarity on cross-seeding using metallo-β-lactamases with sequence identities and sequence similarities as low as 24% and 36%, respectively, but with homologous core folds. Despite the low sequence identities, the results show that micro-cross-seeding matrix screening can increase the number of hits obtained and can shorten crystallization time. It can also help in the identification of new crystallization conditions and different crystal forms

Uma experiência de tradução do conto “Marcha Fúnebre” de Machado de Assis para o japonês

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Pharmacological Inhibition of FTO

<div><p>In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function <i>in vitro</i> and <i>in vivo</i> as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate <i>in vitro</i>. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO’s demethylase activity could be therapeutically useful for the treatment of obesity.</p></div

Chemical structure of IOX3 and IC₅₀ values for FTO and PHD2.

<p>Chemical structure of IOX3 and IC₅₀ values for FTO and PHD2.</p

Oxygen Consumption Rate (OCR), Extracellular Acidification Rate (ECAR) of C2C12, and wild-type and FTO knockout MEFs treated with 1 μM IOX3 or an equivalent amount of vehicle control for 16 hours.

<p>A) OCR and, B) basal ECAR measured in C2C12 cells treated with vehicle (n = 10) and 1 μM IOX3 (n = 10) at baseline and after Oligomycin, FCCP and Rotenone treatment, data normalised to live stain. C) OCR and, D] ECAR measured in <i>Fto</i>^<i>+/+</i> and <i>Fto</i>^<i>-/-</i> MEFs cells treated with vehicle and 1 μM IOX3 (n = 5 per group) at baseline and after Oligomycin, FCCP and Rotenone treatment, data normalised to live stain. Data were analysed using a 2 way ANOVA with Bonferroni post-hoc test. Data is of readings following each compound injection and are expressed as mean ± SEM. E) Expression of FTO, phosphorylated-AMPKα and HIF-1α with representative ACTIN in cells treated with vehicle, 1uM IOX3, control scrambled siRNA or <i>Fto</i> siRNA for 24 hours. N = 3 biological replicates per condition.</p

DEXA and organ weights of vehicle and 60 mg/kg every two days IOX3-treated mice.

<p>A) Bone Mineral Density (BMD), B) Bone Mineral Content (BMC), C) Liver mass, D) Epigonadal white adipose tissue (WAT), E) Abdominal WAT, F) Peri-renal WAT, G) Brown adipose tissue (BAT), H) Calf muscle weight of vehicle (n = 20) and 60 mg/kg every two days IOX3 (n = 20) treated mice. Data analysed by student’s t-test *P<0.05, **P<0.01, ***P<0.001. Data are expressed as mean ± SEM and individual data points are shown.</p

Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of <i>N</i>^ε-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of <i>N</i>-oxalyl-d<i>-</i>tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds

Binding of (5<i>S</i>)‑Penicilloic Acid to Penicillin Binding Protein 3

β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (<i>Pseudomonas aeruginosa</i>) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5<i>S</i>)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5<i>R</i>)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance

Materials challenges of high efficient power generator

Zinc ion-dependent β-lactamases (MBLs) catalyze the hydrolysis of almost all β-lactam antibiotics and resist the action of clinically available β-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-β-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing <i>Enterobacteriaceae,</i> with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops