Crosslinked bifunctional gonadotropin analogs with reduced efficacy

The N-linked oligosaccharides on human choriogonadotropin (hCG) and follitropin (hFSH) alpha-subunit loop 2 (alpha2) have a dominant influence on hormone efficacy. hCG analogs lacking this oligosaccharide retain approximately 40% the efficacy of the fully glycosylated hormone in cyclic AMP accumulation assays. Previous efforts to reduce efficacy further have involved removing the other N-linked oligosaccharides. We found that some intersubunit disulfide crosslinks reduced the efficacies of hCG analogs lacking only the alpha2 oligosaccharide. The least active analog was an hCG/hFSH chimera containing hFSH residues 95-108 in place of hCG residues 101-114 and a disulfide bond between alpha-subunit residue 37 and beta-subunit residue 33. While it bound lutropin receptors 2- to 3-fold better than hCG and follitropin receptors 10-30% as well as hFSH, it had less than 10% and 5% the efficacies of either hormone. This suggests that complete deglycosylation will not be required to produce glycoprotein hormone analogs that have low efficacies.