A genetic, non-transcriptional assay for nuclear receptor ligand binding in yeast

Köhler, Fabian; Zimmermann, André; Hager, Martin H.; Sippel, Albrecht E.

doi:10.1016/j.gene.2004.04.018

Cited by 7 publications

(4 citation statements)

References 35 publications

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“…12 In addition, all of the tested compounds could be detected at nanomolar concentrations and the higher-affinity ones at sub-nanomolar concentrations (Figure 3A). This sensitivity is comparable to that of previously reported chimeric sensors for estrogen binding in yeast 25,26 or in vitro, 27 and to the sensitivity of other simple screening systems, such as recently developed NHR microarrays of coactivator recruitment. 28 Interestingly, for low-affinity ligands, the sensitivity of our system converges with that of highly sensitive in vitro binding assays, 29 and with some transcriptional activation assays in genetically engineered yeast 30,31 and mammalian cells.…”

Section: Construction Of a Second-generation Estrogen-regulatedsupporting

confidence: 82%

Engineered Chimeric Enzymes as Tools for Drug Discovery: Generating Reliable Bacterial Screens for the Detection, Discovery, and Assessment of Estrogen Receptor Modulators

et al. 2007

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Engineered protein-based sensors of ligand binding have emerged as attractive tools for the discovery of therapeutic compounds through simple screening systems. We have previously shown that engineered chimeric enzymes, which combine the ligand-binding domains of nuclear hormone receptors with a highly sensitive thymidylate synthase reporter, yield simple sensors that report the presence of hormone-like compounds through changes in bacterial growth. This work describes an optimized estrogen sensor in Escherichia coli with extraordinary reliability in identifying diverse estrogenic compounds and in differentiating between their agonistic/antagonistic pharmacological effects. The ability of this system to assist the discovery of new estrogen-mimicking compounds was validated by screening a small compound library, which led to the identification of two structurally novel estrogen receptor modulators and the accurate prediction of their agonistic/antagonistic biocharacter in human cells. Strong evidence is presented here that the ability of our sensor to detect ligand binding and recognize pharmacologically critical properties arises from allosteric communication between the artificially combined protein domains, where different ligand-induced conformational changes in the receptor are transmitted to the catalytic domain and translated to distinct levels of enzymic efficiency. To the best of our knowledge, this is one of the first examples of an engineered enzyme with the ability to sense multiple receptor conformations and to be either activated or inactivated depending on the nature of the bound effector molecule. Because the proposed mechanism of ligand dependence is not specific to nuclear hormone receptors, we anticipate that our protein engineering strategy will be applicable to the construction of simple sensors for different classes of (therapeutic) binding proteins.

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Section: Construction Of a Second-generation Estrogen-regulatedsupporting

confidence: 82%

Engineered Chimeric Enzymes as Tools for Drug Discovery: Generating Reliable Bacterial Screens for the Detection, Discovery, and Assessment of Estrogen Receptor Modulators

et al. 2007

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“…It has also been shown that OHT can act synergistically with E 2 in a yeast-transcription assay [192]. Kohler et al observed this also using a genetic, non-transcriptional binding assay and their results suggested that it is only seen at the level of ligand binding [193]. Tyulmenkov and Klinge reported that Tetrahydrochrysene Ketone binds to both E 2 -liganded and unliganded ER and ER [194].…”

Section: Alternative Binding Pocket Of the Ermentioning

confidence: 94%

Estrogen Receptors: Molecular Interactions, Virtual Screening and Future Prospects

Knox¹,

Meegan²,

Lloyd

2006

CTMC

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Identification of the Estrogen Receptor (ER) as a key mediator of the proliferation of breast cancer, and its involvement in pathways leading to osteoporosis and coronary heart disease, has resulted in a surge to discover and design compounds with the ability to modulate its actions (SERMs). Concurrently, a dramatic increase in the number of crystal structures of the ER has led to a more in depth understanding of the governing mechanisms involved in ER modulation. Entwining computational techniques with the availability of 3D structural data, has allowed not only the rational design of potent inhibitors of the ER, but also its incorporation in Virtual Screening (VS) in the search for novel chemotypes that can modulate the ER. An important initial step in the VS process is to filter towards molecules that occupy similar chemical space to a set of known actives prior to docking. We illustrate through Principal Component Analysis (PCA) of 145 descriptors the region of chemical space antiestrogens occupy compared with 'drug-like' space. We also review all available studies involving validation of several docking algorithms utilizing the ER, ultimately focusing on analysis of Enrichment (E) rates and False Positive (FP) rates to illustrate the successes attributed to each docking algorithm. Finally, we relate the recent discovery of non-genomic mechanisms of the ER and subsequently present a model involving a recently identified alternative, second binding-pocket of the ER in our laboratory through cavity analysis that suggests how the same receptor can invoke these, 'classical' and rapid responses concurrently.

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“…Another application of the RRS targets the ligand dependent interaction of steroid hormone receptors with heat shock proteins [35]. This system can be used to detect the binding of steroid ligands to defined nuclear receptors in a non-transcriptional in vivo system for e. g. compound screening.…”

Section: Novel Genetic Interaction Screening Systems In Yeastmentioning

confidence: 99%

Protein‐protein interaction screening with the Ras‐recruitment system

Kruse

Hanke

Vasiliev

et al. 2006

Signal Transduction

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The fast paced progress in genomics yielded a multitude of novel identified genes. Therefore reliable and up scalable methods are required to identify the functions of the encoded proteins. By using protein-protein interaction screening technologies, the function of many proteins has successfully been assigned, and proved in combination with other techniques. We describe the application of an in vivo screening system for protein-protein interactions, the Ras-recruitment system (RRS). The RRS has been used extensively to identify novel interaction partners, also in cases where common systems failed. Therefore we propose the RRS as a valuable alternative interaction screening method that can widely be used among protein classes and that has been further developed by our group into a high-throughput screening system.Protein interaction analysis with Ras-recruitment system 199

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A genetic, non-transcriptional assay for nuclear receptor ligand binding in yeast

Cited by 7 publications

References 35 publications

Engineered Chimeric Enzymes as Tools for Drug Discovery: Generating Reliable Bacterial Screens for the Detection, Discovery, and Assessment of Estrogen Receptor Modulators

Engineered Chimeric Enzymes as Tools for Drug Discovery: Generating Reliable Bacterial Screens for the Detection, Discovery, and Assessment of Estrogen Receptor Modulators

Estrogen Receptors: Molecular Interactions, Virtual Screening and Future Prospects

Protein‐protein interaction screening with the Ras‐recruitment system

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