Structure-based optimisation of orally active & reversible MetAP-2 inhibitors maintaining a tight 'molecular budget'.

Structure-based led optimisation of orally active reversible Methionine Aminopeptidase-2 (MetAP-2) inhibitors utilising a 'molecular budget' medicinal chemistry strategy is described. The key physicochemical parameters of target molecules (cLogP, molecular size and H-bond donor count) were monitored through straightforward and intuitive use of atom count and distribution. The balance between structure-based design and an awareness of the physicochemical properties of the compounds synthesised enabled the rapid identification of a potent molecule with good oral pharmacokinetic (PK) characteristics by making fewer, higher quality compounds. The resulting candidate quality molecule was validated in a mechanistic cellular assay and a rodent secondary immunisation model.

Lymphocyte Activation Gene (LAG)-3 Is Associated With Mucosal Inflammation and Disease Activity in Ulcerative Colitis.

BACKGROUND AND AIMS: Lymphocyte activation gene [LAG]-3 is an immune checkpoint and its expression identifies recently activated lymphocytes that may contribute to inflammation. We investigated the role of LAG-3 by analysing its expression and function in immune cells from blood and tissue of patients with ulcerative colitis [UC]. METHODS: The phenotypic properties of LAG-3+ T cells were determined by flow cytometry, qRT-PCR and single-cell RNA-sequencing. LAG-3+ cells were quantified and correlated with disease activity. The functional effects of LAG-3+ cells were tested using a depleting anti-LAG-3 monoclonal antibody [mAb] in a mixed lymphocyte reaction [MLR]. RESULTS: LAG-3+ cells in the blood were negligible. LAG-3+ lymphocytes were markedly increased in inflamed mucosal tissue and both frequencies of LAG-3+ T cells and transcript levels of LAG3 correlated with endoscopic severity. LAG-3 expression was predominantly on effector memory T cells, and single-cell RNA-sequencing revealed LAG3 expression in activated and cytokine-producing T cell subsets. Foxp3+CD25hi Tregs also expressed LAG-3, although most mucosal Tregs were LAG-3-. Mucosal LAG-3+ cells produced mainly interferon γ [IFNγ] and interleukin-17A. LAG-3+ cell numbers decreased in patients who responded to biologics, and remained elevated in non-responders. Treatment with a depleting anti-LAG-3 mAb led to a reduction in proliferation and IFNγ production in an MLR. CONCLUSIONS: LAG-3+ cells are increased in the inflamed mucosa, predominantly on effector memory T cells with an activated phenotype and their cell numbers positively correlate with disease activity. Depleting LAG-3 eliminates activated proliferating T cells, and hence LAG-3 could be a therapeutic target in UC.

Target engagement and cellular fate of otelixizumab: a repeat dose escalation study of an anti-CD3ε mAb in new-onset type 1 diabetes mellitus patients.

AIMS: This paper describes the pharmacological findings from a study where otelixizumab, an anti-CD3ɛ mAb, was dosed in new onset Type 1 diabetes mellitus (NOT1DM) patients. This is the first time that the full dose-response of an anti-CD3ɛ mAb has been investigated in the clinic. The data have been validated using a previously developed pharmacokinetic/pharmacodynamic (PK/PD) model of otelixizumab to simulate the interplay between drug administration, CD3ɛ target engagement and downmodulation. METHODS: Patients were randomized to control or active treatment with otelixizumab (1:4), administered via infusion over 6 days, in a dose-ascending study consisted of three cohorts (n = 10 per cohort) at doses of 9, 18 or 27 mg respectively. The study allowed quantification of otelixizumab PK, CD3ɛ target engagement and its pharmacodynamic effect (CD3ε/TCR modulation on circulating T lymphocytes). RESULTS: Otelixizumab concentrations increased and averaged to 364.09 (54.3), 1625.55 (72.5) and 2781.35 (28.0) ng ml-1 (Geom.mean, %CV) at the 9, 18 and 27 mg dose respectively. CD3ɛ target engagement was found to be rapid (within the first 30 min), leading to a receptor occupancy of ~60% within 6 h of dosing in all three doses. A dose-response relationship was observed with the two highest doses achieving a ~90% target engagement and consequential CD3ɛ/TCR downmodulation by Day 6. CONCLUSIONS: Data from this study revealed maximum target engagement and CD3ɛ/TCR modulation is achieved at doses of 18, 27 mg of otelixizumab. These findings can be useful in guiding dose selection in clinical trials with anti-CD3ɛ mAbs.

