Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): Development of CRFR2 beta-selective antisauvagine-30

Different truncated and conformationally constrained analogs of corticatrop in-releasing factor (CRF) were synthesized on the basis of the amino acid s equences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [I-125-Tyr(0)] oCRF or [I-125-Tyr(0)]Svg from membrane homogenates of human embryonic kidn ey (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor , type 1 (rCRFR1), or mouse CRF receptor, type 2 beta (mCRFR2 beta). Furthe rmore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cA MP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rC RFR1 cells) or mCRFR2 beta (HEK-mCRFR2 beta cells) was determined. In compa rison with astressin, which exhibited a similar affinity to rCRFR1 (K-d = 5 .7 +/- 1.6 nM) and mCRFR2 beta (K-d = 4.0 +/- 2.3 nM), [DPhe(11), His(12)] Svg((11-40)), [DLeu(11)] Svg((11-40)), [DPhe(11)] Svg((11-40)), and Svg((11 -40)), bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affin ity to mCRFR2 beta than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2 beta. In agreement w ith the results of these binding experiments, [DPhe(11),His(12)]Svg((11-40) ), named antisauvagine-30, was the most potent and selective ligand to supp ress agonist-induced adenylate cyclase activity in HEK cells expressing mCR FR2 beta.