Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing...

Adrenomedullin (AM) is a peptide hormone implicated in blood pressure regulation and in the pathophysiology of important diseases, such as hypertension, cancer, and diabetes. However, nonpeptidic modulators of this peptide that could be used to clinically regulate its actions are not available. We present here an efficient new method to screen a large library of small molecules. This technology was applied to the identification of positive and negative modulators of AM function. A two-tier screening strategy was developed in which the first screening entails disruption of the interaction between the peptide and a neutralizing monoclonal antibody. Selected compounds were further characterized by their ability to modulate second messengers in cells containing specific AM receptors. A parallel screen against gastrin-releasing peptide selected a different subset of molecules, confirming the specificity of the screening method. Identified AM-positive regulators reduced blood pressure in vivo, whereas AM-negative regulators mediated vasoconstriction, as predicted by the vasodilatory activity of AM. Binding of the small molecules to immobilized AM was demonstrated by surface plasmon resonance assays, with K(d) values ranging from 7.76 x 10(-9) to 4.14 x 10(-6) m. Preclinical development of AM modulators may result in useful drugs for the prevention and treatment of hypertension, cancer, and diabetes.