Detection and Quantification of Allosteric Modulation of Endogenous M4 Muscarinic Acetylcholine Receptor Using Impedance-Based Label-Free Technology in a Neuronal Cell Line
In this study, we assessed the ability of an impedance-based label-free technology, xCELLigence, to detect allosteric modulation in a neuronal cell line natively expressing rodent M4 muscarinic acetylcholine receptors. We were able to demonstrate that positive allosteric modulation of the endogenous M4 muscarinic acetylcholine receptor can be detected using this technology. Importantly, the allosteric parameters estimated from the label-free approach are comparable to those estimated from endpoint-based assays. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Chen, A. N. Y., Malone, D. T., Pabreja, K., Sexton, P. M., Christopoulos, A., Canals, M. Tags: Original Research Source Type: research
Repurposing of the Open Access Malaria Box for Kinetoplastid Diseases Identifies Novel Active Scaffolds against Trypanosomatids
We describe the screening of the Malaria Box and triaging of the identified hits against kinetoplastids responsible for human African trypanosomiasis (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and visceral leishmaniasis (Leishmania donovani and Leishmania infantum). The in vitro and in vivo profiling of the most promising active compounds with respect to efficacy, toxicity, pharmacokinetics, and complementary druggable properties are presented and a collaborative model used as a way to accelerate the discovery process discussed. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Kaiser, M., Maes, L., Tadoori, L. P., Spangenberg, T., Ioset, J.-R. Tags: Original Research Source Type: research
A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria
The bacterial translational apparatus is an ideal target for the search of new antibiotics. In fact, it performs an essential process carried out by a large number of potential subtargets for antibiotic action. Moreover, it is sufficiently different in several molecular details from the apparatus of Eukarya and Archaea to generally ensure specificity for the bacterial domain. This applies in particular to translation initiation, which is the most different step in the process. In bacteria, the 30S ribosomal subunit directly binds to the translation initiation region, a site within the messenger RNA (mRNA) 5'-untranslated r...
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Raneri, M., Sciandrone, B., Briani, F. Tags: Original Research Source Type: research
A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus
We describe for the first time a cell-based assay that monitors DenV prM processing within the classical secretory pathway, which was exploited to identify a potential novel drug against DenV. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Stolp, Z. D., Smurthwaite, C. A., Reed, C., Williams, W., Dharmawan, A., Djaballah, H., Wolkowicz, R. Tags: Original Research Source Type: research
Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry
HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)–based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured ...
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Meng, J., Lai, M.-T., Munshi, V., Grobler, J., McCauley, J., Zuck, P., Johnson, E. N., Uebele, V. N., Hermes, J. D., Adam, G. C. Tags: Original Research Source Type: research
High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors
Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z' of 0.55 ± 0.08 across 150 assay plat...
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Plant, H., Stacey, C., Tiong-Yip, C.-L., Walsh, J., Yu, Q., Rich, K. Tags: Original Research Source Type: research
Large Scale Meta-Analysis of Fragment-Based Screening Campaigns: Privileged Fragments and Complementary Technologies
A first step in fragment-based drug discovery (FBDD) often entails a fragment-based screen (FBS) to identify fragment "hits." However, the integration of conflicting results from orthogonal screens remains a challenge. Here we present a meta-analysis of 35 fragment-based campaigns at Novartis, which employed a generic 1400-fragment library against diverse target families using various biophysical and biochemical techniques. By statistically interrogating the multidimensional FBS data, we sought to investigate three questions: (1) What makes a fragment amenable for FBS? (2) How do hits from different fragment screening tech...
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Kutchukian, P. S., Wassermann, A. M., Lindvall, M. K., Wright, S. K., Ottl, J., Jacob, J., Scheufler, C., Marzinzik, A., Brooijmans, N., Glick, M. Tags: Original Research Source Type: research
Just-in-Time Compound Pooling Increases Primary Screening Capacity without Compromising Screening Quality
Compound pooling, or multiplexing more than one compound per well during primary high-throughput screening (HTS), is a controversial approach with a long history of limited success. Many issues with this approach likely arise from long-term storage of library plates containing complex mixtures of compounds at high concentrations. Due to the historical difficulties with using multiplexed library plates, primary HTS often uses a one-compound–one-well approach. However, as compound collections grow, innovative strategies are required to increase the capacity of primary screening campaigns. Toward this goal, we have deve...
