Cav2.2

近日，纽约大学的研究人员通过在潜在新药物库中筛选了约2700万种药物分子，发现了一种能够缓解四种不同类型慢性疼痛的小分子。这种小分子通过与钙离子通道蛋白的内部区域结合来间接调节钙离子通道。值得一提的是，在动物实验中，这种化合物表现出优于现有获批药物的效果，并且没有明显副作用。这项研究现已整理发表在《美国科学院院刊》（PNAS）上。 钙离子通道在疼痛信号的传递中起着核心作用，其中部分机制是通过释放神经递质（如谷氨酸和GABA）。钙离子通道Cav2.2是三种临床上常用药物的作用靶点，其中包括gabapentin和pregabalin，这些药物广泛应用于治疗神经痛和癫痫。其中，gabapentin通过与钙离子通道Cav2.2的外部结构结合来影响通道的活性，进而减轻疼痛。然而，和许多止痛药一样，gabapentin的使用也可能伴随着副作用。 CRMP2是钙离子通道Cav2.2的一个重要调节因子，能够与通道的内部结构结合。研究人员此前发现了一种源自CRMP2的肽段CBD3，这段肽链能使CRMP2从钙离子通道中解耦。当CBD3被引入细胞时，它就像诱饵一样，阻止了CRMP2与钙离子通道内部的结合。这种作用减少了进入钙离子通道的钙离子，降低了神经递质的释放，进而在动物实验中有效减轻了疼痛。 肽类药物通常很难合成，这主要是因为它们的作用时间短暂且容易在胃中分解。研究人员尝试在CBD3肽的基础上开发一种小分子药物，他们从构成CBD3肽的15个氨基酸出发，确定了其中的两个关键氨基酸。研究显示，这两个氨基酸对抑制钙离子流入和减轻疼痛至关重要。研究团队与匹兹堡大学的研究人员合作，利用计算机模拟筛选了一个由2700万种化合物组成的化合物库，以寻找能与CBD3肽的关键氨基酸“匹配”的小分子。 经过模拟筛选，研究人员将化合物库中的候选药物分子范围从数百万种缩小到了77种。接下来，他们对这些化合物进行了实验测试，以验证它们是否能有效减少钙离子流入。为了评估这些化合物的效果，研究团队采用了电生理学方法，这种方法可以测量通过钙离子通道的电流变化，从而确定这些化合物是否降低了钙离子通道的活性。 研究人员在筛选出的化合物中发现了一种特别有前景的候选化合物分子，并将其命名为CBD3063。通过生化测试，研究团队发现CBD3063能够干扰钙离子通道CaV2.2和CRMP2蛋白之间的相互作用。这种干扰减少了钙离子进入通道的量，并且降低了神经递质的释放。随后，研究人员继续使用小鼠模型对CBD3063进行了测试，以探究其对于与受伤相关疼痛的影响。实验结果表明，CBD3063能够有效地减轻雄性和雌性小鼠的疼痛。特别值得一提的是，在一项“头对头”研究中，研究人员发现，为了达到减轻疼痛的效果，所需的CBD3063剂量（1至10 mg）远低于获批药物gabapentin（30 mg，活性对照药物）。 “尽管许多科学家也对同样的化合物库进行了筛选，但他们的方法主要是试图从外部阻断钙离子通道。相比之下，我们关注的目标是CRMP2上的这两个氨基酸，它们位于细胞内部。我们采用的这种间接方法可能是我们获得成功的关键因素。”文章的作者解释道。 为了评估CBD3063在治疗不同类型慢性疼痛方面的潜力，研究团队与多家研究机构的研究人员展开了合作，并在动物模型上进行了多项研究，以探索CBD3063对化疗引起的神经病变、炎症性疼痛和三叉神经痛的治疗效果。这些研究均成功地改善了疼痛症状，效果与活性对照药物相当。然而，与之不同的是，CBD3063的使用没有引发副作用，如镇静、记忆和学习能力的变化，或心率和呼吸的改变。 研究团队正在继续对CBD3063进行深入研究，优化其化学结构，并进行更多测试，以评估其安全性和是否会产生耐药性。未来，研究人员计划将基于CBD3063的药物引入临床试验，为安全且有效地缓解疼痛提供新的治疗选项。 参考资料：[1] Gomez K. et al. “A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain” (2023) 120 (47) e2305215120. DOI:https://doi.org/10.1073/pnas.2305215120 [2] New Compound Outperforms Pain Drug by Indirectly Targeting Calcium Channels，Retrieved November 16th, 2023 from https://www.nyu.edu/about/news-publications/news/2023/november/PNAS-CBD3-compound-pain.html#:~:text=Khanna's%20lab%20then%20tested%20CBD3063,30%20mg)%20to%20reduce%20pain. [3] Small molecule alleviates pain more effectively than gabapentin, minus the side effects，Retrieved November 16th, 2023 from https://www.fiercebiotech.com/research/drug-alleviates-pain-more-effectively-gabapentin-minus-side-effects 内容来源于网络，如有侵权，请联系删除。

无论是自由形式，还是与眼镜蛇毒素calciseptine、氨氯地平，胺碘酮与索非布韦结合，Cav1.2通道都呈现出一致的失活构象。在解析多种药物调控Cav1.2的作用机制的过程中，该研究获得三个重要的 颜宁教授联合武汉大学药学院高帅教授、北京大学宋晨研究员，在 Cell 期刊发表了题为：Structural basis for human Cav1.2 inhibition by multiple drugs and the neurotoxin calciseptine 的研究论文。 该研究揭示了自由状态、多种药物结合、多肽毒素结合的钙离子通道Cav1.2的高分辨结构。 无论是自由形式，还是与眼镜蛇毒素calciseptine、氨氯地平，胺碘酮与索非布韦结合，Cav1.2通道都呈现出一致的失活构象。在解析多种药物调控Cav1.2的作用机制的过程中，该研究获得三个重要的非预期发现： 第一，尽管Cav1.2 与Cav1.3同属于L型钙离子通道，在功能及序列上高度同源，但Cav1.2在部分结构特性上更接近于神经钙离子通道Cav2.2/Cav2.3。具体而言，Cav1.2，Cav2.2，Cav2.3的电压感受结构域II （VSDII）处于down构象；Cav1.1，Cav1.3的VSDII则处于up构象； 第二，该研究解析了眼镜蛇毒素calciseptine致死的分子机制，颠覆了以往对动物毒素调控离子通道功能的认知。经典的动物毒素结合模式一般是通过结合在离子孔结构域阻碍离子运输，或者结合在电压感受结构域变构调控离子通道功能。在本工作中，Calciseptine结合在Cav1.2离子孔外周的肩膀区域，这种崭新的结合模式稳定了Cav1.2通道失活状态的构象，导致蛋白功能抑制。毒素及蛋白定点突变电生理研究进一步验证了该结合模式； 第三，该研究在解析胃肠道解痉剂匹维溴铵调控机制时，发现了Cav1.2通道的全新失活构象。在该构象中，VSDII呈现up状态，离子孔扩大，选择性滤器（SF）构象改变。与此同时，重复单元III的S4-S5与S5两个α螺旋元件融合成单个螺旋，导致细胞内孔（Intracellular gate）变宽。除匹维溴铵外，氨氯地平等其他药物分子不能结合该构象。通过分子动力学模拟证实，这个孔道结构域变大的构象仍不能通透钙离子，通道蛋白处于失活状态。 武汉大学药学院高帅教授、深圳医学科学院颜宁教授、北京大学宋晨研究员为该论文共同通讯作者。武汉大学药学院高帅教授、姚霞教授、清华大学陈姣凤博士为论文共同第一作者。武汉大学药学院为文章第一完成单位。清华大学化学系刘磊教授、圣约翰大学于勇教授为该工作提供了关键技术支持。 这是高帅教授在Nature（2021）、Cell Research（2022）、Cell（2022）、Nature Communications（2022）杂志发表钙离子通道镇痛药物齐考诺肽、抗晕动症药物桂利嗪，丙肝神药索非布韦副反应作用机制后，在该领域的又一系统性、突破性进展，为钙离子通道蛋白新型靶向药物研发奠定了重要的理论基础。

