While stable tertiary structures have been demonstrated for several GI peptides, there is little experimental confirmation that this 3D structure influences the physiological response to any peptide. The investigators hypothesize that tertiary structure of the endocrine peptide PYY influences receptor subtype binding, activation and expression of biological activity. They will test this hypothesis in two ways: 1) Determine if change in tertiary structure alters biological activity and 2) determine if there are unique regions of tertiary structure for specific Y receptor agonists. For the first test, the investigators have designed a PYY analog where a minor primary structure change (formation of a disulfide bond in [Cys6,Cys23}PYY) produced a dramatic alteration in molecular conformation and a 33-fold decrease in Y1 receptor binding from the non-cross linked form. The reduced compound had less affinity than native PYY. A systematic design strategy will be used to identify di-cysteine PYY analogs that closely mimic PYY for Y1, Y2, and V5 receptor binding and 3D-conformation. The reduced analog will be oxidized to determine the consequences of tertiary structure alteration by disulfide bond formation. The influence of tertiary structure will be evaluated by measurements of receptor binding, second messenger activation, circulatory half-life, stability to digestion, and inhibition of pancreatic secretion in the rat. This unique coupling of peptide design strategy and evaluation of structure and multiple effectors of physiological response will provide the first direct quantitation of the role of tertiary structure in the expression of PYY bioactivity. The authors have determined the aqueous solution structure of PYY by NMR. They have also shown that Y1 and Y2 specific agonists ([Pro34]PYY and PYY(3 -3 6)) exhibit distinctly different secondary structures using circular dichroism (CD). For the second test, they will select the best 5-7 agonists that potently bind and activate Y1, Y2 or Y5 subtypes expressed in CHO cells. The tertiary structure will be determined for the selected agonists by NMR and used to predict structural elements that are specific for each receptor subtype. This structural analysis may lead to rational design strategies to produce therapeutic agents acting at these V receptor subtypes.