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Elkin, L. L., Harden, D. G., Saldanha, S., Ferguson, H., Cheney, D. L., Pieniazek, S. N., Maloney, D. P., Zewinski, J., O'Connell, J., Banks, M. Tags: Original Research Source Type: research
Methods for the Creation of Cyclic Peptide Libraries for Use in Lead Discovery
The identification of initial hits is a crucial stage in the drug discovery process. Although many projects adopt high-throughput screening of small-molecule libraries at this stage, there is significant potential for screening libraries of macromolecules created using chemical biology approaches. Not only can the production of the library be directly interfaced with a cell-based assay, but these libraries also require significantly fewer resources to generate and maintain. In this context, cyclic peptides are increasingly viewed as ideal scaffolds and have proven capability against challenging targets such as protein-prot...
Source: Journal of Biomolecular Screening - May 19, 2015 Category: Molecular Biology Authors: Foster, A. D., Ingram, J. D., Leitch, E. K., Lennard, K. R., Osher, E. L., Tavassoli, A. Tags: Review Article Source Type: research
High-Throughput Screening of Formulations to Optimize the Thermal Stability of a Therapeutic Monoclonal Antibody
Monoclonal antibodies (mAbs) are an important class of biotherapeutics. Successful development of a mAb depends not only on its biological activity but also on its physicochemical properties, such as homogeneity and stability. mAb stability is affected by its formulation. Among the many techniques used to study the stability of mAbs, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of the protein unfolding transition (Tm) based on the change in fluorescence intensity of the environmentally sensitive dye SYPRO Orange. With DSF adapted to ...
Source: Journal of Biomolecular Screening - March 24, 2015 Category: Molecular Biology Authors: Niedziela-Majka, A., Kan, E., Weissburg, P., Mehra, U., Sellers, S., Sakowicz, R. Tags: Technical Notes Source Type: research
CHO-S Antibody Titers >1 Gram/Liter Using Flow Electroporation-Mediated Transient Gene Expression followed by Rapid Migration to High-Yield Stable Cell Lines
In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell line...
Source: Journal of Biomolecular Screening - March 24, 2015 Category: Molecular Biology Authors: Steger, K., Brady, J., Wang, W., Duskin, M., Donato, K., Peshwa, M. Tags: Technical Notes Source Type: research
Application of the Mirrorball High-Sensitivity Cytometer to Multiplexed Assays for Antibody Drug Discovery
Highly sensitive, high-throughput assay technologies are required for the identification of antibody therapeutics. Multiplexed assay systems are particularly advantageous because they allow evaluation of several parameters within 1 well, increasing throughput and reducing hands-on laboratory time.
The mirrorball (TTP Labtech), using high-throughput fluorometric microvolume assay technology, offers simultaneous scanning with up to 3 lasers as well as laser scatter detection. This makes the mirrorball especially suitable for the development of highly sensitive and multiplexed assays.
We have developed bead- and cell-based bi...
Source: Journal of Biomolecular Screening - March 24, 2015 Category: Molecular Biology Authors: England, E., Newton, P., Neal, F., Kitching, L., Colley, C., Rossant, C. J. Tags: Technical Notes Source Type: research
Use of the Site-Specific Retargeting Jump-In Platform Cell Line to Support Biologic Drug Discovery
Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this artic...
Source: Journal of Biomolecular Screening - March 24, 2015 Category: Molecular Biology Authors: Butler, R., Hornigold, D., Huang, L., Huntington, C., London, T., Dillon, J., Tigue, N. J., Rossi, A., Naylor, J., Wilkinson, T. Tags: Original Research Source Type: research
Characterization of Bispecific T-cell Engager (BiTE(R)) Antibodies with a High-Capacity T-cell Dependent Cellular Cytotoxicity (TDCC) Assay
We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - March 24, 2015 Category: Molecular Biology Authors: Nazarian, A. A., Archibeque, I. L., Nguyen, Y. H., Wang, P., Sinclair, A. M., Powers, D. A. Tags: Original Research Source Type: research
Versatility of Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assays for Biologics Drug Discovery
Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability ...
Source: Journal of Biomolecular Screening - March 24, 2015 Category: Molecular Biology Authors: Rossant, C. J., Matthews, C., Neal, F., Colley, C., Gardener, M. J., Vaughan, T. Tags: Original Research Source Type: research
