Drug–target identification from total cellular lysate by drug-induced conformational changes

Nishiya, Yoichi; Shibata, Kenji; Saito, Seiji; Yano, Keiichi; Oneyama, Chitose; Nakano, Hirofumi; Sharma, Sreenath V.

doi:10.1016/j.ab.2008.11.034

Cited by 12 publications

(10 citation statements)

References 21 publications

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“…S5) which remained active in the presence of detection reagents. Several inhibitors did not demonstrate activation in the cell-based assays, possibly due to cell impermeability, failure to sufficiently stabilize FLuc from intracellular degradation, or because they destabilized FLuc in a cellular context (Nishiya et al, 2009). Significantly, although many of the same FLuc inhibitory compounds were active in the two cell-based assays, the types of CRCs generated by these compounds could differ between the assays (examples are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter

Thorne

Shen

Lea

et al. 2012

Chemistry & Biology

122

153

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SUMMARY Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report.

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Section: Resultsmentioning

confidence: 99%

Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter

Thorne

Shen

Lea

et al. 2012

Chemistry & Biology

122

153

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“…Crystallographic and biochemical investigations demonstrate that geldanamycin preferentially interacts with Hsp90 in an apo, or open-conformation, that is unfavorable for client protein binding [36,37,38], while WS13 preferentially associates with an ATP-bound conformation of the chaperone and stabilizes the client protein/Hsp interaction [32]. Thus, while recognizing similar Hsp90 species, WS13 and geldanamycin induce distinct conformations in Hsp90 and/or gp96 upon binding, leading to client protein trapping or release, respectively.…”

Section: Discussionmentioning

confidence: 99%

Heat shock protein gp96 regulates Toll-like receptor 9 proteolytic processing and conformational stability

Brooks

Sun

Chiosis

et al. 2012

Biochemical and Biophysical Research Communications

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Nucleic acid-sensing Toll-like receptors (TLRs) initiate innate immune responses to foreign RNA and DNA, yet can detect and respond to host DNA. To avoid autoimmune pathologies, nucleic acid sensing TLRs are tightly regulated. TLR9 primarily resides in the endoplasmic reticulum, traffics to endosomes, is proteolytically processed and responds to DNA. The heat shock protein gp96 is one of several accessory proteins that regulate intracellular trafficking of TLR9. In the absence of gp96, TLR9 fails to exit the endoplasmic reticulum, and therefore gp96-deficient macrophages fail to respond to CpG DNA. However, absence of gp96 precludes studies on potential chaperoning functions of gp96 for TLR9. Here we demonstrate that pharmacologic interference with gp96 function inhibits TLR9 signaling. TLR9 remains associated with gp96 during intracellular trafficking, and gp96-specific inhibitors increase TLR9 sensitivity to proteolytic degradation. We propose that gp96 is critical for both TLR9 egress from the ER, and for protein conformational stability in the endosomal compartment. These studies highlight the importance of examining gp96-specific inhibitors for modulating TLR9 activation, and the treatment autoimmune diseases.

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“…Because degradation of proteins inside the cell is predominantly carried out by supramolecular machines, known as the proteasomes and aggresomes, and is elaborately controlled by posttranslational modifications such as phosphorylation and ubiquitinylation, protein stability in vivo is largely unpredictable. Indeed, in vivo stability of proteins upon drug/ligand binding is highly idiosyncratic in the literature; drug binding has been shown to both increase and decrease proteolytic susceptibility of the target protein (6,47,51,52). For instance, whereas unstable FRB domain and FKBP12 mutants are stabilized by the presence of ligands (6,51) and topoisomerase-1 is destabilized by camptothecin (47), binding of estrogen receptor ligands each affects receptor stability differently (53).…”

Section: Discussionmentioning

confidence: 99%

Target identification using drug affinity responsive target stability (DARTS)

Lomenick¹,

Hao²,

Jonai³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

730

707

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Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.aging ͉ label-free ͉ proteomics ͉ small molecules

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Drug–target identification from total cellular lysate by drug-induced conformational changes

Cited by 12 publications

References 21 publications

Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter

Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter

Heat shock protein gp96 regulates Toll-like receptor 9 proteolytic processing and conformational stability

Target identification using drug affinity responsive target stability (DARTS)

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