High-throughput screening platforms in the discovery of novel drugs for neurodegenerative diseases

© 2021 The Authors. Published by MDPI. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3390/bioengineering8020030Neurodegenerative diseases (NDDs) are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells in the central nervous system (CNS). Identification of viable therapeutic targets and new treatments for CNS disorders and in particular, for NDDs is a major challenge in the field of drug discovery. These difficulties can be attributed to the diversity of cells involved, extreme complexity of the neural circuits, the limited capacity for tissue regeneration, and our incomplete understanding of the underlying pathological processes. Drug discovery is a complex and multidisciplinary process. The screening attrition rate in current drug discovery protocols mean that only one viable drug may arise from millions of screened compounds resulting in the need to improve discovery technologies and protocols to address the multiple causes of attrition. This has identified the need to screen larger libraries where the use of efficient high-throughput screening (HTS) becomes key in the discovery process. HTS can investigate hun-dreds of thousands of compounds per day. However, if fewer compounds could be screened without compromising the probability of success, the cost and time would be largely reduced. To that end, recent advances in computer-aided design, in silico libraries, and molecular docking software combined with the upscaling of cell-based platforms have evolved to improve screening efficiency with higher predictability and clinical applicability. We review, here, the increasing role of HTS in contemporary drug discovery processes, in particular for NDDs, and evaluate the criteria underlying its successful application. We also discuss the requirement of HTS for novel NDD therapies and examine the major current challenges in validating new drug targets and developing new treatments for NDDs.Published versio

Recruitment of the Oncoprotein v-ErbA to Aggresomes

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T3-dependent TRα1 clearance. Our studies highlight v-ErbA\u27s complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments

Rotational Broadening and Doppler Tomography of the Quiescent X-Ray Nova Centaurus X-4

We present high and intermediate resolution spectroscopy of the X-ray nova Centaurus X-4 during its quiescent phase. Our analysis of the absorption features supports a K3-K5V spectral classification for the companion star, which contributes approximately 75 % of the total flux at Halpha. Using the high resolution spectra we have measured the secondary star's rotational broadening to be V_rot*sin(i) = 43 +/- 6 km/s and determined a binary mass ratio of q=0.17 +/- 0.06. Combining our results for K_2 and q with the published limits for the binary inclination, we constrain the mass of the compact object and the secondary star to the ranges 0.49 < M_1 < 2.49 Msun and 0.04 < M_2 < 0.58 Msun. A Doppler image of the Halpha line shows emission coming from the secondary star, but no hotspot is present. We discuss the possible origins of this emission.Comment: 10 pages, 4 figures, accepted by MNRA

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function

The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca 2+ i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca 2+ i release than GLP-1(7-36)-NH 2, providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies

Range-Wide Declines of Northern Spotted Owl Populations in the Pacific Northwest: A Meta-Analysis

The northern spotted owl (Strix occidentalis caurina) inhabits older coniferous forests in the Pacific Northwest and has been at the center of forest management issues in this region. The immediate threats to this federally listed species include habitat loss and competition with barred owls (Strix varia), which invaded from eastern North America. We conducted a prospective meta-analysis to assess population trends and factors affecting those trends in northern spotted owls using 26 years of survey and capture-recapture data from 11 study areas across the owls\u27 geographic range to analyze demographic traits, rates of population change, and occupancy parameters for spotted owl territories. We found that northern spotted owl populations experienced significant declines of 6–9% annually on 6 study areas and 2–5% annually on 5 other study areas. Annual declines translated to ≤35% of the populations remaining on 7 study areas since 1995. Barred owl presence on spotted owl territories was the primary factor negatively affecting apparent survival, recruitment, and ultimately, rates of population change. Analysis of spotted and barred owl detections in an occupancy framework corroborated the capture-recapture analyses with barred owl presence increasing territorial extinction and decreasing territorial colonization of spotted owls. While landscape habitat components reduced the effect of barred owls on these rates of decline, they did not reverse the negative trend. Our analyses indicated that northern spotted owl populations potentially face extirpation if the negative effects of barred owls are not ameliorated while maintaining northern spotted owl habitat across their range

