An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format
This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Bergsdorf, C., Fiez-Vandal, C., Sykes, D. A., Bernet, P., Aussenac, S., Charlton, S. J., Schopfer, U., Ottl, J., Duckely, M. Tags: Original Research Source Type: research
Modulation of NF-{kappa}B Signaling as a Therapeutic Target in Autoimmunity
Autoimmune diseases arise from the loss of tolerance to endogenous self-antigens, resulting in a heterogeneous range of chronic conditions that cause considerable morbidity and mortality worldwide. In Western countries, over 5% of the population is affected by some form of autoimmune disease, with enhanced or inappropriate activation of nuclear factor (NF)–B implicated in a number of these conditions. Although treatment strategies for autoimmunity have improved significantly in recent years, current therapeutics are still not capable of achieving satisfactory disease management in all patients, and as such, the thera...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Herrington, F. D., Carmody, R. J., Goodyear, C. S. Tags: Review Article Source Type: research
Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa
In this study, we have developed and optimized a microfluidic online screening technique coupled to nano–liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are ...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Iyer, J. K., Otvos, R. A., Kool, J., Kini, R. M. Tags: Original Research Source Type: research
Target Identification of Compounds from a Cell Viability Phenotypic Screen Using a Bead/Lysate-Based Affinity Capture Platform
The pharmaceutical industry has been continually challenged by dwindling target diversity. To obviate this trend, phenotypic screens have been adopted, complementing target-centric screening approaches. Phenotypic screens identify drug leads using clinically relevant and translatable mechanisms, remaining agnostic to targets. While target anonymity is advantageous early in the drug discovery process, it poses challenges to hit progression, including the development of backup series, retaining desired pharmacology during optimization, discovery of markers, and understanding mechanism-driven toxicity. Consequently, significa...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Tang, H., Duggan, S., Richardson, P. L., Marin, V., Warder, S. E., McLoughlin, S. M. Tags: Original Research Source Type: research
Bioaffinity Mass Spectrometry Screening
Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS or ESI-FTMS) was used to screen 192 natural product extracts and a 659-member natural product-based fragment library for bindings to a potential malaria drug target, Plasmodium falciparum Rab11a (PfRab11a, PF13_0119). One natural product extract and 11 fragments showed binding activity. A new natural product, arborside E, was identified from the active extract of Psydrax montigena as a weak binder. Its binding activity and inhibitory activity against PfRab11a were confirmed by ESI-FTMS titration experiments and an orthogonal e...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Yang, B., Feng, Y. J., Vu, H., McCormick, B., Rowley, J., Pedro, L., Crowther, G. J., Van Voorhis, W. C., Forster, P. I., Quinn, R. J. Tags: Original Research Source Type: research
Exemplifying the Screening Power of Mass Spectrometry Imaging over Label-Based Technologies for Simultaneous Monitoring of Drug and Metabolite Distributions in Tissue Sections
Mass spectrometry imaging (MSI) provides pharmaceutical researchers with a suite of technologies to screen and assess compound distributions and relative abundances directly from tissue sections and offer insight into drug discovery–applicable queries such as blood-brain barrier access, tumor penetration/retention, and compound toxicity related to drug retention in specific organs/cell types. Label-free MSI offers advantages over label-based assays, such as quantitative whole-body autoradiography (QWBA), in the ability to simultaneously differentiate and monitor both drug and drug metabolites. Such discrimination is ...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Goodwin, R. J. A., Nilsson, A., Mackay, C. L., Swales, J. G., Johansson, M. K., Billger, M., Andren, P. E., Iverson, S. L. Tags: Original Research Source Type: research
The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond
Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction–based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography–coupled MS, an analysis time of 8–10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Haslam, C., Hellicar, J., Dunn, A., Fuetterer, A., Hardy, N., Marshall, P., Paape, R., Pemberton, M., Resemannand, A., Leveridge, M. Tags: Original Research Source Type: research
Coupling Laser Diode Thermal Desorption with Acoustic Sample Deposition to Improve Throughput of Mass Spectrometry-Based Screening
The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)–based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)–tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1'-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4'-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless ...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Haarhoff, Z., Wagner, A., Picard, P., Drexler, D. M., Zvyaga, T., Shou, W. Tags: Original Research Source Type: research
Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability
One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and ...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Gordon, L. J., Allen, M., Artursson, P., Hann, M. M., Leavens, B. J., Mateus, A., Readshaw, S., Valko, K., Wayne, G. J., West, A. Tags: Original Research Source Type: research
A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinsons Disease Using RapidFire Mass Spectrometry
LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson’s disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To da...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Leveridge, M., Collier, L., Edge, C., Hardwicke, P., Leavens, B., Ratcliffe, S., Rees, M., Stasi, L. P., Nadin, A., Reith, A. D. Tags: Original Research Source Type: research
High-Throughput Mass Spectrometric Analysis of Covalent Protein-Inhibitor Adducts for the Discovery of Irreversible Inhibitors: A Complete Workflow
We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desaltin...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Campuzano, I. D. G., San Miguel, T., Rowe, T., Onea, D., Cee, V. J., Arvedson, T., McCarter, J. D. Tags: Original Research Source Type: research
Polypharmacology of Small-Molecule Modulators of the 5-Lipoxygenase Activating Protein (FLAP) Observed via a High-throughput Lipidomics Platform
Leukotrienes (LTs) and related species are proinflammatory lipid mediators derived from arachidonic acid (AA) that have pathological roles in autoimmune and inflammatory conditions, cardiovascular diseases, and cancer. 5-Lipoxygenase activating protein (FLAP) plays a critical accessory role in the conversion of AA to LTA4, and its subsequent conversion to LTC4 by LTC4 synthase. Pharmacological inhibition of FLAP results in a loss of LT production by preventing the biosynthesis of both LTB4 and LTC4, making it an attractive target for the treatment of inflammatory diseases in which LTs likely play a role. Small-molecule (SM...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Song, J., Liu, X., Zhu, J., Tootoonchi, M., Keith, J. M., Meduna, S. P., Dvorak, C. A., Eccles, W., Krawczuk, P. J., Blevitt, J. M., Wu, J., Rao, N. L., Lebsack, A. D., Milla, M. E. Tags: Original Research Source Type: research
Identification of DGAT2 Inhibitors Using Mass Spectrometry
Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labe...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Song, X. S., Zhang, J., Chen, X., Palyha, O., Chung, C., Sonatore, L. M., Wilsie, L., Stout, S., McLaren, D. G., Taggart, A., Imbriglio, J. E., Pinto, S., Garcia-Calvo, M., Addona, G. H. Tags: Original Research Source Type: research
Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors
The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography–mass spectroscopy (LCMS) metho...
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Hopcroft, P. J., Fisher, D. I. Tags: Original Research Source Type: research
Advances in Mass Spectrometry Within Drug Discovery
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - January 15, 2016 Category: Molecular Biology Authors: Wingfield, J., Wilson, I. D. Tags: From the Guest Editors Source Type: research
