Discovery of a stable tripeptide targeting the N-domain of CRF1 receptor

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.