Protein Phosphatase 5-Recruiting Chimeras for Accelerating Apoptosis-Signal-Regulated Kinase 1 Dephosphorylation with Antiproliferative Activity

Abstract

A normal phosphorylation state is essential for the function of proteins. Biased regulation frequently results in morbidity, especially for the hyperphosphorylation of oncoproteins. The hyperphosphorylation of ASK1 at Thr838 leads to a persistently high activity state, which accelerates the course of gastric cancer. Under normal conditions, PP5 specifically dephosphorylates p-ASK1 T838 in cells, thereby weakening ASK1 to a low-basal activity state. However, in tumor types, PP5 shows low activity with a self-inhibition mechanism, making p-ASK1 T838 remain at a high level. Thus, we aim to design phosphatase recruitment chimeras (PHORCs) through a proximity-mediated effect for specifically accelerating the dephosphorylation of p-ASK1 T838 . Herein, we describe DDO3711 as the first PP5recruiting PHORC, which is formed by connecting a small molecular ASK1 inhibitor to a PP5 activator through a chemical linker, to effectively decrease the level of p-ASK1 T838 in vitro and in vivo. DDO3711 shows preferable antiproliferative activity (IC 50 = 0.5 μM) against MKN45 cells through a direct binding and proximity-mediated mechanism, while the ASK1 inhibitor and the PP5 activator, used alone or in combination, exhibit no effect on MKN45 cells. Using DDO3711, PHORCs are identified as effective tools to accelerate the dephosphorylation of POIs and provide important evidence to achieve precise phosphorylation regulation, which will promote confidence in the further regulation of abnormally phosphorylated oncoproteins.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1

“…4a,b), which may influence its phosphorylation on T838 within the signalosome. In doing so, TRX1 likely contributes to ASK1 inhibition 46,47 .…”

“…4a,b), which may influence its phosphorylation on T838 within the signalosome. In doing so, TRX1 likely contributes to ASK1 inhibition 46,47 . Therefore, limiting access to residues of the activation segment regulated by phosphorylation may be thus a key regulatory event of ASK1 function.…”

Proximity-Based Modalities for Biology and Medicine

“…A study reported by Zhang et al found that while the ASK1 inhibitor exhibited no effect on MKN45 cells, the PHORCs, composed of ASK1 inhibitor and a phosphatase activator, could reduce p-ASK1 T838 levels both in vitro and in vivo and demonstrated anticancer activity on MKN45 cancer cell line and MKN45 xenograft mouse model, suggesting the therapeutic potential of PHORCs as anticancer agents. 119 Identifying MGs between PTM enzymes and target proteins can potentially be a strategy to bypass ligand discovery for allosteric sites on the writer/eraser.…”

“…However, to pursue the therapeutic potential of targeted protein PTM, allosteric agonists or nonfunctional ligands against the endogenous writer/eraser are likely required. By tethering allosteric activators of kinases or phosphatase with ligands of the target protein separately, PHICs and PHORCs are two new heterobifunctional modalities that rewire the endogenous kinase or phosphatase to precisely phosphorylate or dephosphorylate target proteins, respectively. A study reported by Zhang et al found that while the ASK1 inhibitor exhibited no effect on MKN45 cells, the PHORCs, composed of ASK1 inhibitor and a phosphatase activator, could reduce p-ASK1 T838 levels both in vitro and in vivo and demonstrated anticancer activity on MKN45 cancer cell line and MKN45 xenograft mouse model, suggesting the therapeutic potential of PHORCs as anticancer agents .…”

“…For example, the acetylation tagging system (AceTAG) was developed to modulate protein acetylation. Phoshorylation-targeting chimeras (PhosTACs), , phosphatase recruitment chimeras (PHORCs), , receptor inhibition by phosphatase recruitment (RIPR), and phosphorylation-inducing chimeric small molecules (PHICS) were designed for precisely controlling protein phosphorylation and dephosphorylation. Other PTMs modulated by proximity-based agents include O -GlcNAcylation via fusion of a target-selective nanobody to an O -GlcNAc transferase to induce O-GlcNAcylation of endogenous α-synuclein, as well as nanobodies conjugated to the split O-GlcNAcase eraser enzyme to induce selective deglycosylation of transcription factors c-Jun and c-Fos (Figure ).…”