BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist

Abstract

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.

Figure 1

Competition binding profile of pNPY, C2-NPY, hPYY_3–36, BIIE0246 and T₄-[NPY_33–36]₄ against specific [¹²⁵I]PYY_3–36 binding sites in rat brain membrane preparations. Data represent the mean±s.e.mean of 4–6 determinations, each performed in triplicate.

Figure 2

Photomicrographs of the autoradiographic distribution of [¹²⁵I]PYY_3–36 binding sites in presence and absence of various concentrations of BIIE0246 in the rat brain. Adjacent coronal rat brain sections were incubated in the presence of 30 pM [¹²⁵I]PYY_3–36 and in the presence of BIIE0246 from 1–1000 nM. Non-specific binding was determined in the presence of 1 μM pNPY. See list of abbreviations for anatomical identification. Scale bar represents 10 mm.

Figure 3

Quantitative autoradiographic data of [¹²⁵I]PYY_3–36 in the presence of increasing concentrations of BIIE0246 (0.1–10,000 nM) in various rat brain regions. See list of abbreviations for anatomical identification.

Figure 4

Photomicrographs of the autoradiographic distribution of [¹²⁵I]PYY_3–36 binding sites in the marmoset monkey brain. Adjacent coronal brain sections were incubated with 30 pM [¹²⁵I]PYY_3–36 and in the presence of 1000 nM BIIE0246. Non-specific binding was determined in the presence of 1 μM pNPY. See list of abbreviations for anatomical identification. Scale bar represents 10 mm.

Figure 5

Photomicrographs of the autoradiographic distribution of [¹²⁵I]PYY_3–36 binding sites in the human brain hippocampal area. Adjacent coronal human brain sections were incubated with 30 pM [¹²⁵I]PYY_3–36 and in the presence of either 100 or 1000 nM BIIE0246. Non-specific binding was determined in the presence of 1 μM pNPY. See list of abbreviations for anatomical identification. Scale bar represents 20 mm.

Figure 6

Concentration-response curves of pNPY in the presence and absence of various concentrations of BIIE0246 in the electrically stimulated rat vas deferens. Data represent the mean±s.e.mean of 4–8 determinations.

Figure 7

Concentration-response curves of pNPY in the presence and absence of various concentrations of BIIE0246 in the isolated rat colon. Data represent the mean±s.e.mean of 4–6 determinations.

Figure 8

Prototypical recording showing changes of tension (g) induced by PYY_3–36, [Leu³¹,Pro³⁴]NPY and hPP in the presence and absence of 1 μM BIIE0246 in the rat colon. W, represents washout.