Inhibitor screening assay for neurexin-LRRTM adhesion protein interaction involved in synaptic maintenance and neurological disorders.

Synaptic adhesion molecules, including presynaptic neurexins (NRXNs) and post-synaptic leucine-rich repeat transmembrane (LRRTM) proteins are important for development and maintenance of brain neuronal networks. NRXNs are probably the best characterized synaptic adhesion molecules, and one of the major presynaptic organizer proteins. The LRRTMs were found as ligands for NRXNs. Many of the synaptic adhesion proteins have been linked to neurological cognitive disorders, such as schizophrenia and autism spectrum disorders, making them targets of interest for both biological studies, and towards drug development. Therefore, we decided to develop a screening method to target the adhesion proteins, here the LRRTM-NRXN interaction, to find small molecule probes for further studies in cellular settings. To our knowledge, no potent small molecule compounds against the neuronal synaptic adhesion proteins are available. We utilized the AlphaScreen technology, and developed an assay targeting the NRXN-LRRTM2 interaction. We carried out screening of 2000 compounds and identified hits with moderate IC50-values. We also established an orthogonal in-cell Western blot assay to validate hits. This paves way for future development of specific high affinity compounds by further high throughput screening of larger compound libraries using the methods established here. The method could also be applied to screening other NRXN-ligand interactions.Peer reviewe

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 50-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe

The effects of glare and inhomogeneous visual fields on contrast detection in the context of driving

An experiment was carried out to investigate how contrast threshold for target detection is affected by the presence of glare and by extraneous light sources using the method of ascending limits. The target was located at either a foveal or a peripheral (10° right) location, glare was adjacent to the foveal location, simulating the headlamps of an oncoming vehicle, and extraneous light sources were at either foveal or peripheral (10° right or left) locations. Contrast threshold for a foveal target without glare was affected mainly by the surrounding local luminance distribution. However, in the presence of glare and also for the peripheral target (both with and without glare) the global luminance distribution matters. Glare increased the contrast needed for detection of the foveal target, but this effect was reduced by the presence of extraneous light sources that were peripheral to the target. For peripheral targets, contrast threshold was also reduced by the presence of extraneous light at a non-target location and this effect was increased in the presence of glare. Glare equations tend to be based on tests using uniform, homogenous fields: these data indicate that, in the presence of extraneous light sources, the influence of glare is over-estimated

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Inhibitor screening assay for neurexin-LRRTM adhesion protein interaction involved in synaptic maintenance and neurological disorders

Abstract Synaptic adhesion molecules, including presynaptic neurexins (NRXNs) and post-synaptic leucine-rich repeat transmembrane (LRRTM) proteins are important for development and maintenance of brain neuronal networks. NRXNs are probably the best characterized synaptic adhesion molecules, and one of the major presynaptic organizer proteins. The LRRTMs were found as ligands for NRXNs. Many of the synaptic adhesion proteins have been linked to neurological cognitive disorders, such as schizophrenia and autism spectrum disorders, making them targets of interest for both biological studies, and towards drug development. Therefore, we decided to develop a screening method to target the adhesion proteins, here the LRRTM-NRXN interaction, to find small molecule probes for further studies in cellular settings. To our knowledge, no potent small molecule compounds against the neuronal synaptic adhesion proteins are available. We utilized the AlphaScreen technology, and developed an assay targeting the NRXN-LRRTM2 interaction. We carried out screening of 2000 compounds and identified hits with moderate IC₅₀-values. We also established an orthogonal in-cell Western blot assay to validate hits. This paves way for future development of specific high affinity compounds by further high throughput screening of larger compound libraries using the methods established here. The method could also be applied to screening other NRXN-ligand interactions

Multiple crystal forms of human MacroD2

Abstract MacroD2 is one of the three human macrodomain proteins characterized by their protein-linked mono-ADP-ribosyl-hydrolyzing activity. MacroD2 is a single-domain protein that contains a deep ADP-ribose-binding groove. In this study, new crystallization conditions for MacroD2 were found and three crystal structures of human MacroD2 in the apo state were solved in space groups P4₁2₁2, P43212 and P4₃, and refined at 1.75, 1.90 and 1.70 Å resolution, respectively. Structural comparison of the apo crystal structures with the previously reported crystal structure of MacroD2 in complex with ADP-ribose revealed conformational changes in the side chains of Val101, Ile189 and Phe224 induced by the binding of ADP-ribose in the active site. These conformational variations may potentially facilitate design efforts of a MacroD2 inhibitor

Crystal structures of Trichoderma reesei beta-galactosidase reveal conformational changes in the active site.

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2Å resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4Å resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain

Development of an inhibitor screening assay for mono-ADP-ribosyl hydrolyzing macrodomains using AlphaScreen technology

Abstract Protein mono-ADP-ribosylation is a posttranslational modification involved in the regulation of several cellular signaling pathways. Cellular ADP-ribosylation is regulated by ADP-ribose hydrolases via a hydrolysis of the protein-linked ADP-ribose. Most of the ADP-ribose hydrolases share a macrodomain fold. Macrodomains have been linked to several diseases, such as cancer, but their cellular roles are mostly unknown. Currently, there are no inhibitors available targeting the mono-ADP-ribose hydrolyzing macrodomains. We have developed a robust AlphaScreen assay for the screening of inhibitors against macrodomains having mono-ADP-ribose hydrolysis activity. We utilized this assay for validatory screening against human MacroD1 and identified five compounds inhibiting the macrodomain. Dose–response measurements and an orthogonal assay further validated four of these compounds as MacroD1 inhibitors. The developed assay is homogenous, easy to execute, and suitable for the screening of large compound libraries. The assay principle can also be adapted for other ADP-ribose hydrolyzing macrodomains, which can utilize a biotin-mono-ADP-ribosylated protein as a substrate

Approaches for Mapping Night-Time Road Environment Lighting Conditions

The integration of the 3D measurement techniques with luminance imaging has increased the potential for mapping night-time road lighting conditions. In this study, we present selected static and mobile approaches for the purpose. The measurement methods include conventional 2D imaging luminance photometry and the integration of the luminance imaging with terrestrial and mobile laser scanning. In addition, we present our initial experiences with performing integrated luminance mapping and photogrammetric reconstruction from drone imagery. All of the presented methods require that the camera is calibrated with a reference luminance source. Our results show the results of luminance calibration and feasibility of 3D luminance point clouds for evaluating road surface luminances. In addition, we discuss the other potential applications, limitations and future research.Peer reviewe

Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition

Abstract Human PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5′-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 in complex with an activating 5′-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD⁺ and histone PARylation factor 1 that changes PARP2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation