Dataset Information

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

ABSTRACT: The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

SUBMITTER: Camara A

PROVIDER: S-EPMC7582957 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

Camara Ali A George Alyssa A Hebner Evan E Mahmood Anika A Paluru Jashun J Mattoo Seema S

International journal of molecular sciences 20200927 19

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification ...[more]

PMID: 32992526

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Project description:The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Dataset Information

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

Publications

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

Similar Datasets

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