Inhibition of O-acetylserine sulfhydrylase by fluoroalanine derivatives

O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.     

Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions

Delineating design principles of biological systems by reconstitution of purified components offers a platform to gauge the influence of critical physicochemical parameters on minimal biological systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymatic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films containing immobilized substrates and reversible inhibitors. Quantitative fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addition of strong inhibitors, consistent with a mathematical model of molecular titration. The results confirm that ultrasensitive and threshold effects at the molecular level can convert a graded input signal to a steep spatial response at macroscopic length scales.     

Ischemia induces aggravation of baseline repolarization abnormalities in left ventricular hypertrophy: a deleterious interaction.

Epidemiological studies show that left ventricular hypertrophy (LVH) and hypertension (HT) in coronary artery disease increases the risk for cardiovascular events including sudden cardiac death (SCD). According to experimental studies, myocardial hypertrophy is associated both with altered electrophysiological properties (including prolonged repolarization) and increased vulnerability to ischemia. However, human data to support a repolarization-related mechanism for the increased SCD risk has not been provided. We therefore studied 187 patients undergoing three-dimensional vectorcardiographic monitoring during coronary angioplasty. Eight parameters reflecting different aspects of ventricular repolarization were used: 1) the ST segment (ST-VM and STC-VM), 2) the T vector (QRS-T angle, Televation, and Tazimuth), and 3) the T vector loop (Tavplan, Teigenv, and Tarea). Data collection was performed at rest and at the time of maximum ischemia during coronary occlusion. The patients were divided into three groups: 33 patients with ECG signs of LVH (18 with HT), 54 with HT but without LVH signs, and 100 patients with neither. Coronary artery disease patients with LVH not only had the most abnormal baseline repolarization (as expected) but also a significantly more pronounced repolarization response during coronary occlusion, whereas HT patients had mean parameter values between LVH patients and those without neither HT nor LVH signs. Because there is a relation between increased SCD risk and repolarization disturbances in various clinical settings, the results of the present study are in agreement with animal data and epidemiological observations, although other factors than disturbed repolarization might be of importance.     

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.     

Biomechanical properties of oesophagus wall under loading

In this investigation, firstly, the biomechanical properties of different parts of oesophagus were determined. Oesophagus stress and strain are the greatest in the cervical part for all age groups. The human oesophagus deforms unevenly, depending on the direction of load in relation to the organ's axis, it exhibits anisotropical behaviour. With the age the values of mechanical parameters of the oesophagus wall reduce, in particular beginning from 45 years of age, but the modulus of elasticity increases. Biomechanical properties of the oesophagus depend on the architecture of its structure. By loading the organ in the circumferential direction, microfibrilae rupture and deformation of the muscular fibres occurs. With increase of load, collagenous fibres straighten and microruptures in collagenous fibrilae occur. With stretching of oesophagus longitudinally, collagenous fibres partially preserve their wavy and helical configuration. Therefore, higher resistance of the oesophageal wall occurs in the longitudinal direction.     

Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions

Delineating design principles of biological systems by reconstitution of purified components offers a platform to gauge the influence of critical physicochemical parameters on minimal biological systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymatic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films containing immobilized substrates and reversible inhibitors. Quantitative fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addition of strong inhibitors, consistent with a mathematical model of molecular titration. The results confirm that ultrasensitive and threshold effects at the molecular level can convert a graded input signal to a steep spatial response at macroscopic length scales.     

Decreased Silica Land–sea Fluxes through Damming in the Baltic Sea Catchment – Significance of Particle Trapping and Hydrological Alterations

We tested the hypothesis that reservoirs with low water residence time and autochthonous production influence river biogeochemistry in eutrophied river systems draining cultivated watersheds. The effect of a single artificial water reservoir and consecutive reservoirs on silica (Si) river fluxes is exemplified by the moderately dammed Vistula River and the heavily regulated Daugava River that are compared with the practically undammed Oder River. The sum of the discharge weighted annual mean biogenic silica (BSi) and dissolved silicate (DSi) concentrations in the rivers Oder, Vistula and Daugava were about 160 μ M (40 + 120 μ M), 150 μ M (20 + 130 μ M) and 88 μ M (6 + 82 μ M), respectively. Assuming BSi and DSi concentrations as observed in the Oder River as typical for eutrophied but undammed rivers, complete trapping of this BSi could have lowered Si fluxes to the Baltic Sea from rivers with cultivated watersheds by 25%. The superimposed effect of hydrological alterations on reduced Si land–sea fluxes is demonstrated by studies in the boreal/subarctic and oligotrophic rivers Kalixälven and Lueälven. The DSi yield of the heavily dammed Luleälven (793 kg km^?2 yr^?1) constituted only 63% of that was found in the unregulated Kalixälven (1261 kg km^?2 yr^?1), despite the specific runoff of the Luleälven (672 mm m^?2 yr^?1) being 19% higher than that of theKalixälven (563 mm m^?2 yr^?1); runoff normalized DSi yield of the former, regulated watershed, was only half the DSi yield of the latter, unperturbed watershed. Based on these findings, it is hypothesized here that perturbed surface water–groundwater interactions are the major reasons for the reduced annual fluctuations in DSi concentrations as also seen in the heavily dammed and eutrophic river systems such as the Daugava and Danube.     

2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved hydrophilicity and selectivity

2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective leads for antimalarial drug development targeting the plasmepsins. Here we report the lead optimization studies with the aim to reduce inhibitor lipophilicity and increase selectivity versus the human aspartic protease Cathepsin D. Exploiting the solvent exposed area of the enzyme provides an option to install polar groups (R¹) the 5-position of 2-aminoquinazolin-4(3H)-one to inhibitors such as carboxylic acid without scarifying enzymatic potency. Moreover, introduction of R¹ substituents increased selectivity factors of compounds in this series up to 100-fold for Plm II, IV vs CatD inhibition. The introduction of flap pocket substituent (R²) at 7-postion of 2-aminoquinazolin-4(3H)-one allows to remove Ph group from THF ring without notably impairing Plm inhibitory potency. Based on these findings, inhibitors were developed, which show Plm II and IV inhibitory potency in low nanomolar range and remarkable selectivity against Cathepsin D along with decreased lipophilicity and increased solubility.