A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat

Abstract

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

GDNF receptor agonist supports dopamine neurons in vitro and protects their function in animal model of Parkinson’s

“…To provide a quantitative estimate on the level of intracellular signaling cascade stimulation in response to BT13, we used luciferase reporter gene-based assay reflecting the level of ERK activation (Sidorova et al , 2010). In line with Western blotting data (Fig 1G-I), RET phosphorylation data (Fig 1A-C, D-F) and published reports (Sidorova et al , 2017), BT13 increased luciferase activity in a concentration dependent manner in reporter cell lines expressing GFRα1/RET (F (6, 14) = 43.78, P < 0.0001, one-way ANOVA) and RET (F (6, 14) = 19.85, P < 0.0001, one-way ANOVA). BT13 in concentrations 25 and 50 μM increased luciferase activity in GFRα1/RET expressing cells by 8.6 fold and 11.5 fold, respectively (P < 0.0001), and in RET expressing cells by 11.9 fold (P =0.0040) and 21.7 fold, respectively (p<0.0001, post hoc comparisons with Dunnett’s test in all cases) (Fig.…”

“…Cells lysates for analysis of RET, pERK and pAKT phosphorylation in response to BT13 were prepared from MG87RET cells transfected with hGFRα1, hGFRα2 and GFP-expressing plasmids as described previously (Sidorova et al , 2017).…”

“…Levels of pERK and pAKT in immortalized cells were analyzed by Western blotting using E4 pERK (1:1000, Santa Cruz Biotechnology Cat# sc-7383, RRID:AB_627545) and pAKT (1:500, Cell Signaling Technology Cat# 9271, RRID:AB_329825) antibodies as described previously (Sidorova et al , 2017). Membranes were probed with GAPDH antibody (1:4000, Millipore Cat# MAB374, RRID:AB_2107445) to confirm equal loading.…”

“…Previously, we discovered a small molecule, BT13, that selectively activates GFL receptor-dependent signaling in immortalized cells, supports sensory neurons and alleviates neuropathy in rats (Sidorova et al, 2017). In the present study, we evaluated the biological effects of BT13 in dopamine system.…”

Molecular and cellular correlates of human nerve regeneration: ADCYAP1 encoding PACAP enhances sensory neuron outgrowth

“…In fact, function blocking antibodies to PAC1 are undergoing clinical testing for migraine treatment 42 and given our findings, care will need to be taken that such treatments do not impair sensory neuron function in the context of coexisting neuropathy. One means of optimising delivery to promote neurite outgrowth of small fibres would be to develop small molecule agonists of PAC1 which can be given locally as has been achieved for other growth promoting molecules such as GDNF 43 .…”