This invention relates to novel humoral hypercalcemic factor (HCF) antagonists having the following general structure: ##STR1## wherein X is 7, 8, 9, 10, or 11, and which encompasses various amino acid substitutions.
Peptide analogues can be used as inhibitors of their naturally occurring peptide counterparts in vivo and in vitro, and may also be useful as inhibitors of other analogous molecules. Peptides bind to their specific cell surface receptors and directly or indirectly cause changes in cell metabolism or physiology. If the cell receptor is blocked, the peptidal effect will also be blocked. A peptide analogue, when administered to a vertebrate such as a mammal, can bind to specific cell surface receptors and thereby block the activity of a corresponding or otherwise related peptide. Peptide analogues are useful in treating various diseases caused by an excess of the naturally occurring peptide and in treating peptide dependent tumors. Peptide analogues that act as antagonists are also useful in vitro in combination with a bioassay for the naturally occurring peptide. One example of this invention relates to the synthesis and use of novel humoral hypercalcemic factor (HCF) analogues that are useful for inhibiting the action of HCF both in vivo and in vitro. Human humoral hypercalcemic factor is designated herein as "hHCF."
Previously, it had been proposed that tumors could secrete parathyroid hormone (PTH) ectopically and cause hypercalcemia of malignancy. However, messenger RNA for PTH was not found in such tumors. Several studies demonstrated that a PTH-like factor, physicochemically and immunologically distinct from PTH and later identified as HCF, is secreted by tumor cells. S. B. Rodan et al., J. Clin. Invest., 72:1511 (1983); A. F. Stewart et al., Proc. Natl. Acad. Sci. U.S.A., 80:1454 (1983); G. J. Strewler et al., J. Clin. Invest., 71:769 (1983). It was also known that this PTH-like factor stimulates adenylate cyclase in PTH target cells, and that this activity can be inhibited by PTH antagonists.
Recently, several investigators isolated and obtained partial amino acid sequences of peptide derived from several different human tumors (lung squamous carcinoma, renal cell carcinoma, and breast carcinoma). J. M. Moseley et al., Proc. Natl. Acad. Sci. U.S.A., 84:5048 (1987); G. J. Strewler et al., J. Clin. Invest., 80:1803 (1987); A. F. Stewart et al., Biochem. Biophys. Res. Commun., 146:672 (1987); M. Mangin et al., Proc. Natl. Acad. Sci. U.S.A., 85:597 (1988). One group published the putative full-length peptide structure (141 amino acids) based on the complementary DNA (cDNA) nucleotide sequence. L. J. Suva et al., Science, 237:893 (1987). This new PTH-like factor has been named human "humoral hypercalcemic factor" and is considered to be related in biological effects to PTH. Thus, HCF is currently considered to be a factor responsible for hypercalcemia of malignancy by its secretion from the tumor and its altering effect on calcium metabolism.
HCF shows considerable homology to the biologically critical NH.sub.2 -terminal region of PTH, a region that is believed to have an alpha helix followed by a beta turn. However, there are significant differences in the peptide sequences between PTH and HCF, and this new factor is the product of a different gene.
Fragments of peptide containing the region specific for binding to the cell surface receptor can be used as inhibitors or blocking agents. For HCF, it is considered that the N-terminal 34 amino acids are sufficient to define binding specificity to the cell surface receptor. The following is the N-terminal 34 amino acid sequence of hHCF: Ala-Val-Ser-Glu-His(5)-Gln-Leu-Leu-His-Asp(10)-Lys-Gly-Lys-Ser-Ile(15)-Gln -Asp-Leu-Arg-Arg-(20)-Arg-Phe-Phe-Leu-His(25)-His-Leu-Ile-Ala-Glu(30)-Ile-H is-Thr-Ala. Standard abbreviations well recognized in the peptide chemistry art are utilized herein.
Extensive structure and activity studies have now led to the design of peptide analogues that have high binding affinity for their respective cell surface receptors but do not stimulate the production of second messenger molecules once bound. HCF analogues with two to thirteen amino acids removed from the N-terminus produce inhibitors that still bind with high affinity to the peptide hormone receptor without causing a change in cyclic AMP concentration.
The novel peptide analogues of this invention are HCF antagonists with six to ten amino acids removed from the N-terminus that contain a lactam bridge five residues (inclusive) apart in the alpha helical region of the peptide. The lactam modification confers rigidity to this area of the peptide structure thereby enhancing its helical nature. The lactam bridge also stabilizes the biologically active conformation of the analogues and improves their resistance to metabolic degradation.
It is an object of the present invention to provide novel peptide analogues that act as antagonists of HCF; a peptide analogue of HCF that will bind with the cell surface HCF receptor can be used to block the effect of the naturally occurring peptide. Thus, it is also an object of the present invention to provide peptide analogues useful for the treatment of hypercalcemia of malignancy.
Another object of the present invention is to provide HCF analogues with enhanced alpha-helical nature and rigidity in the NH.sub.2 -terminal region, and with improved metabolic stability.
Another object of the present invention is to provide HCF analogues wherein amino acid modifications result in binding to the cell surface receptors without activating a second messenger molecule. Still another object of the present invention is to provide methods of inhibiting the action of HCF through the administration of novel HCF analogues. The above and other objects are accomplished by the present invention in the manner more fully described below.