Predicting Physical Time Series Using Dynamic Ridge Polynomial Neural Networks

Forecasting naturally occurring phenomena is a common problem in many domains of science, and this has been addressed and investigated by many scientists. The importance of time series prediction stems from the fact that it has wide range of applications, including control systems, engineering processes, environmental systems and economics. From the knowledge of some aspects of the previous behaviour of the system, the aim of the prediction process is to determine or predict its future behaviour. In this paper, we consider a novel application of a higher order polynomial neural network architecture called Dynamic Ridge Polynomial Neural Network that combines the properties of higher order and recurrent neural networks for the prediction of physical time series. In this study, four types of signals have been used, which are; The Lorenz attractor, mean value of the AE index, sunspot number, and heat wave temperature. The simulation results showed good improvements in terms of the signal to noise ratio in comparison to a number of higher order and feedforward neural networks in comparison to the benchmarked techniques

Measuring health and broader well-being benefits in the context of opiate dependence: the psychometric performance of the ICECAP-A and the EQ-5D-5L

Background Measuring outcomes in economic evaluations of social care interventions is challenging because both health and well-being benefits are evident. The ICEpop CAPability instrument for adults (ICECAP-A) and the five-level EuroQol five-dimensional questionnaire (EQ-5D-5L) are measures potentially suitable for the economic evaluation of treatments for substance use disorders. Evidence for their validity in this context is, however, lacking. Objectives To assess the construct validity of the ICECAP-A and the EQ-5D-5L in terms of convergent and discriminative validity and sensitivity to change on the basis of standard clinical measures (Clinical Outcomes in Routine Evaluation-Outcome Measure, Treatment Outcomes Profile, Interpersonal Support Evaluation List, Leeds Dependence Questionnaire, and Social Satisfaction Questionnaire). Methods A secondary analysis of pilot trial data for heroin users in opiate substitution treatment was conducted. Baseline convergence with clinical measures was assessed using the Pearson correlation coefficient. Discriminative validity was assessed using one-way analysis of variance and stepwise regressions. Sensitivity to changes in clinical indicators was assessed at 3 and 12 months using the standardized response mean statistic and parametric and nonparametric testing. Results Both measures had the same level of construct validity, except for clinical indicators of well-being, for which the ICECAP-A performed better. The ICECAP-A was sensitive to changes in both health and well-being indicators. The EQ-5D-5L had lower levels of sensitivity to change, and a ceiling effect (27%), particularly evident in the dimensions of self-care (89%), mobility (75%), and usual activities (72%). Conclusions The findings support the construct validity of both measures, but the ICECAP-A gives more attention to broader impacts and is more sensitive to change. The ICECAP-A shows promise in evaluating treatments for substance use disorders for which recovery is the desired outcome

Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor

Class B GPCRs can activate multiple signalling effectors with the potential to exhibit biased agonism in response to ligand stimulation. Previously, we highlighted key TM domain polar amino acids that were crucial for the function of the GLP-1 receptor, a key therapeutic target for diabetes and obesity. Using a combination of mutagenesis, pharmacological characterisation, mathematical and computational molecular modelling, this study identifies additional highly conserved polar residues located towards the TM helical boundaries of Class B GPCRs that are important for GLP-1 receptor stability and/or controlling signalling specificity and biased agonism. This includes (i) three positively charged residues (R3.30227, K4.64288, R5.40310) located at the extracellular boundaries of TMs 3, 4 and 5 that are predicted in molecular models to stabilise extracellular loop 2, a crucial domain for ligand affinity and receptor activation; (ii) a predicted hydrogen bond network between residues located in TMs 2 (R2.46176), 6 (R6.37348) and 7 (N7.61406 and E7.63408) at the cytoplasmic face of the receptor that is important for stabilising the inactive receptor and directing signalling specificity, (iii) residues at the bottom of TM 5 (R5.56326) and TM6 (K6.35346 and K6.40351) that are crucial for receptor activation and downstream signalling; (iv) residues predicted to be involved in stabilisation of TM4 (N2.52182 and Y3.52250) that also influence cell signalling. Collectively, this work expands our understanding of peptide-mediated signalling by the GLP-1 receptor