The synthesis and purification of the Light-sensitive Dimerizer Zapalog

12 4 月, 2023

Protein dimerizers, also known as chemical inducers of dimerization (CIDs), are widely used in biochemistry, molecular biology, and cell biology. CIDs are chemical compounds that bind two different proteins and bring them into proximity solely in the presence of the dimerizer. They are commonly used to build protein complexes. Zapalog is photosensitive, cell-permeable, non-toxic, and effective as a non-covalent dimerizer. This article will tell you about the design and Synthesis of the Light-sensitive Dimerizer Zapalog.

Mitochondria are cells’ primary source of ATP, and their motility and localization are fundamental to cellular function. Many such cell biological tools have been developed to control proteins directly. Light-inducible approaches provide a means to control biological systems with a spatial and temporal resolution unmatched by traditional genetic perturbations. Induced dimerization systems differ primarily according to their dimerization trigger.

Chemically induced dimerization (CID) systems are based on small molecules interacting with two identical proteins (homodimerization) or two different proteins (heterodimerization). Optogenetic dimerization systems employ photosensitive proteins that undergo a conformational change upon illumination and induce protein interaction. Chemo-optogenetic dimerization systems use photoactivatable and/or -cleavable small-molecule dimerizers so that proximity can be influenced and/or disrupted by light. Light-induced dimerization systems confer a spatiotemporal resolution that is unmatched by chemical approaches. Both chemical and genetically encoded dimerizers have been valuable for studying the axonal transport of mitochondria and other organelles.

Dimerization

In this study, researchers present the chemical inducers of dimerization of potential value to cell biologists. Zapalog is a photocleavable small-molecule hetero-dimerizer that can repeatedly initiate and instantaneously terminate a physical interaction between two target proteins. Zapalog is a small-molecule dimerizer that undergoes photolysis when exposed to blue light.

FK506 binding proteins (FKBPs) are a family of highly conserved proteins in eukaryotes. FKBPs are involved in diverse cellular functions, including protein folding, cellular signaling, apoptosis, and transcription. The E. coli dihydrofolate reductase (DHFR) destabilizing domain, which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis.

Dimerization can be repeatedly restored with an uncleaved Zapalog. Researchers implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using Zapalog, they tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in the complete restoration of their endogenous motility. Results show that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with Latrunculin A reduces this firmly anchored pool. Upon release from exogenous motors, mitochondria are preferentially recaptured at presynapses.

Design and synthesis of Zapalog

Zapalog is photosensitive, cell-permeable, non-toxic, and effective as a non-covalent dimerizer. Several parameters guided the design of Zapalog:

1) The CID must be membrane permeable, and the linker must be long enough to allow both binding domains to be simultaneously engaged without steric interference.

2) The photocleavable linker must respond to a wavelength that is neither cytotoxic nor interferes with common imaging fluorophores.

3) The compound needs to be synthesized on the milligram scale.

4) The chosen starting point was the hetero-dimerizer TMP-SLF, a cell-permeable CID effective at low micromolar concentrations.

a, Schematic illustration of the Zapalog function

b, Chemical structure of Zapalog before and after photolysis of the DANB moiety by 405nm light

c, Time-lapsed imaging

c’, Quantification of multiple experiments

c”, From assays of YFP translocation to mitochondria

d, YFP-DHFR-Myc translocation to mitochondria

d’, Quantification of data

Zapalog, a photocleavable hetero-dimerizer, can reverse translocate cytosolic yellow fluorescent protein (YFP) to mitochondria. With 405nm light Zapalog in solution underwent the expected photocleavage. Zapalog was not affected by 458nm light, making it suitable for cyan fluorescent protein (CFP) imaging.

To assess in living COS7 cells Zapalog’s ability to function as a hetero-dimerizer and undergo photocleavage, they modified a fluorophore-translocation assay previously established for rapalogs: they tagged the surface of mitochondria with Tom20-mCherry-FKBP and co-expressed cytoplasmic YFP-DHFR-Myc. They used a spinning disk confocal microscope for live imaging cytoplasmic YFP and mitochondrial mCherry at two frames/sec. The addition of Zapalog to the media-induced dimerization of the constructs and YFP rapidly cleared from the cytoplasm and increased mitochondria. They quantified the progression of YFP translocation by measuring cytoplasmic and mitochondrial YFP levels in each cell. They plotted the time from 10% to 90% YFP translocation as a function of Zapalog concentration. Zapalog exhibited equivalent on-rate kinetics to previously reported CIDs and an EC50 of ~100nm. To test the efficiency and speed of reversing the YFP translocation with exposure to 405nm light, they repeated YFP translocation onto mitochondria as above. Still, they removed Zapalog from the media after 10min. YFP recruitment to mitochondria persisted after Zapalog was washed out, evidence of Zapalog retention in the cell. They used a laser scanning confocal to flash 405nm light separately onto each cell sequentially. Exposure of each cell to a single 500msec flash of blue light proved sufficient to dissociate YFP localization from mitochondria in <1sec fully.

Synthesis of Zapalog

Synthesis of Zapalog was performed by Medicilon.

Photolysis of Zapalog in Solution

To achieve partial photolysis, a 40µl sample of 1mM Zapalog in phosphate-buffered saline, pH 7.2, contained in a quartz cuvette, was exposed for 5s to ~15 mW of light from a 0.22 NA fiber optic cable coupled to a 405nm laser. Both purity and photolytic cleavage of Zapalog were confirmed by reverse-phase high-performance liquid chromatography on a 4.6 mm 150 mm Zorbax SB-C8 column (5 mm particle size) in H2O, CH3CN, 0.1% TFA at 1 ml/ min. Samples were monitored at 220 nm and 350 nm. A non-photolyzed Zapalog sample was prepared and ran under the same conditions.

Zapalog is photolyzed by 405nm, but not 458nm light.

a, LC-MS of Zapalog

b, Chromatograms of Zapalog

Conclusion

In this study, researchers have used Zapalog to probe the molecular underpinnings of mitochondrial motility and positioning in the axon by temporarily forcing their motility by adding constitutively active kinesins. Researchers identified and characterized a population of mitochondria restrained by actin-dependent anchoring through these manipulations. They summarize that controlling cellular processes with light can help elucidate their underlying mechanisms.

References:

[1] Mingming Tong, et al. FK506-Binding Proteins and Their Diverse Functions. Curr Mol Pharmacol. 2015;9(1):48-65. doi: 10.2174/1874467208666150519113541.

[2] Hui Peng, et al. Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo. Mol Ther Methods Clin Dev. 2019 Aug 15;15:27-39. doi: 10.1016/j.omtm.2019.08.002.

[3] Amos Gutnick, et al. The light-sensitive dimerizer zapalog reveals distinct modes of immobilization for axonal mitochondria. Nat Cell Biol. 2019 Jun;21(6):768-777. doi: 10.1038/s41556-019-0317-2.

[4] Laura Klewer, et al. Light-Induced Dimerization Approaches to Control Cellular Processes. Chemistry. 2019 Sep 25;25(54):12452-12463. doi: 10.1002/chem.201900562.