Temporal pharmacokinetic/pharmacodynamic interaction between human CD3ε antigen-targeted monoclonal antibody otelixizumab and CD3ε binding and expression in human peripheral blood mononuclear cell static culture.

Otelixizumab is a monoclonal antibody (mAb) directed to human CD3ε, a protein forming part of the CD3/T-cell receptor (TCR) complex on T lymphocytes. This study investigated the temporal interaction between varying concentrations of otelixizumab, binding to human CD3 antigen, and expression of CD3/TCR complexes on lymphocytes in vitro, free from the confounding influence of changing lymphocyte frequencies observed in vivo. A static in vitro culture system was established in which primary human peripheral blood mononuclear cells (PBMCs) were incubated over an extended time course with titrated concentrations of otelixizumab. At each time point, free, bound, and total CD3/TCR expression on both CD4+ and CD8+ T cells and the amount of free otelixizumab antibody in the supernatant were measured. The pharmacokinetics of free otelixizumab in the culture supernatants was saturable, with a shorter apparent half-life at low concentration. Correspondingly, a rapid, otelixizumab concentration-, and time-dependent reduction in CD3/TCR expression was observed. These combined observations were consistent with the phenomenon known as target-mediated drug disposition (TMDD). A mechanistic, mathematical pharmacokinetic/pharmacodynamic (PK/PD) model was then used to characterize the free otelixizumab-CD3 expression-time relationship. CD3/TCR modulation induced by otelixizumab was found to be relatively fast compared with the re-expression rate of CD3/TCR complexes following otelixizumab removal from supernatants. In summary, the CD3/TCR receptor has been shown to have a major role in determining otelixizumab disposition. A mechanistic PK/PD model successfully captured the PK and PD in vitro data, confirming TMDD by otelixizumab.

Alpha-ethyltryptamines as dual dopamine-serotonin releasers.

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.

Hybrid dopamine uptake blocker-serotonin releaser ligands: a new twist on transporter-focused therapeutics.

As part of our program to study neurotransmitter releasers, we report herein a class of hybrid dopamine reuptake inhibitors that display serotonin releasing activity. Hybrid compounds are interesting since they increase the design potential of transporter related compounds and hence represent a novel and unexplored strategy for therapeutic drug discovery. A series of N-alkylpropiophenones was synthesized and assessed for uptake inhibition and release activity using rat brain synaptosomes. Substitution on the aromatic ring yielded compounds that maintained hybrid activity, with the two disubstituted analogues (PAL-787 and PAL-820) having the most potent hybrid activity.

A CD3-specific antibody reduces cytokine production and alters phosphoprotein profiles in intestinal tissues from patients with inflammatory bowel disease.

BACKGROUND & AIMS: T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients. METHODS: Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls. Isolated lamina propria mononuclear cells (LPMCs) and mucosal tissue explants were incubated with otelixizumab for 24 or 48 hours. Production of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. Levels of 36 cytokines and chemokines and phosphorylation of 39 receptor tyrosine kinases and signaling molecules were measured using protein arrays. Immunoblot analysis was used to analyze T-cell transcription factors. RESULTS: Incubation of intestinal tissues or LPMCs with otelixizumab reduced production of interferon gamma, interleukin (IL)-17A, and other inflammatory cytokines and chemokines, simultaneously increasing production of IL-10. Mucosal biopsy specimens from patients with IBD retained inflammation-associated tyrosine phosphoprotein profiles ex vivo. Incubation of the inflamed tissue with otelixizumab reduced phosphorylation of these proteins to levels observed in control tissues. Otelixizumab also markedly reduced phosphorylation of proteins associated with T-cell receptor activation. Neutralization of IL-10 blocked the anti-inflammatory effects of otelixizumab. CONCLUSIONS: We observed anti-inflammatory effects of anti-CD3 in inflamed intestinal tissues from patients with IBD. The antibody appears to down-regulate T-cell activation via IL-10.

