The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF₁R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF₁R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF₁R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF₁R antagonists. In an effort to develop novel CRF₁R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF₁R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF₁R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF₁R as suggested by data using a constitutively active chimera of CRF₁R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

促肾上腺皮质激素释放因子（CRF）及其CRF1受体（CRF ₁ R）在维持体内平衡中起着重要作用。CRF / CRF ₁ R单元功能异常与多种疾病相关，例如焦虑和抑郁。已显示非肽CRF ₁ R选择性拮抗剂对实验动物具有抗焦虑和抗抑郁作用。但是，由于几种副作用，它们中的任何一种目前都没有在临床上使用，因此表明需要开发其他更合适的CRF ₁ R拮抗剂。为了开发新颖的CRF ₁我们设计，合成并化学表征了CRF的两个三肽类似物R拮抗剂，即（R）-LMI和（S）-LMI，其Leu分别为R（或D）或S（或L）构型。他们的设计基于CRF ₁ R / CRF复合物的N细胞外结构域（N域）的晶体结构，采用了一系列相关的计算方法。对人血浆中合成肽稳定性的实验评估表明，（R）-LMI在蛋白水解上比（S）-LMI更稳定。基于这一发现，选择（R）-LMI进行药理学表征。我们发现（R）-LMI是一种CRF拮抗剂，抑制（1）CRF刺激的表达CRF _1的HEK 293细胞中cAMP的积累R，（2）脂肪细胞产生白介素，（3）RAW 264.7细胞的增殖速率。（R）-LMI可能阻止通过与CRF的N结构域相互作用激动剂动作_1个R作为由数据使用CRF的组成型活性的嵌合体建议₁ R.我们建议，（R）-LMI可以用作最佳铅化合物在新型CRF拮抗剂的合理设计中。