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

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 ?1 (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.Peer reviewe

Small-Molecule Ligands that Bind the RET Receptor Activate Neuroprotective Signals Independent of but Modulated by Coreceptor GFR alpha 1

Glial cell line-derived neurotrophic factor (GDNF) binds the GFR alpha 1 receptor, and the GDNF-GFR alpha 1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFR alpha 1-RET signaling complex, agents that bind and activate RET directly and independently of GFR alpha 1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFR alpha 1 coexpression. However, RET activation by these ligands is constrained by GFR alpha 1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor a, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.Peer reviewe

p75NTR antagonists attenuate photoreceptor cell loss in murine models of retinitis pigmentosa

ProNGF signaling through p75NTR has been associated with neurodegenerative disorders. Retinitis pigmentosa (RP) comprises a group of inherited retinal dystrophies that causes progressive photoreceptor cell degeneration and death, at a rate dependent on the genetic mutation. There are more than 300 mutations causing RP, and this is a challenge to therapy. Our study was designed to explore a common mechanism for p75NTR in the progression of RP, and assess its potential value as a therapeutic target. The proNGF/p75NTR system is present in the dystrophic retina of the rd10 RP mouse model. Compared with wild-type (WT) retina, the levels of unprocessed proNGF were increased in the rd10 retina at early degenerative stages, before the peak of photoreceptor cell death. Conversely, processed NGF levels were similar in rd10 and WT retinas. ProNGF remained elevated throughout the period of photoreceptor cell loss, correlating with increased expression of α2-macroglobulin, an inhibitor of proNGF processing. The neuroprotective effect of blocking p75NTR was assessed in organotypic retinal cultures from rd10 and RhoP mouse models. Retinal explants treated with p75NTR antagonists showed significantly reduced photoreceptor cell death, as determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and by preservation of the thickness of the outer nuclear layer (ONL), where photoreceptor nuclei are located. This effect was accompanied by decreased retinal-reactive gliosis and reduced TNFα secretion. Use of p75NTR antagonist THX-B (1,3-diisopropyl-1-[2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea) in vivo in the rd10 and RhoP mouse models, by a single intravitreal or subconjunctival injection, afforded neuroprotection to photoreceptor cells, with preservation of the ONL. This study demonstrates a role of the p75NTR/proNGF axis in the progression of RP, and validates these proteins as therapeutic targets in two different RP models, suggesting utility irrespective of etiology.Fil: Platón-Corchado, María. Consejo Superior de Investigaciones Científicas; EspañaFil: Barcelona, Pablo Federico. Mc Gill University. Lady Davis Research Intitute; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Jmaeff, Sean. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Marchena, Miguel. Consejo Superior de Investigaciones Científicas; EspañaFil: Hernández-Pinto, Alberto M.. Consejo Superior de Investigaciones Científicas; EspañaFil: Hernández-Sánchez, Catalina. Consejo Superior de Investigaciones Científicas; EspañaFil: Saragovi, H. Uri. Mc Gill University. Lady Davis Research Intitute; CanadáFil: de la Rosa, Enrique J. Centro de Investigaciones Biológicas; Españ

p75NTR and Its Ligand ProNGF Activate Paracrine Mechanisms Etiological to the Vascular, Inflammatory, and Neurodegenerative Pathologies of Diabetic Retinopathy

In many diseases, expression and ligand-dependent activity of the p75NTR receptor can promote pericyte and vascular dysfunction, inflammation, glial activation, and neurodegeneration. Diabetic retinopathy (DR) is characterized by all of these pathological events. However, the mechanisms by which p75NTR may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75NTR is upregulated very early in glia and in pericytes to mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood–retina barrier breakdown, edema, and neuronal death. In a mouse model of oxygen-induced retinopathy, mimicking proliferative DR, p75NTR-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75NTR or antagonists of the ligand proNGF suppresses each distinct phase of pathology, ameliorate disease, and prevent disease progression. Thus, our study documents novel disease mechanisms and validates druggable targets for diabetic retinopathy.This work was supported by the Canadian Institutes of Health Research and the Foundation to Fight Blindness to H.U.S. and the Canadian Diabetes Association to P.S

Alternative splicing of a receptor Intracellular domain yields different ectodomain conformations, enabling Isoform-Selective Functional Ligands

Events at a receptor ectodomain affect the intracellular domain conformation, activating signal transduction (out-to-in conformational effects). We investigated there verse direction (in-to-out) where the intracellular domain may impact on ectodomain conformation. The primary sequences of naturally occurring TrkC receptor isoforms(TrkC-FL and TrkC.T1) only differ at the intracellular domain. However, owing to their differential association with Protein Disulfide Isomerase the isoforms have different disulfide bonding and conformations at the ectodomain. Conformations were exploited to develop artificial ligands, mAbs, and small molecules, with isoform-specific binding and biased activation. Consistent, the physiological ligandsNT-3 and PTP-sigma bind both isoforms, but NT-3 activates all signaling pathways,whereas PTP-sigma activates biased signals. Our data support an "in-to-out" model controlling receptor ectodomain conformation, a strategy that enables heterogeneityin receptors, ligands, and bioactivity. These concepts may be extended to the many wild-type or oncogenic receptors with known isoforms.Fil: Brahimi, Fouad. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Galan, Alba. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Jmaeff, Sean. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Barcelona, Pablo Federico. Mc Gill University. Lady Davis Research Intitute; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: De Jay, Nicolas. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Dejgaard, Kurt. McGill University; CanadáFil: Young, Jason C.. McGill University; CanadáFil: Kleinman, Claudia Laura. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Thomas, David Y.. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Saragovi, H. Uri. Mc Gill University. Lady Davis Research Intitute; Canad