4-氨基哌啶类新化合物阻断N型钙离子通道的分子机制研究
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摘要
长期以来，人类为了提高生活质量，缓解慢性疼痛，一直在寻找高效的非阿片类镇痛药。随着疼痛与N型钙离子通道之间关系的深入研究，人们发现，如果能研究出特异性、非阿片类、可口服、非肽类的N型钙通道阻断剂对解决慢性疼痛将是个突破。因此，近年来非肽类小分子化合物逐渐成为N型VDCCs阻断剂研究的新方向。为此，本所药物合成室以4-氨基哌啶为母核，合成了一系列非肽类小分子化合物。本研究的目的是拟从这一系列新化合物中发现出具有特异性N型钙通道阻断作用的化合物，并详细地探讨其对N型钙通道作用的分子机制，以寻找出更加理想的治疗慢性疼痛的新型化合物。
在爪蟾卵母细胞瞬时表达人N型VDCCs的模型上，采用了双电极电压钳技术，初步评价了59个化合物。而后详细观察了ZC88及ZC101对N型VDCCs的作用机制。
ZC101对爪蟾卵母细胞瞬时表达人N型VDCCs(α_(1B)／β_(1b)／α_2δ)的阻断作用呈明显的浓度依赖抑制但不具有电压依赖抑制，其IC_(50)为2.3±0.29μM；功能亚基与不同辅助亚基构成的重组体，ZC101对其阻断作用及可逆性均未发生改变，ZC101可能是作用于功能亚基来发挥阻断作用的；ZC101使通道电压依赖稳态失活曲线左移，这可能加速了通道的关闭；ZC101还作用于通道的静息态；ZC101对I_(Na)、I_(k(DR))、I_(k(A))及R、L、P／Q型VDCCs不具有抑制作用；
在HEK-293细胞瞬时表达人N型VDCCs的模型上，采用膜片钳记录技术详细观察了ZC88对N型VDCCs(α_(1B)／β_(1b)／α_2δ)的作用机制。结果发现，ZC88及ZC1(对照)对N型VDCCs的阻断作用呈浓度依赖抑制；IC_(50)分别为0.45±0.09μM和0.34±0.04μM。ZC88的阻断作用具有可逆性而ZC1不具有可逆性。ZC88及ZC1呈电压依赖抑制N型VDCCs；ZC88及ZC1使通道的电压依赖稳态失活曲线左移；ZC88加速了通道的衰减；ZC88对I_(Na)、I_(k(DR))、I_(k(A))及R、L、P／Q型VDCCs不具有抑制作用。而ZC1对L型钙通道具有抑制作用；配体竞争结合实验证实ZC88及ZC101与阿片受体不具有亲和力。
ZC101及ZC88在小鼠醋酸扭体模型及STZ诱导的大鼠糖尿病性神经痛模型上均具有理想的镇痛作用。
基于上述研究，本文的主要结论为：4-氨基哌啶类新化合物的作用机制首次在分子水平上进行了深入研究，ZC88及ZC101有可能成为N型钙通道特异性阻断剂，这可能是为临床找到了新的非阿片类、无成瘾性、可口服、选择性良好的潜在镇痛药物，可能对解决慢性疼痛具有重要的现实意义。
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 Ca~(2+) 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)/α_2δ) 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)/α_2δ) 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.
在爪蟾卵母细胞瞬时表达人N型VDCCs的模型上，采用了双电极电压钳技术，初步评价了59个化合物。而后详细观察了ZC88及ZC101对N型VDCCs的作用机制。
ZC101对爪蟾卵母细胞瞬时表达人N型VDCCs(α_(1B)／β_(1b)／α_2δ)的阻断作用呈明显的浓度依赖抑制但不具有电压依赖抑制，其IC_(50)为2.3±0.29μM；功能亚基与不同辅助亚基构成的重组体，ZC101对其阻断作用及可逆性均未发生改变，ZC101可能是作用于功能亚基来发挥阻断作用的；ZC101使通道电压依赖稳态失活曲线左移，这可能加速了通道的关闭；ZC101还作用于通道的静息态；ZC101对I_(Na)、I_(k(DR))、I_(k(A))及R、L、P／Q型VDCCs不具有抑制作用；
在HEK-293细胞瞬时表达人N型VDCCs的模型上，采用膜片钳记录技术详细观察了ZC88对N型VDCCs(α_(1B)／β_(1b)／α_2δ)的作用机制。结果发现，ZC88及ZC1(对照)对N型VDCCs的阻断作用呈浓度依赖抑制；IC_(50)分别为0.45±0.09μM和0.34±0.04μM。ZC88的阻断作用具有可逆性而ZC1不具有可逆性。ZC88及ZC1呈电压依赖抑制N型VDCCs；ZC88及ZC1使通道的电压依赖稳态失活曲线左移；ZC88加速了通道的衰减；ZC88对I_(Na)、I_(k(DR))、I_(k(A))及R、L、P／Q型VDCCs不具有抑制作用。而ZC1对L型钙通道具有抑制作用；配体竞争结合实验证实ZC88及ZC101与阿片受体不具有亲和力。
ZC101及ZC88在小鼠醋酸扭体模型及STZ诱导的大鼠糖尿病性神经痛模型上均具有理想的镇痛作用。
基于上述研究，本文的主要结论为：4-氨基哌啶类新化合物的作用机制首次在分子水平上进行了深入研究，ZC88及ZC101有可能成为N型钙通道特异性阻断剂，这可能是为临床找到了新的非阿片类、无成瘾性、可口服、选择性良好的潜在镇痛药物，可能对解决慢性疼痛具有重要的现实意义。
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 Ca~(2+) 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)/α_2δ) 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)/α_2δ) 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.
引文
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88．刘国凯，黄宇光，罗爱伦．加巴喷丁用于神经病理性疼痛治疗的研究进展．中国临床药理学与治疗学．2003，3-12．
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88．刘国凯，黄宇光，罗爱伦．加巴喷丁用于神经病理性疼痛治疗的研究进展．中国临床药理学与治疗学．2003，3-12．