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P物质（Substance P）的定义

P物质（SP）是一种十一肽，在外周和中枢神经系统中都很丰富，它通常与经典的神经递质之一（最常见的是血清素（5-HT））共定位【1】。

Substance P (SP) an undecapeptide, is abundant both in the periphery and in the CNS, where it is usually co-localised with one of the classical neurotransmitters, most commonly serotonin (5- HT) 【1】.

P物质（Substance P）的相关肽

SP属于一种神经肽家族，称为速激肽，具有共同的C端序列：Phe-X-Gly-Leu-Met-NH2。三种最常见的速激肽是SP，神经激肽A（NKA）和神经激肽B（NKB）；它们的生物学作用是通过指定为NK1，NK2和NK3的特定细胞表面受体介导的，SP是NK1受体的首选激动剂，NK2受体的NKA和NK3受体的NKB【2】。

SP belongs to a family of neuropeptides known as tachykinins that share the common C-terminal sequence: Phe-X-Gly-Leu-Met-NH2. The three most common tachykinins are SP, neurokinin A (NKA), and neurokinin B (NKB); their biologic actions are mediated through specific cell-surface receptors designated NK1, NK2, and NK3, with SP the preferred agonist for NK1 receptors, NKA for NK2 receptors, and NKB for NK3 receptors 【2】.

P物质（Substance P）的发现

SP最初是由von Euler和Gaddum于1931年发现的，是一种在体外引起肠道收缩的组织提取物；在随后的几十年中，进一步研究了其生物学作用和组织分布【3】。

SP was originally discovered in 1931 by von Euler and Gaddum as a tissue extract that caused intestinal contraction in vitro; its biologic actions and tissue distribution were further investigated over subsequent decades 【3】.

P物质（Substance P）的结构特征

SP是一种11个残基的神经肽，序列为Arg-Pro-Lys-Pro-Gln-Glin-Phe-Gly-Leu-Met-NH2）【4】。在一项研究中，将SP的C端和N端片段与亲本分子进行比较，以了解它们的能力：（a）收缩分离的豚鼠回肠，（b）诱导大鼠流涎，（C）激发单个猫背角神经元，以及（d）通过小鼠颅内注射诱导抓挠。与七肽一样小的C末端片段在所有测定系统上都是有效的SP激动剂。含有五个氨基酸或更少的C末端片段最多只有弱活性。N末端片段在分离的豚鼠回肠上完全无活性。然而，在大鼠唾液和中枢神经系统测定中，N末端片段能够产生弱的SP样活性【5】。获得的结果表明，虽然SP的羧基末端对于肽的支气管活性至关重要，但氨基末端肽（多达四个残基）的丢失实际上增强了支气管收缩剂对肽的反应。这种增强的部分原因似乎是SP和SP5-11的酶促降解差异所致。数据表明，二肽基氨肽酶切割SP可以增强其生物活性【6】。SP类似物：Senktide（琥珀酰-[Asp6，Me-Phe8]SP-（6-11））是NK-3（SP-N）受体的选择性类似物，其效力是SP的20-100倍，比驻留在肌肉细胞上的NK-1（SP-P）受体的选择性类似物强约1000倍【7】。在小鼠鞘内注射后，研究了五种SP类似物对神经激肽（NK）1受体激动剂如SP，physalaemin和（p-Glu6，Pro9）-SP（6-11）（septide）诱导的舔，咬和抓挠反应的影响。肽产生类似SP的行为反应，其效力约为D-Pro9类似物D-七肽的25倍。较低剂量的（D-Arg1，D-Pro2,4，D-Phe7，D-His9，Leu11）-SP比（D-Phe7，D-His9，Leu11）-SP（6-11）显着降低了Septide诱导的反应。相反，（D-Arg1，D-Pro2,4，D-Phe7，D-His9）-SP（0.5-1.0 nmol）和（D-Phe7，D-His9）-SP（6-11）（0.5-2.0 nmol）仅抑制SP诱导的行为反应，而不抑制藻毒素或七肽诱导的反应。这项研究的结果表明，NK-1受体激动剂不一定受到含有D-His 的SP类似物的相同程度的影响【8】。P物质[D-Arg1，D-Phe5，D-Trp7,9，Leu11]SP（SpD）和[Arg6，D-Trp7,9，NmePhe8]P物质的类似物可抑制神经肽刺激的Ca2+动员，酪氨酸磷酸化和ERK激活。至关重要的是，SpD和[Arg6，D-Trp7,9，NmePhe8]SP在体内和体外抑制SCLC细胞生长并刺激SCLC细胞凋亡。SP类似物最初被表征为“广谱神经肽拮抗剂”【9】。

SP is an 11-residue neuropeptide with the sequence Arg-Pro-Lys-Pro-Gln-Glin-Phe-Gly-Leu-Met-NH2) 【4】. In a study, the C- and N-terminal fragments of SP were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity 【5】. The results obtained, indicated that while the carboxy terminal of SP is essential for peptide bronchoactivity, loss of amino terminal peptides (up to four residues) actually enhances bronchoconstrictor responses to the peptide. Part of this enhancement appears to result from differences in the enzymatic degradation of SP and SP5-11. The data suggest that cleavage of SP by dipeptidyl aminopeptidases could enhance its bioactivity 【6】. SP analogs: Senktide (succinyl-[Asp6,Me-Phe8]SP-(6-11)), a selective analog for the NK-3 (SP-N) receptor, is 20-100 times more potent than SP and about 1000-fold more potent than the selective analogs for the NK-1 (SP-P) receptor, which resides on muscle cells 【7】. Effects of five SP analogs on the licking, biting and scratching response induced by neurokinin (NK) 1 receptor agonists such as SP, physalaemin and (p-Glu6,Pro9)-SP (6-11) (septide) were studied after intrathecal injections in mice. Peptide brought about a SP-like behavioral response, and was approximately 25 times more potent than the D-Pro9 analog, D-septide. Septide-induced response was significantly reduced by lower doses of (D-Arg1, D-Pro2,4, D-Phe7, D-His9, Leu11)-SP than (D- Phe7, D-His9, Leu11)-SP (6-11). In contrast, (D-Arg1, D-Pro2,4, D-Phe7, D-His9)-SP (0.5-1.0 nmol) and (D-Phe7, D-His9)-SP (6-11) (0.5-2.0 nmol) inhibited only SP-induced behavioral response, but not physalaemin- or septide-induced response. The results of this study indicate that NK-1 receptor agonists are not necessarily affected to a same degree by SP analogs containing D-His 8. Analogues of substance P, [D-Arg1,D-Phe5,D-Trp7,9,Leu11] SP (SpD) and [Arg6,D-Trp7,9,NmePhe8]substance P can inhibit neuropeptide-stimulated Ca2+ mobilization, tyrosine phosphorylation, and ERK activation . Crucially, SpD and [Arg6,D-Trp7,9,NmePhe8] SP inhibit SCLC cell growth in vivo and in vitro and stimulate SCLC cell apoptosis. SP analogues were characterized originally as "broad spectrum neuropeptide antagonists" 【9】.

P物质（Substance P）的作用方式

SP受体是一种G蛋白偶联受体，在许多方面类似于精神病学中其他经过充分研究的受体，特别是单胺受体2。SP与其受体的相互作用激活了Gq，Gq又激活了磷脂酶C，将磷脂酰肌醇二磷酸分解为肌醇三磷酸（IP3）和二酰基甘油（DAG）。IP3作用于肌浆网中的特定受体以释放细胞内的Ca2+，而DAG通过蛋白激酶C作用于打开质膜中的L型钙通道。细胞内[Ca2+]的升高诱导组织反应。与SP所见的一系列不同的行动，有许多治疗可能性【10】。

The SP receptor is a G protein-coupled receptor, in many respects similar to other well-studied receptors in psychiatry, particularly monoamine receptors 2.  The interaction of SP with its receptor activates Gq, which in turn activates phospholipase C to break down phosphatidyl inositol bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts on specific receptors in the sarcoplasmic reticulum to release intracellular stores of Ca2+, while DAG acts via protein kinase C to open L-type calcium channels in the plasma membrane. The rise in intracellular [Ca2+] induces the tissue response. With an array of actions as diverse as that seen with SP, there is scope for numerous therapeutic possibilities 【10】.

P物质（Substance P）的功能

在中枢神经系统中，SP与情绪障碍，焦虑，压力，强化，神经发生，神经毒性和疼痛的调节有关。在消化道中，SP和其他一些速激肽是调节运动活动，离子和液体分泌以及血管功能的神经递质【11，12】。

In the central nervous system, SP is associated with the regulation of mood disorders, anxiety, stress, reinforcement, neurogenesis, neurotoxicity and pain.  In the digestive tract, SP, along with some other tachykinins, are neurotransmitters that regulate motor activity, secretion of ions and fluid, as well as vascular functions 【11，12】.

