Ž . European Journal of Pharmacology 374 1999 435–443 www.elsevier.nlrlocaterejphar Correlation between binding characteristics and functional antagonism in human glioma cells by tachykinin NK receptor antagonists 1 Carla Palma ) , Federica Nardelli, Stefano Manzini Department of Pharmacology, Menarini Ricerche, Via Tito Speri, 10, 00040 Pomezia, Rome, Italy Received 18 January 1999; received in revised form 28 April 1999; accepted 30 April 1999 Abstract Binding characteristics and functional antagonism exerted by two structurally related tachykinin NK receptor antagonists, MEN 1 ŽŽ . w Ž . x  a Ž . a Ž . Ž . 4 . 11467 1 R,2 S -2 N 1 H indol-3-yl-carbonyl -1-N- N p-tolylacetyl -N methyl -D-3- 2-naphthyl alanyl diaminocyclohexane and Ž 2 wŽ . Ž . x Ž . . FK888 N -4 R -4-hydroxy-1- 1-methyl-1 H-indol-3-yl carbonyl-L-prolyl -N-methyl-N-phenylmethyl-L-3- 2-naphthyl alaninamide , w 3 x were investigated in the human astrocytoma cell line U373 MG. In radioligand binding studies, conducted with H substance P and intact cells at 378C, MEN 11467 bound to tachykinin NK receptors in an irreversible manner with a K value of 1.2 "0.5 nM while FK888 1 i bound in competitive manner with a K value of 4.6 "2.2 nM. Receptor blockade by both antagonists resulted in a powerful and i complete inhibition of functional responses induced by substance P, such as accumulation of the second messenger inositol monophos- phate or interleukin-6 release. However, MEN 11467 showed a greater potency for blocking functional responses than FK888 despite their similar affinity for human tachykinin NK receptors. Moreover, MEN 11467 was more potent in inhibiting late rather than early 1 phases of substance P-induced inositol monophosphate accumulation and its antagonism was enhanced by drug preincubation and barely affected by removal of unbound drug from the external medium, suggesting that MEN 11467 bound in a tight manner to the receptor. Such behaviour was not observed with the competitive and rapidly reversible antagonist FK888. These data indicate that the small differences in the chemical structure of MEN 11467 and FK888 determine the different binding characteristics at the tachykinin NK 1 receptor and which are responsible for the greater potency of MEN 11467 for blocking functional responses. The K value obtained in i binding studies may be inadequate to reveal the real potency of tachykinin NK receptor antagonists. q 1999 Elsevier Science B.V. All 1 rights reserved. w 3 x Keywords: H Substance P binding; Inositol phosphate; Tachykinin NK receptor antagonist; Glioma cell, human 1 1. Introduction The tachykinin NK receptor mediates a wide range of 1 physiological and potentially pathological responses to Ž . tachykinins Quartara and Maggi, 1998 . On the basis of this fact, a great deal of research has been carried out to identify and develop a specific, selective and long-lasting antagonist of this class of receptor as a potential new drug for the treatment of tachykinin-related pathologies. In the last decades a wide range of tachykinin NK receptor 1 antagonists have been described with heterogeneous chem- ical structures and types of pharmacological antagonism Ž . Quartara and Maggi, 1998 . To characterize their phar- macological profile, most tachykinin NK receptor antago- 1 ) Corresponding author. Tel.: q0039-06-91184463; fax: q0039-06- 9100220 nists have been studied in similar experimental models ranging from binding studies in cells expressing human Ž tachykinin NK receptors IM-9, U373, transfected Chi- 1 Ž .. nese hamster ovary CHO to the assessment of in vitro or in vivo blockade of substance P-induced functional re- Ž sponses Johnson and Johnson, 1992; Heuillet et al., 1993; . Goso et al., 1994; Sagan et al., 1997 . However, compara- ble studies to assess the affinity and the characteristics of interaction with the receptor in binding experiments and the blockade of functional responses induced by substance P stimulation in the same experimental model, and in particular with the human tachykinin NK receptor type, 1 are lacking. To investigate this issue we decided to study, in the human astrocytoma cell line U373 MG, the binding interaction and the antagonism of the functional cellular response to substance P of two tachykinin NK receptor 1 Ž 2 wŽ . antagonists: the peptide FK888 N -4 R -4-hydroxy- 0014-2999r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0014-2999 99 00334-9