Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): Development of CRFR2β-selective antisauvagine-30

Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125 I-Tyr0 ]oCRF or [125 I-Tyr0 ]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2β (mCRFR2β). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2β (HEK-mCRFR2β cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (K d = 5.7 ± 1.6 nM) and mCRFR2β (K d = 4.0 ± 2.3 nM), [d Phe11 ,His12 ]Svg(11–40) , [d Leu11 ]Svg(11–40) , [d Phe11 ]Svg(11–40) , and Svg(11–40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2β than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2β. In agreement with the results of these binding experiments, [d Phe11 ,His12 ]Svg(11–40) , named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2β.