Isoxazole 9 (ISX9), a small molecule targeting Axin, activates Wnt/β-catenin signalling and promotes hair regrowth

by Selleckchem | Jul 05 2023

Background and purpose: Isoxazole 9 (ISX9) is a neurogenesis-promoting small molecule compound that can up-regulate the expression of NeuroD1 and induce differentiation of neuronal, cardiac and islet endocrine progenitors. So far, the molecular mechanisms underlying the action of ISX9 still remain elusive.

Experimental approach: To identify a novel agonist of the Wnt/β-catenin, a cell-based SuperTOPFlash reporter system was used to screen known-compound libraries. An activation effect of ISX9 on the Wnt/β-catenin pathway was analysed with the SuperTOPFlash or SuperFOPFlash reporter system. Effects of ISX9 on Axin1/LRP6 interaction were examined using a mammalian two-hybrid system, co-immunoprecipitation, microscale thermophoresis, emission spectra and mass spectrometry assays. The expression of Wnt target and stemmness marker genes were evaluated with real-time PCR and immunoblotting. In vivo hair regeneration abilities of ISX9 were analysed by immunohistochemical staining, real-time PCR and immunoblotting in hair regrowth model using C57BL/6J mice.

Key results: In this study, ISX9 was identified as a novel agonist of the Wnt/β-catenin pathway. ISX9 targeted Axin1 by covalently binding to its N-terminal region and potentiated the LRP6-Axin1 interaction, thereby resulting in the stabilization of β-catenin and up-regulation of Wnt target genes and stemmness marker genes. Moreover, the topical application of ISX9 markedly promoted hair regrowth in C57BL/6J mice and induced hair follicle transition from telogen to anagen via enhancing Wnt/β-catenin pathway.

Conclusions and implications: Taken together, our study unravelled that ISX9 could activate Wnt/β-catenin signalling by potentiating the association between LRP6 and Axin1, and may be a promising therapeutic agent for alopecia treatment.

Comments:

The background and purpose of the study you described involve investigating the molecular mechanisms of a compound called ISX9, which is known to promote neurogenesis and induce differentiation of neuronal, cardiac, and islet endocrine progenitors. The specific focus of the study was to understand how ISX9 affects the Wnt/β-catenin pathway, a signaling pathway involved in various cellular processes, including hair follicle development.

The researchers used a cell-based SuperTOPFlash reporter system to screen known-compound libraries in search of a novel agonist of the Wnt/β-catenin pathway. They found that ISX9 exhibited an activation effect on this pathway. To further explore the mechanism of action, the researchers conducted various assays. They examined the effects of ISX9 on the interaction between two key proteins, Axin1 and LRP6, using a mammalian two-hybrid system, co-immunoprecipitation, microscale thermophoresis, emission spectra, and mass spectrometry assays. The results indicated that ISX9 covalently bound to the N-terminal region of Axin1, leading to potentiation of the LRP6-Axin1 interaction. This, in turn, stabilized β-catenin, a key component of the Wnt/β-catenin pathway, resulting in the up-regulation of Wnt target genes and stemness marker genes.

The researchers also investigated the effects of ISX9 in vivo using a hair regrowth model in C57BL/6J mice. They analyzed the hair regeneration abilities of ISX9 through immunohistochemical staining, real-time PCR, and immunoblotting. The findings demonstrated that topical application of ISX9 significantly promoted hair regrowth and induced the transition of hair follicles from the resting phase (telogen) to the growth phase (anagen) by enhancing the Wnt/β-catenin pathway.

In conclusion, this study revealed that ISX9 acts as a novel agonist of the Wnt/β-catenin pathway by enhancing the association between LRP6 and Axin1. By stabilizing β-catenin, ISX9 up-regulates Wnt target genes and stemness marker genes. The findings suggest that ISX9 has potential as a therapeutic agent for the treatment of alopecia (hair loss) by activating the Wnt/β-catenin pathway.