Novel effects of ectoine, a bacteria-derived natural tetrahydropyrimidine, in experimental colitis.

Evidence suggests an important role of intestinal barrier dysfunction in the etiology of inflammatory bowel disease (IBD). Therefore stabilizing mucosal barrier function constitutes a new therapeutic approach in its management. Ectoine is a compatible solute produced by aerobic chemoheterotrophic and halophilic/halotolerant bacteria, where it acts as osmoprotectant and effective biomembrane stabilizer, protecting the producing cells from extreme environmental stress. Since this natural compound was also shown to prevent inflammatory responses associated with IBD, its potential usefulness was studied in a model of colitis. Groups of rats were treated orally with different doses of ectoine (30-300 mg/kg) or sulfasalazine (reference drug) daily for 11 days. On day 8 colitis was induced by intracolonic instillation of 2,4,6-trinitrobenzenesulfonic acid, when overt signs of lesions develop within the next 3 days. On day 12, blood was withdrawn from the retro-orbital plexus of the rats and the animals were sacrificed. The colon was excised and examined macroscopically and microscopically. Relevant parameters of oxidative stress and inflammation were measured in serum and colon homogenates. Induction of colitis led to marked weight loss, significant histopathological changes of the colon, and variable changes in levels of myeloperoxidase, reduced glutathione, malondialdehyde, and all inflammatory markers tested. Treatment with ectoine ameliorated the inflammatory changes in TNBS-induced colitis. This effect was associated with reduction in the levels of TNF-α, IL-1ß, ICAM-1, PGE2 and LTB4. The findings suggest that intestinal barrier stabilizers from natural sources could offer new therapeutic measures for the management of IBD.

Compatible solutes: ectoine and hydroxyectoine improve functional nanostructures in artificial lung surfactants.

Ectoine and hydroxyectoine belong to the family of compatible solutes and are among the most abundant osmolytes in nature. These compatible solutes protect biomolecules from extreme conditions and maintain their native function. In the present study, we have investigated the effect of ectoine and hydroxyectoine on the domain structures of artificial lung surfactant films consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and the lung surfactant specific surfactant protein C (SP-C) in a molar ratio of 80:20:0.4. The pressure-area isotherms are found to be almost unchanged by both compatible solutes. The topology of the fluid domains shown by scanning force microscopy, which is thought to be responsible for the biophysical behavior under compression, however, is modified giving rise to the assumption that ectoine and hydroxyectoine are favorable for a proper lung surfactant function. This is further evidenced by the analysis of the insertion kinetics of lipid vesicles into the lipid-peptide monolayer, which is clearly enhanced in the presence of both compatible solutes. Thus, we could show that ectoine and hydroxyectoine enhance the function of lung surfactant in a simple model system, which might provide an additional rationale to inhalative therapy.

A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581 T.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581(T) thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4,061,296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-L-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production.

The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes.

Compatible solutes are small organic osmolytes responsible for osmotic balance and at the same time compatible with the cellular metabolism. Here, we have investigated the effect of the compatible solutes, ectoine and hydroxyectoine, on the fluid-rigid domain structure of lipid monolayer and bilayer membranes. Mainly saturated dipalmitoyl-phosphatidylcholine membranes exhibiting a clear le/lc phase transition were used. Fluorescence microscopy showed that ectoines added to the aqueous subphase expand and fluidize the lipid monolayers especially at surface pressures below 30mN/m. The domain structure at the le/lc phase transition is sensitively modified leading to smaller but more numerous domains in the presence of ectoines. Hydroxyectoine was more efficient than ectoine. These results are explained by the replacement theory assuming that the ectoines are likely to be expelled from the membrane surface thus favoring the hydration of the lipid membrane. This effect reduces the line tension, which is the interfacial energy at the domain edges leading to reduced domain sizes and increased number of rigid domains. Isotherms of negatively charged phosphatidylglycerol membranes show a similar expansion, while unsaturated lipids are less affected. Mixed phosphatidylcholine/phosphatidylglycerol membranes exhibit the same effect on the line tension increasing the tendency for a phase separation. This could be shown also in bilayer vesicles, where the compatible solutes have only a minor effect on the lipid main phase transition in pure DPPC membranes but reduce the extent of the pretransition. In mixed DPPC/DPPG bilayer membranes ectoines cause a phase separation leading to the enrichment of expanded DPPC domains. In conclusion, our study gives for the first time evidence that ectoines have an effect on lipid membranes increasing the hydration of the surface and thus increasing the mobility of the lipid head groups and fluidizing the lipid layer accordingly. This increased fluidity may be of advantage for cell membranes to withstand extreme conditions like temperature or osmotic pressure and might also accelerate cellular repair mechanisms.

The pyrostatins A and B do not inhibit N-acetyl-beta-D-glucosaminidase.

