The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.
Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.
Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881
Kinase activity-based assays are cost-effective and widely used to drive early drug discovery and lead identification, however, they typically require purified, active kinase preparations to be produced that are shown to phosphorylate a known substrate. While it is possible to satisfy these requirements for well-studied kinases, there are many kinases which lack one or more of these requirements, such as CDK8 and STK33, making drug discovery efforts difficult. To solve for these assay development problems, we have developed a TR-FRET binding assay platform which allows for characterization of compounds with kinases that are not sufficiently purified, completely lack activity, or do not have a known substrate. These new assay options for addressing kinase assay requirements in early drug discovery should enable more targets to be screened sooner, resulting in compounds being made available for lead optimization faster. The ability to detect and characterize binding of compounds to non-activated kinases is also a benefit to the lead optimization process, as compounds that bind preferentially to kinases in their non-activated state, such as Gleevec®, are generally preferred due to their selectivity. In this poster, we present solutions for kinases that are difficult to address with activity assays and provide a comparison of compound affinities for active and non-activated forms of a diverse set of kinases to enable faster lead optimization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3688.
Kinase activity-based assays are cost-effective and widely used to drive early drug discovery and lead identification, however, they typically require purified, active kinase preparations be produced that are shown to phosphorylate a known substrate. While it is possible to satisfy these requirements for the well-studied kinases, there are many kinases which lack one or more of these requirements, such as CDK8 and STK33, making drug discovery efforts difficult. To solve for these assay development problems, we have developed a TR-FRET-based binding assay platform which allows for characterization of compounds with kinases that are not sufficiently purified or do not demonstrate measurable activity against putative or potential substrates. This assay addresses kinase assay requirements early in the drug discovery process, and should enable more targets to be screened sooner, resulting in compounds being made available for lead optimization faster. The ability to detect and characterize binding of compounds to non-activated kinases is also a benefit to the lead optimization process, as compounds that bind preferentially to kinases in their non-activated state, such as Imatinib, are sometimes preferred due to their selectivity. In this poster, we present solutions for kinases that are difficult to address with activity assays and provide a comparison of compound affinities for active and non-activated forms of a diverse set of kinases to enable faster lead optimization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C95.
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