| Currently, neuropathic pain treatment is a major challenge. Due to the complexmechanism of neuropathic pain, there is still no ideal clinical medicine to effectivelytreat it. Current therapy strategies mainly consist of antidepressants, antiepilepticdrugs, local anesthetics, opioids and nonsteroidal anti-inflammatory drugs. Theclinical application of these drugs is often unsatisfactory because of the side effects.Thus, it is very important to unravel the pathogenesis of neuropathic pain and toidentify the new potential targets in the case of finding new compounds of treatingneuropathic painRecent studies found that glial including microglia and astrocytes activation wasclosely related with pain. Numerous studies show that glial cells play an importantrole in the occurrence and development of neuropathic pain. So far, in manyneuropathic pain models the phenotype and function of glials was changed, whichoccurs in the spinal cord, peripheral nerves and some brain regions. The inhibition ofglials can prevent or treat neuropathic pain, suggesting that glials may play animportant role in the development of neuropathic pain.Studies have shown that the expression of P2X4receptor was significantlyincreased in activated microglia after nerve injury, which indicated that P2X4receptorplayed a key role in the occurrence of neuropathic pain. Tsuda et al found that themechanical pain threshold of rats was gradually decreased after nerve injury, and theprotein level of P2X4receptor in the damage side of spinal dorsal was graduallyincreased. In addition, immunohistochemical experiments showed that P2X4receptorshad higher expression in microglia. The mechanical pain threshold was decreased inpain models of rats caused by autoimmune neuritis, chronic constriction injure andformalin, meanwhile the expression of P2X4receptor was gradually increased in theserats. These studies suggest that the expression of P2X4receptors in activatedmicroglia may play an important role in the development of nerve injury induced painand inflammation occurrence.Tsuda et al found that spinal cord injury induced the decrease of mechanical painthreshold and activation of microglia, which can be reversed by P2X receptorsantagonist TNP-ATP but not PPADS. Furthermore, intrathecal injection of microgliaactivated by ATP treatment in rats induced the decrease of mechanical withdrawalthreshold, which can also be reversed by TNP-ATP but not by PPADS. Since P2X4receptor could be antagonized by TNP-ATP but not PPADS, we concluded thatneuropathic pain was mainly associated with P2X4receptors. In addition, afterintrathecal injection of P2X4receptor antisense oligonucleotide, hyperalgesia and P2X4receptor expression in spinal microglia was simultaneously reduced in rats.Furthermore, in P2X4receptor knockout mice, the role of this receptor in thedevelopment of neuropathic pain was also confirmed.Taken together, the above data inferred that blockade of P2X4receptors might bebeneficial to the treatment of neuropathic pain. In the present study, the followingresearch has been done.Objective On the basis of P2X4receptor and its antagonist, we have designed newP2X4receptor antagonists through traditional computer-aided drug design andconditional drug design, and then we screened active antagonists from thesecompounds. Next, we evaluated the effect of these antagonists on neuropathic pain.Methods:1Several neuropathic pain models, including CFA model in rats, SNI model in ratsand mice, have been established. The expression of P2X4receptors and BDNF weredetected by qRT-PCR and western blot in different regions including homolateral,contralateral thalamus, amygdala, hypothalamus, medulla oblongata, spinal cord andstriatum at different times after the animal model formed.2Based on the zfP2X4receptor crystal structure, we construct hP2X4receptor throughhomology modeling. Thus, structure-based drug design and ligand-based drug designhad been used. As for structure-based drug design, the homology modeling crystalstructure was used to find the possible active binding sites. In terms of ligand-baseddrug design, pharmacophore models of TNP-ATP had been built. According to thecurrent P2X4receptor antagonists, we taped new compounds skeleton by skeletontransition. After computer aided screening, the compounds had been further screenedin cell and animal level.3The compounds was screened in SNI mice. HEK293-pEGFP-N1-rP2X4, a stablytransfected cell line, was established. Next, These compounds which were primarilyscreened in SNI mice were further screened via patch-clamp on the cell line. Weprimarily evaluated the compound which is effective in mice and on the cell.Results:1At8h after rats were injected CFA, the mRNA levels of P2X4receptor in thalamus,spinal cord and dorsal root ganglion were significantly increased, and the mRNAlevels of BDNF, a downstream signaling molecule of P2X4receptor, in the spinalcord was also significantly increased. At4d after the establishment of SNI model, theprotein expression of P2X4receptor in contralateral amygdala was significantlyincreased. At7d, the protein expression of P2X4receptor in striatum, amygdala andcontralateral hypothalamus was significantly increased. At4d after the establishmentof SNI model in mice, the mRNA levels of P2X4receptor in the homolateralamygdala and hypothalamus were increased. At7d, the mRNAlevel of P2X4receptorin homolateral amygdala was increased. These results suggested that P2X4receptorsplayed an important role in the neuropathic pain.2Structure-based drug design: based on the structure analysis, three possible active sites of P2X4receptor were identified and600compounds which could potentiallycombine with these three sites were screened through virtual screening. Ligand-baseddrug design: according to the structure of present antagonists of P2X4receptor,3333compounds were designed via a variety of methods ranging from the establishment ofpharmacophore model to skeleton transition.396and114compounds had been screened through structure-based and ligand-baseddrug design, respectively. Of these compounds,24compounds have been synthesizedthereafter.4In SNI model mouse, TNP-ATP, L-01-01, L-01-02, L-01-10, L-01-21and L-01-22showed the effective of analgesia. HEK293-pEGFP-N1-rP2X4stably transfected cellline was established. On this cell line, patch-clamp technique was applied to prove thefunction of TNP-ATP, and3compounds were validated preliminarily. TNP-ATP andL-01-01completely blocked ATP-activated P2X4receptor current, and L-01-05partially blocked ATP-activated P2X4receptor current with the inhibition ratio of41.90%.Conclusion: P2X4receptor was closely related with pathogenesis of neuropathic pain.Based on structure-based and ligand-based drug design,3933compounds weredesigned and24compounds were selected through visual screening. L-01-01,L-01-02, L-01-10, L-01-21and L-01-22were found to decrease the pain sensitivity inSNI model mouse; In HEK293-pEGFP-N1-rP2X4stably transfected cell line, L-01-01and L-01-05were found to be potentially antagonized the P2X4receptors. L-01-01was effective in SNI mice and on HEK293-pEGFP-N1-rP2X4stably transfected cellline. But it influenced the mice spontaneous activities, it may have the sedative andhypnotic effects.
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