Table 7.

Binding parameters for bitopic ligands and their constituent parts in the absence or presence of PD81,723 at CHOhA₁R membranes, derived from competition association assays

Compound	kon (M−1·min−1)a	koff (min−1)a	RT (min)b	KD (nM)c
LUF5519	2.0 ± 0.4 × 106	1.19 ± 0.19	0.8 ± 0.1	595 ± 92
+10 μM PD81,723	2.4 ± 1.4 × 106	0.38 ± 0.05*	2.6 ± 0.4*	158 ± 41*
+33 μM PD81,723	9.1 ± 1.1 × 106**	0.46 ± 0.07*	2.2 ± 0.2**	51 ± 6**
LUF6232 (linker N = 4)	6.0 ± 0.3 × 105	0.22 ± 0.11	4.5 ± 1.3	367 ± 106
+10 μM PD81,723	2.8 ± 0.5 × 106*	0.19 ± 0.04	5.3 ± 0.6	68 ± 11*
+33 μM PD81,723	1.0 ± 0.2 × 107**	0.21 ± 0.04	4.8 ± 0.5	21 ± 3*
LUF6234 (linker N = 5)	5.3 ± 0.7 × 104	0.052 ± 0.007	19 ± 3	981 ± 65
+10 μM PD81,723	3.1 ± 0.1 × 106***	0.40 ± 0.05**	2.5 ± 0.3**	129 ± 13***
+33 μM PD81,723	7.8 ± 0.9 × 106***	0.35 ± 0.05**	2.9 ± 0.2**	45 ± 5***
LUF6258 (linker N = 9)	0.8 ± 0.3 × 104	0.021 ± 0.008	48 ± 11	2625 ± 810
+10 μM PD81,723	3.0 ± 1.4 × 104	0.021 ± 0.011	48 ± 15	700 ± 284
+33 μM PD81,723	5.8 ± 3.4 × 104	0.023 ± 0.014	43 ± 15	397 ± 194
LUF7161	2.7 ± 0.7 × 105	1.21 ± 0.32	0.8 ± 0.2	4481 ± 985
+10 μM PD81,723	3.8 ± 0.9 × 106*	0.87 ± 0.21	1.1 ± 0.1	229 ± 45*
+33 μM PD81,723	4.7 ± 0.9 × 106**	0.74 ± 0.15	1.4 ± 0.2	157 ± 25*

Data are shown as mean ± SEM of three separate experiments each performed in duplicate.

^*P < 0.05,

^**P < 0.01,

^***P < 0.001 compared with the values in the absence of an allosteric modulator; Student's t-test.

^ak_on (k₃) and k_off (k₄) of unlabelled A₁ receptor agonists in the absence or presence of an allosteric modulator (PD81,723 or BC-1) were determined in [³H]-DPCPX (2.6 nM) competition association assays.

^bRT (residence time) = 1/k_off.

^cKinetic K_D = k_off/k_on; k_on (k₃) and k_off (k₄) values of unlabelled antagonists were generated from [³H]-DPCPX (2.6 nM) competition association assays at 25°C.