A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction

Benicchi, Tiziana; Iozzi, Sara; Svahn, Andreas; Axelsson, Hanna; Mori, Elisa; Bernocco, Simonetta; Cappelli, Federico; Caramelli, Chiara; Fanti, Paola; Genesio, Eva; Maccari, Laura; Markova, Natalia; Micco, Iolanda; Porcari, Valentina; Schultz, Johan; Fecke, Wolfgang

doi:10.1177/1087057112447873

Cited by 11 publications

(7 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In regard to the first strategy, a high throughput screen measuring the effect of ~60,000 compounds on soluble TWEAK binding to the Fn14 extracellular domain was conducted by Benicchi and colleagues (129). Fifteen primary hit compounds were identified and subsequent counterassays revealed that two were potent but partial inhibitors and two were weak inhibitors (overall hit rate of 0.007%).…”

Section: Tweak/fn14 Axis-targeted Therapeutics: Additional Approachesmentioning

confidence: 99%

TWEAK/Fn14 Axis-Targeted Therapeutics: Moving Basic Science Discoveries to the Clinic

et al. 2013

View full text Add to dashboard Cite

The TNF superfamily member TWEAK (TNFSF12) is a multifunctional cytokine implicated in physiological tissue regeneration and wound repair. TWEAK is initially synthesized as a membrane-anchored protein, but furin cleavage within the stalk region can generate a secreted TWEAK isoform. Both TWEAK isoforms bind to a small cell surface receptor named Fn14 (TNFRSF12A) and this interaction stimulates various cellular responses, including proliferation and migration. Fn14, like other members of the TNF receptor superfamily, is not a ligand-activated protein kinase. Instead, TWEAK:Fn14 engagement promotes Fn14 association with members of the TNFR associated factor family of adapter proteins, which triggers activation of various signaling pathways, including the classical and alternative NF-κB pathways. Numerous studies have revealed that Fn14 gene expression is significantly elevated in injured tissues and in most solid tumor types. Also, sustained Fn14 signaling has been implicated in the pathogenesis of cerebral ischemia, chronic inflammatory diseases, and cancer. Accordingly, several groups are developing TWEAK- or Fn14-targeted agents for possible therapeutic use in patients. These agents include monoclonal antibodies, fusion proteins, and immunotoxins. In this article, we provide an overview of some of the TWEAK/Fn14 axis-targeted agents currently in pre-clinical animal studies or in human clinical trials and discuss two other potential approaches to target this intriguing signaling node.

show abstract

Section: Tweak/fn14 Axis-targeted Therapeutics: Additional Approachesmentioning

confidence: 99%

TWEAK/Fn14 Axis-Targeted Therapeutics: Moving Basic Science Discoveries to the Clinic

et al. 2013

View full text Add to dashboard Cite

show abstract

“…This risk can be mitigated by prefractionation and upfront removal of fractions with a propensity for non-stoichiometric inhibition. [70] In conclusion, state-of-the-art lead finding flowcharts appropriately combine readout-specific, target-specific and general counterscreens [55,89] to deal with the huge hit lists originating from large libraries and high hit rates. The efficacy of this approach is demonstrated by an extreme case study in which the hit list was completely annihilated by such filtering.…”

Section: Experimental Identification Of Nonstoichiometric Inhibitors mentioning

confidence: 98%

“…readout-specific and redox counterscreens. [89] The most likely reason is that the throughput of devices for dynamic [44] or static [45] light scattering is limited. NMR is also used primarily to characterize compounds synthesized during a medicinal chemistry program [50] or to confirm results by higher-throughput methods [29] rather than for screening hits at an early stage of the flowchart.…”

Section: Experimental Identification Of Nonstoichiometric Inhibitors mentioning

confidence: 99%

Non-stoichiometric inhibition in integrated lead finding – a literature review

Klumpp

2015

Expert Opinion on Drug Discovery

View full text Add to dashboard Cite

Over the last few years, awareness of non-stoichiometric inhibitors within screening libraries and HTS hit lists has considerably increased, not only in the pharmaceutical industry but also in the academic drug discovery community. This has resulted in a variety of methods to detect and handle such compounds. These range from in silico approaches to flag suspicious compounds, and counterassays to measure non-stoichiometric inhibition, to biophysical methods that positively demonstrate stoichiometric binding. In addition, novel technologies to verify target engagement within cells are becoming available. While still a time- and resource-consuming nuisance, non-stoichiometric inhibitors therefore do not fundamentally jeopardize the discovery of low molecular weight lead and drug candidates. Rather, they should be viewed as a manageable issue that with appropriate expertise can be overcome through integration of the above-mentioned approaches.

show abstract

“…As an example of target-based primary screening, Benicchi et al 115 described a novel homogeneous time-resolved fluorescence assay to identify small-molecule inhibitors of the TWEAK-Fn14 protein-protein interaction, which is involved in activation of the NF-κB pathway in models of microglia-dependent CNS inflammation. Even though it has not yet been done, this target-based primary assay could be followed by a secondary microglial activation PA to assess the functional efficacy of identified hit compounds.…”

Section: Microglia Activation Assaysmentioning

confidence: 99%

Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery

2014

View full text Add to dashboard Cite

Complex biological processes such as inflammation, cell death, migration, proliferation, and the release of biologically active molecules can be used as outcomes in phenotypic assays during early stages of drug discovery. Although target-based approaches have been widely used over the past decades, a disproportionate number of first-in-class drugs have been identified using phenotypic screening. This review details phenotypic assays based on inhibition of microglial activation and their utility in primary and secondary screening, target validation, and pathway elucidation. The role of microglia, both in normal as well as in pathological conditions such as chronic neurodegenerative diseases, is reviewed. Methodologies to assess microglia activation in vitro are discussed in detail, and classes of therapeutic drugs known to decrease the proinflammatory and cytotoxic responses of activated microglia are appraised, including inhibitors of glutaminase, cystine/glutamate antiporter, nuclear factor κB, and mitogen-activated protein kinases.

show abstract

A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction

Cited by 11 publications

References 41 publications

TWEAK/Fn14 Axis-Targeted Therapeutics: Moving Basic Science Discoveries to the Clinic

TWEAK/Fn14 Axis-Targeted Therapeutics: Moving Basic Science Discoveries to the Clinic

Non-stoichiometric inhibition in integrated lead finding – a literature review

Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery

Contact Info

Product

Resources

About