Development Of Neuropathic Pain Inhibitor Targeting UNC5H2 Mediated Myelinated Nerve Sprouting

Neuropathic pain is a common and serious disease usually caused by nervous system damage or pathology.After nerve injury,axon guidance factor Netrin-1 and its receptor UNC5H2 participate in the growth of myelinated nerve fibers in the spinal dorsal horn,leading to the occurrence and development of neuropathic pain.Therefore,inhibiting UNC5H2 is expected to be an effective method for treating neuropathic pain.However,there is currently a lack of research on UNC5H2 inhibitors.Hence,this study attempts to efficiently and accurately screen for UNC5H2 inhibitors from a candidate compound library based on computational modeling and experimentally validate them,in order to provide novel strategies for the development as well as candidate compounds for neuropathic pain treatment.Method: In this study,we first constructed a compound library by collecting compounds from literature and databases.Then,we determined the optimal binding conformation of the UNC5H2 /Netrin-1 complex,as well as its corresponding fixed region,using ZDOCK softwear.Next,we used a multi-component,multi-target interaction prediction model based on deep learning algorithms and large-scale biomedical data to screen compounds that interact with UNC5H2.Then,we used a molecular docking method to examine the binding of the initially screened compounds with UNC5H2,and evaluated their potential to inhibit the binding between UNC5H2 and Netrin-1 through atomic-level docking,molecular superposition docking,hotspot docking,and examination of their ability to block the binding of UNC5H2 and Netrin-1,to identify the compounds with the best therapeutic potential for neuropathic pain.Subsequently,we established a neuropathic pain model and used a paw filament test to observe the changes in the mechanical withdrawal threshold and thermal withdrawal latency to verify the therapeutic effect of the candidate compounds.We also used Western Blotting technique to measure the expression of UNC5H2 in the L4-L6 spinal dorsal horn tissue of rats before and after treatment with the screened compounds,to evaluate the effects of the compounds on the binding between UNC5H2 and Netrin-1.Results: This study collected a total of 10,126 compounds,including traditional Chinese medicine(TCM)compounds,clinical drugs,and biologically active small molecules,as the candidate drug library from literature,Drugbank and tsbiochem databases.Four best conformations of UNC5H2 bound to Netrin-1 were identified,along with their corresponding fixed regions(Glu355-Ser364,Leu345-Met354,Arg348-Glu355,and Ala340-Arg349),and hotspot residues.These results indicated that the IG1 region of UNC5H2 was a crucial area for binding with Netrin-1.Using a multi-component-multi-target interaction prediction model,5,651 compounds were predicted to interact with UNC5H2.Further screening using a molecular docking model resulted in the identification of 89 compounds with good binding potential to UNC5H2,based on criteria such as interaction sites between compounds and proteins locating in IG1 region,docking binding energy <-5 kcal/mol,the amount of interactions≥ 5,compliance with four or more of Lipinski’s five rules,and number of conformations meeting criterion 1 ≥ 9 out of 10 conformations.Ten candidate compounds were selected based on the number of molecules that bound with UNC5H2 and the IG1 region being greater than 10 and 7,respectively,with a binding energy of less than 5 kcal/mol.The ability of the candidate compounds to block the formation of the UNC5H2/Netrin-1 complex was investigated,and the results showed that SB-705498 and lithospermoside were effective inhibitors of UNC5H2.Mechanical pain and thermal pain experiments showed that lithospermoside and SB-705498 both had significant therapeutic effects.However,lithospermoside had a slower onset of action than SB-705498 at the same dose and a weaker effect at a low dose(3 mg/kg),whereas its therapeutic effect was superior to that of SB-705498 at a high dose(12 mg/kg).Furthermore,Western blotting analysis showed that both SB-705498 and lithospermoside could downregulate the expression of UNC5H2,with lithospermoside being more significant than SB-705498,suggesting that these two compounds may exert their therapeutic effects on neuropathic pain by inhibiting UNC5H2.Conclusion: This study focused on UNC5H2 as a target for neuropathic pain and screened candidate compounds,including lithospermoside and SB-705498,through a multi-component/multi-target interaction prediction model and molecular docking techniques.Animal experiments validated the significant therapeutic effects of lithospermoside and SB-705498 on neuropathic pain.The findings of this study provide a reference for the treatment and mechanism research of neuropathic pain.