P物质（Substance P）的相关文献

1. Argyropoulos SV, Nutt DJ (2000). Substance P antagonists: novel agents in the treatment of depression. Expert Opin Investig Drugs,  9(8):1871-1875.
2. Book: Substance P and Related Tachykinins. Chapter 13: Neuropsychopharmacology: By Nadia MJ, Kramer MS.
3. Senba E, Tohyama M (1985). Origin and fine structure of substance P-containing nerve terminals in the facial nucleus of the rat:an immunohistochemical study. Exp Brain Res., 57(3):537-546.
4. Seidel MF, Tsalik J, Vetter H, Müller W (2007). Substance P in Rheumatic Diseases. Current Rheumatology Reviews, 3:17-30.
5. Piercey MF, Dobry PJ, Einspahr FJ, Schroeder LA, Masiques N (1982) Use of substance P fragments to differentiate substance P receptors of different tissues. Regulatory Peptides, 3(5-6):337-349.
6. Shore SA, Drazen JM (1988). Airway responses to substance P and substance P fragments in the guinea pig. Pulm Pharmacol., 1(3):113-118.
7. Hanani M, Chorev M, Gilon C, Selinger Z (1988). The actions of receptor-selective substance P analogs on myenteric neurons: an electrophysiological investigation. European journal of pharmacology, 153(2-3):247-253.
8. Sakurada T, Yamada T, Tan-no K, Manome Y, Sakurada S, Kisara K, Ohba M (1991). Differential effects of substance P analogs on neurokinin 1 receptor agonists in the mouse spinal cord. J Pharmacol Exp Ther.,  259:205-210
9. MacKinnon AC, Waters C, Jodrell D, Haslett C, Sethi T (2001). Bombesin and Substance P Analogues Differentially Regulate G-protein Coupling to the Bombesin Receptor. J. Biol. Chem.,   276(30):28083-28091..
10. Khawaja AM, Rogers DF (1996). Tachykinins: receptor to effector. Int J Biochem Cell Biol.,  28(7):721-738.
11.  Leeman SE, Mroz EA (1974).  Substance P. Life Sci., 15(12):2033–2044.
12. Wiesenfeld-Hallin Z, Xu XJ (1993). The differential roles of substance P and neurokinin A in spinal cord hyperexcitability and neurogenic inflammation. Regul Pept., 46(1-2):165-173

二硫键广泛存在与蛋白结构中，对稳定蛋白结构具有非常重要的意义，二硫键一般是通过序列中的2个Cys的巯基，经氧化形成。
 

形成二硫键的方法很多：空气氧化法，DMSO氧化法，过氧化氢氧化法等。
 

二硫键的合成过程，  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
   

如果多肽中只含有1对Cys，那二硫键的形成是简单的。多肽经固相或液相合成，然后在pH8-9的溶液中进行氧化。      
 

当需要形成2对或2对以上的二硫键时，合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成，但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线：
 

二硫键反应条件选择    
 

 二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基（-SH）被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键，可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大，空间结构复杂，结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外，多肽结构中还原态的巯基化学性质活泼，容易发生其他的副反应，而且肽链上其他侧链 也可能会发生一系列修饰，因此，肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素， 反应机理也比较复杂，既可能是自由基反应，也可能是离子反应。      

反应条件有多种选择，比如空气氧化，DMSO氧化等温和的氧化过程，也可以采用H2O2，I2， 汞盐等激烈的反应条件。
 

空气氧化法： 空气氧化法形成二硫键是多肽合成中最经典的方法，通常是将巯基处于还原态的多肽溶于水中，在近中性或弱碱性条件下（PH值6.5-10），反应24小时以上。为了降低分子之间二硫键形成的可能，该方法通常需要在低浓度条件下进行。
 

碘氧化法：将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘进行氧化，反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时，氧化条件要控制的更精确，氧化完后，立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时，通常有两种情况：
 

自然随机成环:       序列中的Cys之间随机成环，与一对二硫键成环条件相似；
 

定点成环：       定点成环即序列中的Cys按照设计要求形成二硫键，反应过程相对复杂。在 固相合成多肽之前，需要提前设计几对二硫键形成的顺序和方法路线，选择不同的侧链 巯基保护基，利用其性质差异，分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。