Researchers studied the effects of their compound, CBD3063, in multiple models of acute and chronic pain. A small molecule alleviates four different types of acute and chronic pain in rodents, so far with none of the side effects associated with widely prescribed non-opioid painkiller gabapentin. In an article published Nov. 13 in Proceedings of the National Academy of Sciences, researchers from New York University described how they discovered their compound, dubbed CBD3063, and tested it in models of injury- and chemotherapy-induced neuropathy, trigeminal nerve pain and inflammatory pain. They hope to eventually take it to the clinic. “The compound can be given [via] multiple routes, it works in different species—mice and rats—and in males and females,” senior author Rajesh Khanna, Ph.D., who is also working on a gene therapy for chronic pain, told Fierce Biotech Research in an interview. “No toxicity has been seen, and it works across an array of pain conditions. That’s really the crux of this paper.” CBD3063, gabapentin and pregabalin all ultimately exert their effects through their interactions with a calcium channel called Cav2.2 on pain-sensing neurons, but they do so via different mechanisms. Where gabapentin acts on the outside of the channel, CBD3063 alters it from the inside by stopping a protein called CRMP2 from binding to it. Decoupling the two keeps calcium from entering the channel, which ultimately leads to less neurotransmitter release—and pain reduction. Khanna’s lab first discovered the peptide that would become the basis for CBD3063 back in 2011. Since then, the team has worked on formulating it into a small molecule, figuring out that they could take the number of necessary amino acids down from 15 to just two and pairing those with a protein taken from the HIV virus to help it get into cells. As part of the new study, they worked with the University of Pittsburgh to run a large screen of 27 million existing drug compounds to see whether any had the same composition. The results led them to CBD3063. After a barrage of cell studies to show that the compound did indeed inhibit CRMP2 and was highly selective for Cav2.2—an important consideration, as drugs for arrhythmias and hypertension target similar channels—the researchers tested it in double-blinded experiments on a mouse model of nerve injury, assessing it head-to-head with gabapentin. They found that mice who received the compound were less debilitated by pain than untreated mice. On top of that, CBD3063 worked at a fraction of gabapentin’s effective dose.  “We were getting the same amount of relief [with 1 to 10 milligrams per kilogram of CBD3063] that the clinically available compound gabapentin was producing at 30 milligrams per kilogram,” Khanna said. “That’s very important, because now we are comparing it to a clinical benchmark.” The researchers then conducted experiments on rodents that had nerve damage caused by treatment with paclitaxel, a chemotherapy drug. After giving four doses of paclitaxel over eight days, the researchers waited for the animals to develop nerve damage and an aversion to cold, another common symptom experienced by people who undergo chemotherapy. When the animals developed symptoms two or three weeks later, the team gave them single doses of CBD3063. “We saw a beautiful reversal of both nerve pain and cold aversion,” Khanna said. Next, the researchers looked at how well the drug could control orofacial pain. Until this set of experiments, the scientists had been giving the drug through the animals’ abdomens via what’s known as intraperitoneal delivery. This time, they tried giving it intranasally, thinking that a nasal spray formulation would be ideal for this kind of scenario. “It also bypasses the blood-brain barrier, and it goes right into the regions it needs to,” Khanna said. Once again, the drug worked: It relieved pain consistent with the expected half-life of the compound in such a formulation, he said, which is a little more than three hours. His team then moved to their final models. An assessment of whether CBD3063 could relieve chronic pain from inflammatory conditions like arthritis showed that single injections of the drug into mice with paw inflammation relieved their pain for at least four hours afterward. And an experiment on rats with a chronic nerve injury showed that they didn’t develop tolerance to the drug even with multiple injections over a two-week period. But the researchers weren’t done yet: They wanted to be sure that CBD3063 didn’t block out unrelated types of pain or replicate gabapentin’s side effects, which include memory loss, mood changes, lethargy and more. Experiments on uninjured mice showed that the drug had no effect on types of pain that were unrelated to the type of neuropathy it was designed to treat. Meanwhile, gabapentin seemed to make mice less responsive to important acute pain signals. As for side effects, the scientists found that abdominal injections of CBD3063 had no impact on rodents’ memory or cognition—unlike gabapentin, which dramatically diminished their ability to recognize objects. Their compound also had no effects on the animals’ cardiovascular function, which case studies suggest may be impacted by gabapentin. “This demonstrates that CBD3063 preserves that adaptive role of pain without causing any kind of neurological side effects,” Khanna said. “That’s really the benefit.” There’s still a long road to the clinic. The researchers will need to do many more experiments to understand the drug’s long-term effects as well as whether tolerance eventually arises. And while CBD3063 has never been used for pain relief, it is not technically a new compound. Thus, Khanna’s team will need to substantially transform and optimize it before they can patent it, then will need to partner with a pharmaceutical company to take it into development, he said. “I don’t want to provide any false hope, because the path to humans is three to five years away at the very least,” Khanna said. “In order to get there, there’s a huge journey ahead.”