A semantic sensor web for environmental decision support applications.

Sensing devices are increasingly being deployed to monitor the physical world around us. One class of application for which sensor data is pertinent is environmental decision support systems, e.g., flood emergency response. For these applications, the sensor readings need to be put in context by integrating them with other sources of data about the surrounding environment. Traditional systems for predicting and detecting floods rely on methods that need significant human resources. In this paper we describe a semantic sensor web architecture for integrating multiple heterogeneous datasets, including live and historic sensor data, databases, and map layers. The architecture provides mechanisms for discovering datasets, defining integrated views over them, continuously receiving data in real-time, and visualising on screen and interacting with the data. Our approach makes extensive use of web service standards for querying and accessing data, and semantic technologies to discover and integrate datasets. We demonstrate the use of our semantic sensor web architecture in the context of a flood response planning web application that uses data from sensor networks monitoring the sea-state around the coast of England.

An e-Research approach to Web-scale music analysis.

The growing quantity of digital recorded music available in large-scale resources such as the Internet archive provides an important new resource for musical analysis. An e-Research approach has been adopted in order to create a very substantive web-accessible corpus of musical analyses in a common framework for use by music scholars, students and beyond, and to establish a methodology and tooling that will enable others to add to the resource in the future. The enabling infrastructure brings together scientific workflow and Semantic Web technologies with a set of algorithms and tools for extracting features from recorded music. It has been used to deliver a prototype system, described here, that demonstrates the utility of LINKED DATA for enhancing the curation of collections of music signal data for analysis and publishing results that can be simply and readily correlated to these and other sources. This paper describes the motivation, infrastructure design and the proof-of-concept case study and reflects on emerging e-Research practice as researchers embrace the scale of the Web.

Ligand modulation of the Epstein-Barr virus-induced seven-transmembrane receptor EBI2: identification of a potent and efficacious inverse agonist.

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding assays, the potency of this compound was 2.6-53.6 nm, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe(111) at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC(50). In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.

Synthesis and monoamine transporter binding properties of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes.

A series of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2beta-[3'-(substituted benzyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (3a-h) showed high binding affinities for the dopamine transporter (DAT). The IC(50) values ranged from 5.9 to 22nM. On the other hand, the 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (4a-h), with IC(50) values ranging from 65 to 173nM, were approximately 3- to 25-fold less potent than the corresponding 2beta-[3'-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3Beta-(4-Methylphenyl)-2beta-[3'-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC(50) of 5.9nM at the DAT and K(i)s of 454 and 113nM at the NET and 5-HTT, respectively, was the most potent and DAT-selective analog. Molecular modeling studies suggested that the rigid conformation of the isoxazole side chain in 4a-h might play an important role on their low DAT binding affinities.

Conformationally constrained analogues of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716): design, synthesis, computational analysis, and biological evaluations.

Structure-activity relationships (SARs) of 1 (SR141716) have been extensively documented, however, the conformational properties of this class have received less attention. In an attempt to better understand ligand conformations optimal for receptor recognition, we have designed and synthesized a number of derivatives of 1, including a four-carbon-bridged molecule (11), to constrain rotation of the diaryl rings. Computational analysis of 11 indicates approximately 20 kcal/mol energy barrier for rotation of the two aryl rings. NMR studies have determined the energy barrier to be approximately 18 kcal/mol and suggested atropisomers could exist. Receptor binding and functional studies with these compounds displayed reduced affinity and potency when compared to 1. This indicates that our structural modifications either constrain the ring systems in a suboptimal orientation for receptor interaction or the introduction of steric bulk leads to disfavored steric interactions with the receptor, and/or the relatively modest alterations in the molecular electrostatic potentials results in disfavored Coulombic interactions.