The compounds pyrostatin A and B, isolated from Streptomyces sp. SA-3501 have been reported as N-acetyl-beta-D-glucosaminidase inhibitors with inhibition constants in the micromolar range. Recently, a comparison of NMR spectral data of the pyrostatins has led to a structural revision of the pyrostatins. It was shown that the pyrostatins A and B are identical to the ectoines 5-hydroxectoine and ectoine, respectively. Ectoines are known as compatible osmolytes in many halophilic and stress-tolerant bacteria. We have performed enzymatic experiments demonstrating that neither ectoine nor 5-hydroxyectoine exhibit an inhibitory effect on N-acetyl-beta-D-glucosaminidase. The previously reported inhibition of N-acetyl-beta-D-glucosaminidase by pyrostatins A and B may thus be due to the contamination of the compound preparations with a strong N-acetyl-beta-D-glucosaminidase inhibitor with an inhibition constant (Ki) in the nanomolar range, as has been reported in other Streptomyces species.

Inhibition of beta-amyloid peptide aggregation and neurotoxicity by alpha-d-mannosylglycerate, a natural extremolyte.

The aggregation of soluble beta-amyloid (Abeta) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The use of naturally occurring small molecules for inhibiting protein aggregation has recently attracted many interests due to their effectiveness for treating protein folding diseases such as AD, Parkinson's, Huntington's disease, and other amyloidosis diseases. alpha-d-Mannosylglycerate (MG), a natural extremolyte identified in microorganisms growing under extremely high temperatures up to 100 degrees C, had been shown to protect proteins against various stress conditions such as heat, freezing, thawing, and drying. Here, we report the effectiveness of MG on the suppression of Alzheimer's Abeta aggregation and neurotoxicity to human neuroblastoma cells. According to our study--carried out by using thioflavin-T induced fluorescence, atomic force microscopy, and cell viability assay--MG had significant inhibitory effect against Abeta amyloid formation and could reduce the toxicity of amyloid aggregates to human neuroblastoma cells while MG itself was innocuous to cells. On the other hand, the structural analogs of MG such as alpha-d-mannosylglyceramide, mannose, methylmannoside, glycerol, showed negligible effect on Abeta aggregate formation. The results suggest that MG could be a potential drug candidate for treating Alzheimer's disease.

Ectoine as a natural component of food: detection in red smear cheeses.

Ectoine is a compatible solute accumulated in halophilic bacteria in response to high salt concentrations and offers protection from osmotic stress. The occurrence of compatible solutes is widespread among bacteria, yet ectoine has never been detected in foods. The use of an ectoine producing microorganism (Brevibacterium linens) in the surface ripening of red smear cheeses led to the question whether ectoine can be found in cheese. Therefore we examined samples from a variety of cheese manufacturers and different types of red smear cheeses for the presence of ectoine using HPLC and HPLC/MS analysis. Ectoine solely appears in the rind and was detected up to 178 mg/200 g red smear cheese, depending on several factors like ripening status and conditions throughout the cheese production process (e.g. salt concentrations of used brine baths).

Discovery of a potent CDK2 inhibitor with a novel binding mode, using virtual screening and initial, structure-guided lead scoping.

Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.

Extremolytes: Natural compounds from extremophiles for versatile applications.

Extremophilic microorganisms have adopted a variety of ingenious strategies for survival under high or low temperature, extreme pressure, and drastic salt concentrations. A novel application area for extremophiles is the use of "extremolytes," organic osmolytes from extremophilic microorganisms, to protect biological macromolecules and cells from damage by external stresses. In extremophiles, these low molecular weight compounds are accumulated in response to increased extracellular salt concentrations, but also as a response to other environmental changes, e.g., increased temperature. Extremolytes minimize the denaturation of biopolymers that usually occurs under conditions of water stress and are compatible with the intracellular machinery at high (>1 M) concentrations. The ectoines, as the first extremolytes that are produced in a large scale, have already found application as cell protectants in skin care and as protein-free stabilizers of proteins and cells in life sciences. In addition to ectoines, a range of extremolytes with heterogenous chemical structures like the polyol phosphates di-myoinositol-1,1'-phosphate, cyclic 2,3-diphosphoglycerate, and alpha-diglycerol phosphate and the mannose derivatives mannosylglycerate (firoin) and mannosylglyceramide (firoin-A) were characterized and were shown to have protective properties toward proteins and cells. A range of new applications, all based on the adaptation to stress conditions conferred by extremolytes, is in development.

Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site.

The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5-3.0 Angstroms resolution and that of neamine at 2.8 Angstroms resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Angstroms resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.

Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer's beta-amyloid.

beta-Amyloid peptide (Abeta) is the major constituent of senile plaques, the key pathological feature of Alzheimer's disease. Abeta is physiologically produced as a soluble form, but aggregation of Abeta monomers into oligomers/fibrils causes neurotoxic change of the peptide. In nature, many microorganisms accumulate small molecule chaperones (SMCs) under stressful conditions to prevent the misfolding/denaturation of proteins and to maintain their stability. Hence, it is conceivable that SMCs such as ectoine and hydroxyectoine could be potential inhibitors against the aggregate formation of Alzheimer's Abeta, which has not been studied to date. The current work shows the effectiveness of ectoine and hydroxyectoine on the inhibition of Abeta42 aggregation and toxicity to human neuroblastoma cells. The characterization tools used for this study include thioflavin-T induced fluorescence, atomic force microscopy and cell viability assay. Considering that ectoine and hydroxyectoine are not toxic to cellular environment even at concentrations as high as 100 mM, the results may suggest a basis for the development of ectoines as potential inhibitors associated with neurodegenerative diseases.

Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2'-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics.

The bacterial ribosome comprises 30 S and 50 S ribonucleoprotein subunits, contains a number of binding sites for known antibiotics and is an attractive target for selection of novel antibacterial agents. On the 30 S subunit, for example, the A site (aminoacyl site) close to the 3'-end of 16 S rRNA is highly important in the decoding process. Binding by some aminoglycoside antibiotics to the A site leads to erroneous protein synthesis and is lethal for bacteria. We targeted the A site on purified 30 S ribosomal subunits from Escherichia coli with a set of overlapping, complementary OMe (2'-O-methyl) 10-mer oligoribonucleotides. An equilibrium dialysis technique was applied to measure dissociation constants of these oligonucleotides. We show that there is a single high-affinity region, spanning from A1493 to C1510 (Kd, 29-130 nM), flanked by two lower-affinity regions, within a span from U1485 to G1516 (Kd, 310-4300 nM). Unexpectedly, addition of the aminoglycoside antibiotic paromomycin (but not hygromycin B) caused a dose-dependent increase of up to 7.5-fold in the binding of the highest affinity 10-mer 1493 to 30 S subunits. Oligonucleotides containing residues complementary to A1492 and/or A1493 showed particularly marked stimulation of binding by paromomycin. The results are consistent with high-resolution structures of antibiotic binding to the A site and with greater accessibility of residues of A1492 and A1493 upon paromomycin binding. 10-mer 1493 binding is thus a probe of the conformational switch to the 'closed' conformation triggered by paromomycin that is implicated in the discrimination by 30 S subunits of cognate from non-cognate tRNA and the translational misreading caused by paromomycin. Finally, we show that OMe oligonucleotides targeted to the A site are moderately good inhibitors of in vitro translation and that there is a limited correlation of inhibition activity with binding strength to the A site.

Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic "hot spots".

The targeting of RNA for the design of novel anti-viral compounds has until now proceeded largely without incorporating direct input from structure-based design methodology, partly because of lack of structural data, and complications arising from substrate flexibility. We propose a paradigm to explain the physical mechanism for ligand-induced refolding of trans-activation response element (TAR RNA) from human immunodeficiency virus 1 (HIV-1). Based upon Poisson-Boltzmann analysis of the TAR structure, as bound by a peptide derived from the transcriptional activator protein, Tat, our hypothesis shows that two specific electrostatic interactions are necessary to stabilise the conformation. This result contradicts the belief that a single argininamide residue is responsible for stabilising the TAR fold, as well as the conventional wisdom that electrostatic interactions with RNA are non-specific or dominated by phosphates. We test this hypothesis by using NMR and computational methods to model the interaction of a series of novel inhibitors of the in vitro RNA-binding activities for a peptide derived from Tat. A subset of inhibitors, including the bis-guanidine compound rbt203 and its analogues, induce a conformation in TAR similar to that brought about by the protein. Comparison of the interactions of two of these ligands with the RNA and structure-activity relationships observed within the compound series, confirm the importance of the two specific electrostatic interactions in the stabilisation of the Tat-bound RNA conformation. This work illustrates how the use of medicinal chemistry and structural analysis can provide a rational basis for prediction of ligand-induced conformational change, a necessary step towards the application of structure-based methods in the design of novel RNA or protein-binding drugs.

Structural basis for contrasting activities of ribosome binding thiazole antibiotics.

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region. The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin. We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA. The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.

Structure-based design of agents targeting the bacterial ribosome.

Rational structure-based drug design has been applied to the antibiotic thiostrepton, in an attempt to overcome some of its' limitations. The identification of a proposed binding fragment allowed construction of a number of key fragments, which were derivatised to generate a library of potential antibiotics. These were then evaluated to determine their ability to bind to the L11 binding domain of the prokaryotic ribosome and inhibit bacterial protein translation.

The complex of a designer antibiotic with a model aminoacyl site of the 30S ribosomal subunit revealed by X-ray crystallography.

The ribosome is an important target for aminoglycoside antibiotics; however, the clinical effectiveness of aminoglycosides has diminished due to bacterial resistance mechanisms. Here we report the X-ray structure of a novel synthetic aminoglycoside bound to the A site of the ribosome, its target for manifestation of activity. The structure validates the in silico design paradigms for the antibiotic and reveals the molecular interactions made by this novel antibiotic in prokaryotes.

RNA as a drug target.

In the antiviral and antibacterial area, increasing drug resistance means that there is an ever growing need for novel approaches towards structures and mechanisms which avoid the current problems. The huge increase in high resolution structural data is set to make a dramatic impact on targeting RNA as a drug target. The examples of the RNA binding antibiotics, particularly, the totally synthetic oxazolidinones, should help persuade the skceptics that clinically useful, selective drugs can be obtained from targeting RNA directly.