Study On Molecular Mechanism Of Novel 4-amino Pepidine Derivatives On N-type Ca～(2+) Channels

Many diseases have chronic pain as an important symptom. So, Searching high effective and non-opioid analgesic agent is our goal for people. In recent years, drug discovery efforts focused on novel potent analgesic agents with no addict and new action mechanism.Therefore, there are great progresses in the researches of novel analgesic agents targeted N-type calcium channel.Recently, drug discovery efforts have been focused on small molecule N-type calcium channel blockers for analgesia. To look for no addictive analgesia and higher selective blockers for N-type Ca²⁺ channels, a series of new compounds were designed and synthesized in our institute by optimizing the 4-amino-piperidine template. Here, we observed the effects of a series of new derivates on N-type VDCCs expressed in oocytes and HEK-293 cells. Meanwhile, in vivo pharmacology was evaluated by STZ-induced diabetic neuropathic pain and mouse acetic writhing.In our experiment, the effects of 59 new 4-amino-piperidine derivates on N-type calcium channel expressed in oocytes were observed by two-electrode voltage clamp technique firstly. Then, we further observed the effects of ZC101 on N-type VDCCs (α_1B/β_1b/α₂δ) expressed in oocytes. ZC101 inhibited the channels with concentration-dependence manner and the effect of ZC101 was completely reversed. To evaluate mechanism of action ZC101, We constructed the four recombinant of subunits expressed in Xenopus oocytes.The results showed that inhibitory effects of ZC101 on four recombinant of subunits were similar. The I-V relationship showed the inhibitory effect of ZC101 on N-type I_Ba was voltage-independence. The steady-state inactivation relationship was shifted to more negative potentials after exposure to ZC101. ZC101 could block the expressed N-type calcium channels at resting states. ZC101 high selectively targeted N-type calcium channels, other than I_Na, I_k(DR),I_k(A) and R, L, P/Q-type calcium channels.We further observed the effects of ZC88 and ZC1 on N-type VDCCs (α_1B/β_1b/α₂δ) expressed in HEK-293 cells by patch clamp technique. The result showed that ZC88 and ZC1 inhibited the channel current with concentration-dependence manner and the inhibition effect of ZC88 was partly reversed after wash-out for 5 min. However, the inhibition effect of ZC1 was irreversed. The I-V relationship also demonstrated the inhibitory effects of ZC88 and ZC1 on N-type I_Ba were voltage-dependence inhibition. The steady-state inactivation relationship was shifted to more negative potentials after exposure to ZC88 and ZC1. ZC88 accelerated the decay of N-type VDCCs. ZC88 selectively targeted N-type calcium channels, other than I_Na, I_k(DR), I_k(A) and R, L, P/Q-type calcium channels. Then, ZC1 blocked L-type calcium channels expressed in oocytes. In competitive radioactive ligand binding test, which suggests ZC88 and ZC101 don't bind opioid receptor.ZC88 and ZC101 produced obvious analgesia on mouse acetic acid writhing test and STZ-induced diabetic neuropathic pain model.In conclusion: Molecular mechanism of action ZC88 and ZC101 on N-type VDCCs was observed detailed firstly. It would be find no addict, highly selective, non-peptide and potential analgesic. It is very important for chronic pain patient to find potential analgesic.