Short-chain pseudopeptide bombesin receptor antagonists with enhanced binding affinities for pancreatic acinar and Swiss 3T3 cells display strong antimitotic activity

The high inhibitory potency of the previously developed bombesin antagonist [Leu¹³,ψCH₂NHLeu¹⁴]bombesin (analogue I) (IC₅₀ values of 30 and 18 nM for inhibition of bombesin-stimulated amylase secreation from guinea pig acinar cells and Swiss 3T3 cell growth, respectively) diminished considerably when shorter chain lengths were examined. For instance, [Leu¹³,ψCH₂NHLeu¹⁴]bombesin-(5-14),[Leu¹³,ψCH₂NHLeu¹⁴]bom esin-(6-14), and [Leu⁹,ψCH₂NHLeu¹⁰]neuromedin C had IC₅₀ values of 150, 150, and 280 nM, respectively. Incorporation of a D-Phe residue at position 6 of [Leu¹³,ψCH₂NHLeu¹⁴]bombesin did not significantly change the various biological parameters. However, its presence in [Leu¹³,ψCH₂NHLeu¹⁴]bombesin-(6-14) and at position 2 of ψ-neuromedin C(2-10) resulted in about 10-fold increases in potency up to and above that of the original antagonist. For instance, [D-Phe⁶,Leu¹³,ψCH₂NHLeu¹⁴]bombesin-(6-14) and des-Gly¹-[D-Phe²,Leu⁹,ψCH₂NHLeu¹⁰]neuromedin C exhibited IC₅₀ values of 5 and 28 nM, respectively. Analogues based on the litorin sequence which contains an NH₂-terminal pyroglutamic acid residue at the bombesin position 6 equivalent were also quite potent. The ability of various analogues to interact with bombesin receptors on pancreatic acini correlated reasonably well with potencies derived from inhibition of bombesin-stimulated growth of Swiss 3T3 cells. Additional studies of NH₂- and COOH-terminal structure-activity relationships resulted in the synthesis of [D-Phe⁶,Leu¹³,ψCH₂NHPhe¹⁴]bombesin-(6-14), which was particularly effective in inhibiting 3T3 cell growth at high picomolar concentration (IC₅₀ = 0.72 nM and K(i) = 3.1 nM for 3T3 cells; IC₅₀ = 7.5 nM and K(i) = 9.9 nM for acini). Detailed investigations with one of the most potent antagonists, [D-Phe⁶,Leu¹³,ψCH₂NHLeu¹⁴]bombesin-(6-14) (K(i) = 14 nM for acini cells and 7.1 for 3T3 cells), demonstrated that this analogue was a competitive inhibitor of bombesin and that this activity was specific for the bombesin recpetor. Thus, inhibitory potencies have been improved generally up to 25 times over previously reported structures; and, given that bombesin itself has a K(i) of 1.2 nM for 3T3 cell binding, some of these analogues are extraordinarily high affinity receptor antagonists. They can also be synthesized more readily and offer fewer proteolytic degradation sites than the original pseudopeptide and should be excellent candidates for in vivo studies aimed at inhibition of bombesin-dependent human small cell lung carcinoma growth.