Insights into the Complex Formed by Matrix Metalloproteinase-2 and Alloxan Inhibitors: Molecular Dynamics Simulations and Free Energy Calculations

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S1′ specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S1′ subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC50 = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases

The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene

Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 158,724 bp in size, comprising inverted repeats of 25,925 bp and single-copy regions of 88,576 bp and 18,298 bp. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease comple

Small molecules, big targets: drug discovery faces the protein-protein interaction challenge.

Protein-protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Recent work has begun to show that, with the right tools, certain classes of PPI can yield to the efforts of medicinal chemists to develop inhibitors, and the first PPI inhibitors have reached clinical development. In this Review, we describe the research leading to these breakthroughs and highlight the existence of groups of structurally related PPIs within the PPI target class. For each of these groups, we use examples of successful discovery efforts to illustrate the research strategies that have proved most useful.JS, DES and ARB thank the Wellcome Trust for funding.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nrd.2016.2

Ligand-Dependent Conformations and Dynamics of the Serotonin 5-HT2A Receptor Determine Its Activation and Membrane-Driven Oligomerization Properties

From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization

Correlations between powder wettability and part colour in the High Speed Sintering process

High Speed Sintering (HSS) is a powder bed fusion additive manufacturing technology that relies on inkjet printing of an infrared (IR) radiation absorbing material (RAM) onto a polymer powder bed in order to produce cross-sections which can be selectively sintered using an IR lamp. The RAM used in HSS is carbon black, which is suspended in a petroleum-based carrier fluid to form an ink. The use of carbon black in this way means that any parts currently produced by HSS are inevitably some shade of black or grey depending on the amount and distribution of RAM on the part surface. As well as affecting aesthetics, the effect of printing different densities of RAM has previously been shown to influence part sintering and subsequently mechanical properties, which demonstrates the importance of RAM distribution in determining the final properties of parts made using HSS. Anecdotally, it has also been observed that parts produced with the same type, amount and printed density of ink, but using different polymer powders, can be significantly different colours. This suggests that the wetting interaction between the ink and the polymer powder is also significant factor in determining the distribution of RAM on a part surface. However, to date there has been no investigation into the nature of this relationship. By measuring part colour, in this work we quantify differences in RAM distribution when different polymers are processed via HSS using the same printed density of ink, with parts made from commercially available powders of poly(propylene) (PP), pol(ether-b-amide) (PEBA), poly(styrene) (PS), poly(amide)-11 (PA11), and poly(amide)-12 (PA12) having Relative Luminance (i.e. brightness) of (5.3 0.2)%, (5.5 0.5)%, (12 1)%, (15 2)%, and (18 1)%, respectively. The contact angles of the ink on the powder beds were measured in a simple model system, and these were found to be (46.1 0.8)°, (80 3)°, 82°, (90 2)°, and (93 2)° for PP, PEBA, PS, PA11, and PA12, respectively. With the exception of one anomalous data point for PEBA, it is demonstrated that part Relative Luminance can be correlated to contact angle, and that we can describe that relationship to good precision using a semi-empirical model. Knowledge about the effect of ink wettability on RAM distribution should prove useful for continuing the optimisation of the parts obtained by HSS

Interaction of saddle girth construction and tension on respiratory mechanics and gas exchange during supramaximal treadmill exercise in horses

Objective To determine the effect of girth construction and tension on respiratory mechanics and gas exchange during supramaximal treadmill exercise in horses. Methods Six healthy detrained Thoroughbred horses were exercised on a treadmill inclined at 10% at 110% VO(2)max. Horses were instrumented for respiratory mechanics and gas exchange studies, and data were recorded during incremental exercise tests. The animals were exercised for 2 min at 40% VO(2)max, and samples and measurements were collected at 1 min 45 sec. After 2 min, speed was increased to that estimated at 110% VO(2)max and data was collected at 45 sec, 90 sec and every 30 sec thereafter at this speed until the horses fatigued. Horses were run on three occasions with the same racing saddle and saddle packing but using two different girths, either an elastic girth: (EG) or a standard canvas girth (SCG) which is nonelastic. A run with 5 kg tension applied to a standard canvas girth was the control for each horse, with additional runs at 15 kg using either the standard canvas girth or using the elastic girth, The runs were randomised and tensions applied were measured at end exhalation whilst at rest. Results Increasing girth tension was not associated with changes in respiratory mechanical or gas exchange properties. Although girths tightened to 15 kg tension had short run to fatigue times this was not found to be significantly different to girths set at 5 kg resting tension. Girth tensions declined at end exhalation in horses nearing fatigue. Conclusions Loss in performance associated with high girth tensions is not due to alteration of respiratory mechanics. Loss in performance may be related to inspiratory muscles working at suboptimal lengths due to thoracic compression or compression of musculature around the chest. However, these changes are not reflected in altered respiratory mechanical or gas exchange properties measured during tidal breathing during supramaximal exercise. Other factors may hasten the onset of fatigue when horses exercise with tight girths and further studies are required to determine why excessively tight girths affect performance

