Small-molecule agonists of the RET receptor tyrosine kinase activate biased trophic signals that are influenced by the presence of GFRa1 co-receptors

Sean Jmaeff,Yulia Sidorova,Hinyu Nedev,Mart Saarma,H Uri Saragovi

doi:10.1074/jbc.ra119.011802

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET-GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

Highlights

PTP activity/inhibition assays are as we reported previously [4]
The tagged catalytic domain or the full length of the following phosphatases were used for the screen: GST–PTP1B aa 1–321, GST–LAR–D1D2, GST–Sigma–D1D2, GST–MKPx aa 1–184, and His-SHP-1 aa 1–595
Enzyme reactions were performed in assay buffer 50 mM HEPES, pH 7.0, for PTP1B, LAR, Sigma, and MKPx and buffer 50 mM Bis-Tris, pH 6.3, 2 mM EDTA for SHP-1

Summary

Introduction

PTP activity/inhibition assays are as we reported previously [4]. Enzyme reactions were performed in assay buffer 50 mM HEPES, pH 7.0, for PTP1B, LAR, Sigma, and MKPx and buffer 50 mM Bis-Tris, pH 6.3, 2 mM EDTA for SHP-1. DiFMUP (Invitrogen) was used as substrate for all assays, in black 96-well plates (Corning) in a final volume of 100 ␮l at 25 °C. The reaction was monitored by measuring the fluorescence (excitation wavelength, 358 nm; emission, 455 nm) with the Varioskan plate reader (Thermo Electron). Km was determined from rates at various substrate concentrations using Michaelis–Menten equation from GraphPad Prism software. A substrate concentration equivalent to the Km value for each PTP was used for the screening of the compound. Phosphatases and compounds were preincubated 2–5 min prior to addition of DiFMUP for enzyme inhibition quantificati

Methods

Results

Discussion

Conclusion