TABLE 1.
Affinities of various single or double substituted PACAP-related analogs for PAC1, VPAC1-R and VPAC2 receptors.
| Peptide Number | Peptide structure | IC50 (nM) |
||||
|---|---|---|---|---|---|---|
| PAC1-R |
VPAC1-R |
VPAC2-R |
||||
| PAC1-R | VPAC1- R/PANC-1 |
T47D | VPAC2- R/PANC-1 |
Sup-T1 | ||
| PACAP27 | 2.82 ± 0.17 | 0.23 ± 0.09 | 0.20 ± 0.07 | 4.79 ± 0.20 | 24.5 ± 1.0 | |
| PACAP38 | 0.28 ± 0.02 | 1.35 ± 0.04 | 1.29 ± 0.05 | 1.00 ± 0.11 | 6.03 ± 0.30 | |
| VIP | >1,000 | 0.14 ± 0.01 | 0.20 ± 0.02 | 6.03 ± 0.30 | 16.6 ± 1.0 | |
| P42 | [Iaa1PACAP38 | 0.28 ± 0.02 | 2.10 ± 0.09 | 2.45 ± 0.12 | 1.00 ± 0.07 | 3.16 ± 0.21 |
| P44 | [Iac1]PACAP38 | 1.63 ± 0.10 | 5.01 ± 0.13 | 3.80 ± 0.11 | 9.40 ± 0.71 | 32.4 ± 1.3 |
| P43 | [Iaa1,d-Ser2]PACAP38 | 2.19 ± 0.16 | 23.4 ± 0.9 | 12.9 ± 0.4 | 21.9 ± 0.9 | 25.7 ± 1.4 |
| P27 | [N-Ac-His1,Pip3]PACAP38 | 46.8 ± 3.3 | 316 ± 9 | 288 ± 16 | 37.2 ± 1.9 | 87 ± 2 |
| P45 | [Iaa1,Ala22]PACAP38 | 1.74 ± 0.73 | 16.3 ± 0.4 | 9.12 ± 0.57 | 676 ± 34 | >1,000 |
| P46 | [Iac1,Ala22]PACAP38 | 7.41 ± 0.47 | 53.7 ± 2.7 | 37.2 ± 3.0 | >1,000 | >1,000 |
| P47 | [Pya1,Ala22]PACAP38 | 83.2 ± 3.8 | 186 ± 8 | 162 ± 8 | 490 ± 28 | 692 ± 17 |
| P15 | [Aib2]PACAP38 | 4.68 ± 0.29 | 7.08 ± 0.35 | 4.37 ± 0.22 | 2.04 ± 0.11 | 3.16 ± 0.15 |
| P4 | [d -Ser 2 ]PACAP27 | 54.9 ± 2.4 | 8.51 ± 0.47 | 4.68 ± 0.20 | 31.6 ± 1.6 | 72.4 ± 6.5 |
| P16 | [d -Ser 2 ]PACAP38 | 7.94 ± 0.50 | 417 ± 19 | 170 ± 8 | 14.8 ± 0.9 | 63.1 ± 1.6 |
| P14 | [Aib2,Lys(Palmitoyl)38]PACAP38 | 6.31 ± 0.22 | 14.5 ± 1.1 | 3.16 ± 0.72 | 55 ± 4 | 13.2 ± 0.2 |
| P13 | [d-Ser2,Lys(Palmitoyl)38]PACAP38 | 7.94 ± 0.48 | 11.5 ± 0.9 | 3.90 ± 0.29 | 15.8 ± 0.1 | 13.2 ± 0.5 |
| P26 | [Pip3]PACAP38 | 48.9 ± 2.6 | 575 ± 43 | 468 ± 56 | 74.1 ± 2.5 | 257 ± 4 |
| P5 | [Pro3]PACAP27 | >1,000 | 467 ± 28 | 149 ± 12 | 316 ± 22 | >1,000 |
| P18 | [Pro3]PACAP38 | 79.4 ± 2.4 | 302 ± 9 | 151 ± 9 | 83.2 ± 2.5 | 229 ± 13 |
| P6 | [Sar3]PACAP27 | >1,000 | 251 ± 9 | 83.2 ± 2.9 | 631 ± 42 | >1,000 |
| P19 | [Sar3]PACAP38 | 32.4 ± 0.8 | 288 ± 13 | 263 ± 8 | 91.2 ± 5.4 | 214 ± 13 |
| P25 | [Pro3,Lys34]PACAP38 | 32 ± 2 | 44.7 ± 2.7 | 64.6 ± 4.3 | 38 ± 2 | 107 ± 4 |
| P22 | [Sar4]PACAP27 | 69 ± 4 | 675 ± 37 | 575 ± 27 | 30.2 ± 0.7 | 302 ± 15 |
| P2 | [Me-Lys15]PACAP27 | >1,000 | 398 ± 19 | 339 ± 25 | 327 ± 25 | >5000 |
| P17 | [Ala22]PACAP38 | 3.72 ± 0.12 | 17.4 ± 0.4 | 20.4 ± 1.3 | >1,000 | >1,000 |
| P24 | [Lys34]PACAP38 | 1.38 ± 0.06 | 0.96 ± 0.05 | 1.10 ± 0.10 | 2.42 ± 0.18 | 4.20 ± 0.16 |
| P12 | [Lys(palmitoyl)38]PACAP38 | 6.31 ± 0.26 | 8.13 ± 0.46 | 4.40 ± 0.20 | 21.4 ± 0.5 | 5.89 ± 0.40 |
| P50 | [Pro3]VIP | >1,000 | 457 ± 16 | 692 ± 28 | >1,000 | >1,000 |
The indicated cell type was incubated with 50 pM 125I-labeled peptide and various concentrations of the unlabeled PACAP27 and PACAP38 analog as described under Materials and Methods. The IC50 was the concentration causing half-maximal inhibition of the saturable binding, calculated using the curve-fitting program KaleidaGraph. In each experiment each value was determined in duplicate, and values given are means and S.E.M. from at least three separate experiments. Abbreviations: VIP, vasoactive intestinal peptide; PAC1-R, PAC1-receptor; VPAC1-R, VPAC1-receptor; VPAC2-R, VPAC2-receptor; PAC1-R stably transfected into NIH 3T3 cells; VPAC1-R and VPAC2-R stably transfected into PANC1 cells; T47D breast cancer cells and Sup-T1 lymphoblastoma cells containing native VPAC1-Rand VPAC2-R respectively, others-see Fig. 1 legend.