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Protein regulation approaches have expanded into the field of dephosphorylation, and Phosphorylation Targeting Chimeras (PhosTACs) are novel bifunctional small molecules that target phosphorylation regulation by recruiting phosphatases.

Advances in PhosTACs

Many diseases are associated with proteins hyperphosphorylation, which is dynamically controlled by kinases and phosphatases. Given the off-target effects and increasing resistance of kinase inhibitors, a new generation of drug phosphorylation-targeted chimeras (PhosTACs) has been proposed. In 2021, Crews group proposed the concept of PhosTACs and successfully performed targeted dephosphorylation of proteins via PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to the target proteins PDCD4 and FOXO3a. Recently, a PhosTAC that recruits the dephosphorylase PP2A to target dephosphorylation of Tau for therapeutic purposes was reported.

Fig. 1 PhosTAC targets PDCD4 and FOXO3a for dephosphorylation (Chen, 2021)

What is the Target of PhosTAC?

Tau is a microtubule-associated protein that has important implications for microtubule assembly and stability, and its abnormal aggregation is associated with the formation of several neurodegenerative diseases. It has been found that hyperphosphorylation of Tau is frequently found in the brains of Alzheimer's patients and that Tau phosphorylation at Thr231 affects the binding of Tau to microtubule proteins, which in turn affects downstream effects. In addition, hyperphosphorylation of Tau induces aberrant aggregation, and Tau phosphorylation at Ser262 and Ser356 impairs HSP70 protein recognition, thus preventing its degradation by the proteasome system. In conclusion, Tau hyperphosphorylation is considered as a potential therapeutic target for neurodegenerative diseases.

How to Design PhosTACs?

• Validation of PP2A-PhosTAC-Tau ternary complex formation

Since there is no specific ligand molecule for PP2A, to test the feasibility of PhosTACs to induce dephosphorylation, the FKBP12F36V and HaloTag fusion protein system, which is well established in the PROTAC technology, was used. First, cell lines with FKBP12F36V-PP2A and HaloTag-Tau fusion proteins were constructed, and a heterobifunctional molecule PhosTAC7 consisting of a FKBP12F36V ligand and a HaloTag ligand was synthesized, and non-HaloTag-binding PhosTAC7F was used as an inactive control. Halotrap pull-down experiments showed that PhosTAC attached with Halotag bound to Tau protein and then co-precipitated with PP2A indicating the formation of a PP2A-PhosTAC-Tau ternary complex. And the induced expression of PhosTAC triggered the elimination of significant Tau protein phosphorylation at Thr231 position.

• Construction of PhosTAC that directly binds Tau

Using the Tau tracer PI-2620 as a ligand instead of the HaloTag system, a series of PhosTACs were constructed by connecting PI-2620 to the FKBP12 ligand via different PEG linkers. Western Blot results showed that the efficiency of Tau dephosphorylation at Thr231 position was optimal when the PEG unit of PhosTAC was 8 (Tau2-8), with 50% dephosphorylation efficiency at 1 μM treatment.

Fig. 2 Design of Tau-targeted PhosTAC and expression of Tau-Halotag-fluorescent protein fusion gene. (Hu, 2023)

• Investigation of the dephosphorylation kinetics of PhosTAC

Cellular experiments showed that PhosTAC (Tau2-8) achieved 50% dephosphorylation within 2 h of 1 μM treatment, and the dephosphorylation efficiency was about 75% after 24 h. The catalytic property of PhosTAC was confirmed by elution experiments, PhosTAC (Tau2-8) remained potent in dephosphorylation within 24 h of changing to normal medium culture. It was confirmed that PhosTAC has the catalytic activity similar as PROTAC.

References

1. Chen, P., et al. Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs), ACS Chem. Biol., 2021, 16, 12, 2808-2815.
2. Hu, Z., et al. Targeted Dephosphorylation of Tau by Phosphorylation Targeting Chimeras (PhosTACs) as a Therapeutic Modality, J. Am. Chem. Soc., 2023, 145, 7, 4045-4055.