ISOLATION OF GLUCAGON ANTAGONISTS BY RANDOM MOLECULAR MUTAGENESIS ANDSCREENING

Glucagon has an important role in the regulation of glucose homeostasi s, and glucagon antagonists may be effective therapeutic agents in the control of diabetes mellitus. We were able to identify a number of an alogs with antagonist activity by creating libraries of mutant glucago n coding sequences, expressing them in a yeast (Saccharomyces cerevisi ae) secretion system, and screening for clones that produce analogs th at inhibit the glucagon stimulation of rat hepatocyte membrane adenyla te cyclase. These libraries were constructed by allowing random misinc orporation during the synthesis of oligonucleotides that contained the complete coding sequence for mammalian glucagon or for an analog (des His1-glucagon) that had partial antagonist activity. We developed and used a simplified screening assay to test culture broths from >3500 in dividual transformant yeast clones for their ability to inhibit glucag on-dependent adenylate cyclase activity. Ultimately, >20 different ana logs with antagonist activity were identified by recovering and sequen cing plasmid DNA from yeast strains that were positive in the screenin g assay, Interestingly, several analogs were identified repeatedly in independent yeast clones and certain amino acid substitutions occurred in more than one analog. This clustering of randomly isolated mutatio ns clearly delineates the regions of the glucagon molecule that are im portant for designing improved glucagon antagonists. A subset of the a ntagonists identified in yeast broth were produced by peptide synthesi s to confirm their activities as pure compounds.