Novel RET agonist for the treatment of experimental neuropathies

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) alleviate symptoms of experimental neuropathy, protect and stimulate regeneration of sensory neurons in animal models of neuropathic pain, and restore their functional activity. However, clinical development of GFL proteins is complicated by their poor pharmacokinetic properties and multiple effects mediated by several receptors. Previously, we have identified a small molecule that selectively activates the major signal transduction unit of the GFL receptor complex, receptor tyrosine kinase RET, as an alternative to GFLs, for the treatment of neuropathic pain. We then introduced a series of chemical changes to improve the biological activity of these compounds and tested an optimized compound named BT44 in a panel of biological assays. BT44 efficiently and selectively stimulated the GFL receptor RET and activated the intracellular mitogene-activated protein kinase/extracellular signal-regulated kinase pathway in immortalized cells. In cultured sensory neurons, BT44 stimulated neurite outgrowth with an efficacy comparable to that of GFLs. BT44 alleviated mechanical hypersensitivity in surgery- and diabetes-induced rat models of neuropathic pain. In addition, BT44 normalized, to a certain degree, the expression of nociception-related neuronal markers which were altered by spinal nerve ligation, the neuropathy model used in this study. Our results suggest that the GFL mimetic BT44 is a promising new lead for the development of novel disease-modifying agents for the treatment of neuropathy and neuropathic pain.Peer reviewe

Characterization of small molecule GDNF family ligand mimetic BT44 for the treatment of neuropathic pain

Neuropaattinen kipu on on hermojen vahingoittumisesta johtuva krooninen sairaus, joka vaikuttaa miljoonien ihmisten elämään maaimanlaajuisesti. Parantavaa hoitoa ei ole keksitty, ja kivun hallinnassa käytetään masennuslääkkeitä ja opioideja, jotka vaikuttavat kivun välittymiseen mutta eivät vahingoittuneisiin neuroneihin. GDNF-perheeseen kuuluvat gliasoluperäinen hermokasvutekijä (GDNF) ja artemiini ovat tutkimuksissa entisöineet vahingoittuneita neuroneita. Näiden hermokasvutekijöiden käyttö klinikoissa on rajoittunutta, koska GDNF:n ja artemiinin annostelu ja tuotanto ovat haasteellisia. Tästä johtuen olemme kehittäneet GDNF-perheen ligandeja matkivan molekyylin nimeltä BT44. Molekyylin karakterisointi alkoi in vitro-kokeilla, joissa tutkimme BT44:n kykyä indusoida lusiferaasin produktiota GDNF:n ja artemiinin reseptoreita (GFRalpha1 ja RET sekä GFRalpha3 ja RET) ekspressoivissa soluissa. Tutkimme myös BT44:n indusoimaa RET fosforylaatiota ja MAPK/ERK ja Pi3-K/Akt-prosessien aktivaatiota. Lisäksi vertasimme BT44:n ja artemiinin kykyä muodostaa neuriitteja soluviljellyistä rotan spinaaliganglioista. In vitro-kokeet osoittivat BT44:n aktivoivan GDNF:n ja artemiinin reseptoreita, indusoivan RET-fosforylaatiota, aktivoivan MAPK/ERK ja Pi3-K/Akt-prosesseja, ja stimuloivan neuriittien kasvua soluviljellyistä spinaaliganglioista. Näiden lupaavien tulosten jälkeen siirryimme in vivo-kokeisiin rotilla. Käytimme SNL-mallia, jossa spinaalihermo sidotaan, ja aiheuttaa kiputilan rotassa. BT44 lievensi mekaanista nosiseptiota ja kylmäkipua SNL-rotissa. Lisäksi havaitsimme BT44:n normalisoivan kipumarkkereita SNL-rottien kudoksissa. Tulokset ovat verrannollisia aikaisempien tutkimusten kanssa. Näiden lupaavien tulosten johdosta ehdotamme BT44:kehittämistä lupaavana ja innovatiivisena neuropaattisen kivun hoitokeinona.Neuropathic pain is pain caused by injury or damage to the nervous system. This adverse condition affects millions of people in all parts of the world, and no known cure has been developed. Existing treatments are mainly anti-depressants or opioids that alleviate symptoms instead of repairing damaged neurons. Glial cell line-derived neurotrophic factor (GDNF) and artemin, belonging to GDNF family ligands, have been shown to restore damaged neurons. However due to the poor pharmaceutical properties of these proteins, such as difficult administration and expensive production, their transition to clinics is complicated. That is why we have been developing small molecule GFL-mimetics as an alternative. One of these mimetics is a compound named BT44. Characterization of BT44 began with in vitro experiments, where we tested the compound’s ability to activate luciferase reporter gene in cells expressing GDNF (GFRalpha1 and RET) and artemin (GFRalpha3 and RET) receptors, as well as ability to induce RET phosphorylation and activate intracellular MAPK/ERK and Pi3-K/Akt pathways. Furthermore, we tested stimulation of neurite outgrowth by the compound from cultured dorsal root ganglion neurons. In a similar manner to GDNF and artemin, BT44 was shown to activate GFRalpha1/RET and GFRalpha3/RET receptors and induce RET phosphorylation and intracellular signaling, in addition to stimulating neurite outgrowth from cultured DRG neurons. Because of the promising in vitro results, we moved on to in vivo testing in rat spinal nerve ligation (SNL) model of neuropathic pain. Similarly to artemin, BT44 was able to alleviate mechanical nociception and cold allodynia in SNL rats. In addition, we found that BT44 normalized to a certain degree nociception-related markers influenced by SNL in the tissues of experimental animals, which emulates previously published results for artemin. To summarize, our results indicate that BT44 is effective in neuronal restoration and pain alleviation, suggesting it for further development as innovative neuropathic pain treatment