Maximum O2 uptake, O2 debt and deficit, and muscle metabolites in Thoroughbred horses.

This study determined maximal O2 uptake (VO2max), maximal O2 deficit, and O2 debt in the Thoroughbred racehorse exercising on an inclined treadmill. In eight horses the O2 uptake (VO2) vs. speed relationship was linear until 10 m/s and VO2max values ranged from 131 to 153 ml.kg-1.min-1. Six of these horses then exercised at 120% of their VO2max until exhaustion. VO2, CO2 production (VCO2), and plasma lactate (La) were measured before and during exercise and through 60 min of recovery. Muscle biopsies were collected before and at 0.25, 0.5, 1, 1.5, 2, 5, 10, 15, 20, 40, and 60 min after exercise. Muscle concentrations of adenosine 5'-triphosphate (ATP), phosphocreatine (PC), La, glucose 6-phosphate (G-6-P), and creatine were determined, and pH was measured. The O2 deficit was 128 +/- 32 (SD) ml/kg (64 +/- 13 liters). The O2 debt was 324 +/- 62 ml/kg (159 +/- 37 liters), approximately two to three times comparative values for human beings. Muscle [ATP] was unchanged, but [PC] was lower (P less than 0.01) than preexercise values at less than or equal to 10 min of recovery. [PC] and VO2 were negatively correlated during both the fast and slow phases of VO2 during recovery. Muscle [La] and [G-6-P] were elevated for 10 min postexercise. Mean muscle pH decreased from 7.05 (preexercise) to 6.75 at 1.5 min recovery, and the mean peak plasma La value was 34.5 mmol/l.(ABSTRACT TRUNCATED AT 250 WORDS

The levonorgestrel intrauterine device in Australia: analysis of prescribing data 2008-2012

BACKGROUND: Unplanned pregnancy is a significant problem in Australia. Local data pertaining to use of the levonorgestrel-releasing intra-uterine device (LNG-IUD), and associated factors are limited. The aim of this analysis was to calculate prescribing rates of the LNG-IUD in Australia, including trends in prescribing and associations with socio-demographic factors, in order to increase understanding regarding potential use. METHODS: We examined prescriptions for the LNG-IUD recorded in the national Pharmaceutical Benefits Scheme (PBS) from 2008 to 2012. Prescribing trends were examined according to patient age, remoteness of residential location, and proximity to relevant specialist health services. Associations between these factors and prescription rates were examined using poisson regression. Analyses were stratified by 5-year age-groups. RESULTS: Age-adjusted prescription rates rose from 11.50 per 1000 women aged 15-49 (95% CI: 11.41-11.59) in 2008 to 15.95 (95% CI:15.85-16.01) in 2012. Prescription rates increased most among 15-19-year-olds but remain very low at 2.76 per 1000 women (95% CI: 2.52-3.01). Absolute increases in prescriptions were greatest among 40-44-year-olds, rising from 16.73 per 1000 women in 2008 (95% CI: 16.12-17.34) to 23.77 in 2012 (95% CI: 22.58-24.29). Rates increased significantly within all geographical locations (p < 0.01). Non-metropolitan location was significantly associated with increased prescribing rates, the association diminishing with increasing age groups. CONCLUSIONS: Prescription of LNG-IUD in Australia is very low, especially among young women and those in major cities. Service providers and young women may benefit from targeted education outlining use of the LNG-IUD, strengthened training and referral pathways. Disparities in prescription according to location require further investigation