Using photolabile ligands in drug discovery and development

Dormán, György; Prestwich, Glenn D.

doi:10.1016/s0167-7799(99)01402-x

Cited by 389 publications

(282 citation statements)

References 72 publications

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“…Key to this approach is our utilization of the well-established idea of derivatizing the capillary wall with covalently bound acrylamide, for which multiple approaches exist (21-23). Similarly, we have used an established approach to photoimmobilization of proteins, with UV-activatable benzophenone (24,25).…”

mentioning

confidence: 99%

Isoelectric focusing technology quantifies protein signaling in 25 cells

O'Neill¹,

Bhamidipati²,

Bi³

et al. 2006

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

187

234

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A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. Highresolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.cell signaling ͉ immunoassay ͉ phosphorylation ͉ Western blot ͉ microfluidic

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mentioning

confidence: 99%

Isoelectric focusing technology quantifies protein signaling in 25 cells

O'Neill¹,

Bhamidipati²,

Bi³

et al. 2006

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

187

234

View full text Add to dashboard Cite

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“…27 Photochemical activation may be a single-cycle process, when irradiation of the photocleavable functional group causes activation, or even a multicycle process, when biologically inactive and active states can be acessed and reversed with different wavelenghts. 33 For extracellular stimulation of Dictyostelium cells with complex and rapidly switching signals, the light-induced release of such compounds was successfully demonstrated. 34,35 In the present work, we used the [7-(dimethylamino)coumarin-4-yl]methyl (DMACM) ester of 8-Br-cGMP, a photolabile cGMP analogue that is known to be membrane permeable, poorly hydrolyzed by phosphodiesterases, stably soluble in aqueous buffer solution, and rapidly released upon illumination.…”

Section: Discussionmentioning

confidence: 99%

Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells

et al. 2013

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Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. Insight, innovation, integrationSecond messengers like cGMP play a central role in the regulatory pathways of living cells. Here, we demonstrate that an increase in intracellular cGMP can trigger not only myosin II but also actin responses in motile amoeboid cells. In previous studies, an increase in intracellular cGMP was induced indirectly via membrane receptor stimulation. In the present work, we employ, for the first time, the direct light-induced release of cGMP from a caged precursor to raise the cytosolic cGMP level in Dictyostelium cells that carry fluorescent markers for filamentous actin and myosin II. Based on this advanced combination of live cell photo-uncaging and multi-color confocal microscopy, we could identify a link between cGMP and actin dynamics in motile amoeboid cells.

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“…In the current analogues, benzophenone was employed as the photoprobe for labeling of proteins because it is readily activated with long-wavelength UV light, is stable in most of the chemical reactions used during the synthesis of the probe, and generally yields covalent products modified at a single site. 21 In the four probes reported herein, the benzophenone moiety is linked to the end of a Δ 9 cis hydrocarbon chain. These analogues were characterized via the covalent modification of rat plasma proteins.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of Photoactivatable Analogues of Lysophosphatidic Acid and Covalent Labeling of Plasma Proteins

Baker

Tigyi

et al. 2005

J. Org. Chem.

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Lysophosphatidic acid bearing a benzophenone group in either the sn-1 or sn-2 chain of an oleoyltype ester or oleyl-type ether chain and 32 P in the phosphate group were synthesized. The benzophenone moiety was introduced by selective hydroboration of the double bond of enyne 11 at low temperature, followed by Suzuki reaction with 4-bromobenzophenone. The key intermediates for the preparation of ester-linked LPA 1 and 3 were obtained in one pot by a modified DIBAL-H reduction of orthoformate intermediate 22. These probes were shown to covalently modify a single protein target in rat plasma containing albumin and several protein targets in rat plasma containing a low level of albumin.

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Using photolabile ligands in drug discovery and development

Cited by 389 publications

References 72 publications

Isoelectric focusing technology quantifies protein signaling in 25 cells

Isoelectric focusing technology quantifies protein signaling in 25 cells

Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells

Synthesis of Photoactivatable Analogues of Lysophosphatidic Acid and Covalent Labeling of Plasma Proteins

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