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Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable PhotoPROTACs.

ABSTRACT: Off-tissue effects are persistent issues of modern inhibition-based therapies. By merging the strategies of photopharmacology and small-molecule degraders, we introduce a novel concept for persistent spatiotemporal control of induced protein degradation that potentially prevents off-tissue toxicity. Building on the successful principle of bifunctional all-small-molecule Proteolysis Targeting Chimeras (PROTACs), we designed photoswitchable PROTACs (photoPROTACs) by including ortho-F₄-azobenzene linkers between both warhead ligands. This highly bistable yet photoswitchable structural component leads to reversible control over the topological distance between both ligands. The azo-cis-isomer is observed to be inactive because the distance defined by the linker is prohibitively short to permit complex formation between the protein binding partners. By contrast, the azo-trans-isomer is active since it can engage both protein partners to form the necessary and productive ternary complex. Importantly, due to the bistable nature of the ortho-F₄-azobenzene moiety employed, the photostationary state of the photoPROTAC is persistent, with no need for continuous irradiation. This technique offers reversible on/off switching of protein degradation that is compatible with an intracellular environment and, therefore, could be useful in experimental exploration of biological signaling pathways-such as those crucial for oncogenic signal transduction. Additionally, this strategy may be suitable for therapeutic intervention to address a variety of diseases. By enabling reversible activation and deactivation of protein degradation, photoPROTACs offer advantages over conventional photocaging strategies that irreversibly release active agents.

SUBMITTER: Pfaff P

PROVIDER: S-EPMC6813558 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable PhotoPROTACs.

Pfaff Patrick P Samarasinghe Kusal T G KTG Crews Craig M CM Carreira Erick M EM

ACS central science 20190917 10

Off-tissue effects are persistent issues of modern inhibition-based therapies. By merging the strategies of photopharmacology and small-molecule degraders, we introduce a novel concept for persistent spatiotemporal control of induced protein degradation that potentially prevents off-tissue toxicity. Building on the successful principle of bifunctional all-small-molecule Proteolysis Targeting Chimeras (PROTACs), we designed photoswitchable PROTACs (<b>photoPROTACs</b>) by including <i>ortho-</i>F ...[more]

PMID: 31660436

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Project description:Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore-attached to the protein through a protonated Schiff base-and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.

Dataset Information

Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable PhotoPROTACs.

Publications

Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable PhotoPROTACs.

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