Effects of coancestry on accuracy of individual assignments to population of origin: examples using Great Lakes lake trout (Salvelinus namaycush).

Methods for assigning individuals to population of origin are widely used in ecological genetics, resources management, and forensics. Characteristics of genetic data obtained from putative source populations that enhance accuracy of assignment are well established. How non-independence within and among unknown individuals to be classified [i.e., gene correlations within individual (inbreeding) and gene correlations among individuals within group (coancestry)] affect assignment accuracy is poorly understood. We used empirical data for six microsatellite loci and offspring from full-sib crosses of hatchery strains of lake trout (Salvelinus namaycush; Salmonidae) representing known levels of coancestry (mean theta = 0.006 and 0.06) within families to investigate how gene correlations can affect assignment. Additional simulations were conducted to further investigating the influence of allelic diversity (2, 6 or 10 alleles per locus) and inbreeding (F = 0.00, 0.05, and 0.15) on assignment accuracy for cases of low and high inter-population variance in allele frequency (mean F (st) = 0.01 and 0.1, respectively). Inbreeding had no effect on accuracy of assignments. In contrast, variance in assignment accuracy across replicated simulations, and for each empirical case study increased with increasing coancestry, reflecting non-independence of probabilities of correct assignment among members of kin groups. Empirical estimates of assignment error rates should be interpreted with caution if appreciable levels of coancestry are suspected. Additional emphasis should be placed on sampling designs (spatially and temporally) that define or minimize the potential for sampling related individuals.

Conformational characteristics of the interaction of SR141716A with the CB1 cannabinoid receptor as determined through the use of conformationally constrained analogs.

Interest in cannabinoid pharmacology increased dramatically upon the identification of the first cannabinoid receptor (CB1) in 1998 and continues to expand as additional endocannabinoids and cannabinoid receptors are discovered. Using CB1 receptor (CB1R) systems, medicinal chemistry programs began screening libraries searching for cannabinoid ligands, ultimately leading to the discovery of the first potent cannabinoid receptor antagonist, SR141716A (Rimonabant). Its demonstrated efficacy in treating obesity and facilitating smoking cessation, among other impressive pharmacological activities, has furthered the interest in cannabinoid receptor antagonists as therapeutics, such that the number of patents and publications covering this class of compounds continues to grow at an impressive rate. At this time, medicinal chemistry approaches including combinatorial chemistry, conformational constraint, and scaffold hopping are continuing to generate a large number of cannabinoid antagonists. These molecules provide an opportunity to gain insight into the 3-dimensional structure-activity relationships that appear crucial for CB1R-ligand interaction. In particular, studies in which conformational constraints have been imposed on the various pyrazole ring substituents of SR141716A provide a direct opportunity to characterize changes in conformation/conformational freedom within a single class of compounds. While relatively few conformationally constrained molecules have been synthesized to date, the structure-activity information is often more readily interpreted than in studies where entire substituents are replaced. Thus, it is the focus of this mini-review to examine the structural properties of SR141716A, and to use conformationally constrained molecules to illustrate the importance of conformation and conformational freedom to CB1R affinity, selectivity, and efficacy.

Modification of the gold-weight implant for insertion into the upper eyelid in facial palsy.

Gold weight has been used as a lid-loading device in facial palsy for more than 50 years. This technique is frequently associated with certain complications like ulceration, migration, extrusion, etc. We have devised a new-shaped gold weight to reduce the rate of occurrence of these complications. This new implant has a large central fenestration. We believe that this shape reduces anterior surface contact and promotes fibrous healing through fenestration. This will keep the implant anchored in its position, hence, reducing the risk of ulceration, migration, and extrusion. We have been using this kind of implant for the last 2 years without any complication noted so far.