. Author manuscript; available in PMC: 2014 Sep 1.

Published in final edited form as: Bioorg Med Chem Lett. 2013 Jul 2;23(17):10.1016/j.bmcl.2013.06.065. doi: 10.1016/j.bmcl.2013.06.065

Table 1. Binding affinities and functional activities of bifunctional peptide derivatives at δ/μ opioid receptors and NK1 receptors.

	Opioid agonist activities									NK1 antagonist activities

	radioligand binding assays			[³⁵S] GTPγS		binding assays		MVD (δ)	GPI (μ)	radioligand binding assays			GPI

	hDOR^a^,^b	rMOR^a^,^c	K_i(μ) /K_i(δ)	hDOR^a		rMOR^a		Opioid agonist		hNK1^d^,^e	rNK1^d^,^f	K_i(hNK1) /K_i(rNK1)	Substance P antagonist

no	K_i (nM)^g	K_i (nM)^g		EC₅₀ (nM)^h	E_max (%)ⁱ	EC₅₀ (nM)^h	E_max (%)ⁱ	IC₅₀ (nM)^j	IC₅₀ (nM)^j	K_i (nM)^g	K_i (nM)^g		Ke (nM)^k
1^l	2.8	36	13	2.9	45	32	42	22	360	0.082	0.29	3.5	25
2	25	150	6.0	2.5	26	72	51	12	1200	0.034	1.1	32	380
3	5.0	110	22	0.26	41	54	52	5.1	6.8%@1μM	0.066	7.8	118	2.0
4	3.1	63	20	5.5	28	100	27	6.9	42%@1μM	0.12	9.8	82	12
5	13	76	5.8	190	25	120	30	8.5	25%@1μM	0.88	3.6	4.1	29
6^m	50	180	3.6	35	16	140	26	400	520	0.0023	1.6	696	3.6
7	2.9	23	7.9	4.8	60	14	54	26	45	0.09	0.29	3.2	Not tested
8	0.6	33	55	1.1	36	0.80	51	9.1	390	0.0090	0.089	9.9	2.0
9	5.7	15	2.6	76	56	37	47	85	260	0.097	1.6	16	1.8
10	4.7	0.29	0.062	27	87	10	36	21	26	0.0057	4.2	737	59
Biphalinⁿ	2.6	1.4	0.54	1.1	83			2.7	8.8
DAMGO						37	150
L-732,138										0.73	130	180	250

Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the DOR and MOR. See reference 3 for detailed procedures.

[³H]DPDPE; K_d = 0.45 ± 0.1 nM.

[³H]DAMGO; K_d = 0.50 ± 0.1 nM.

Competition analyses were carried out using membrane preparations from transfected CHO cells that constitutively expressed rat or human NK1 receptors. See reference 3 for detailed procedures.

[³H]Substance P; K_d = 0.16 ± 0.03 nM.

[³H] Substance P; K_d = 0.40 ± 0.17 nM.

The data were collected from at least two independent experiments performed in duplicate. The K_i values are calculated using the Cheng and Prusoff equation to correct for the concentration of the radioligand used in the assay. See reference 3 for detailed procedures.

The EC₅₀ values were determined from the non-linear regression analysis of data collected from at least two independent experiments performed in duplicate. See reference 3 for detailed procedures.

ⁱ

[Total bound − Basal]/[Basal − Non-specific] × 100.

Concentration at 50% inhibition of muscle contraction at electrically stimulated isolated tissues (n = 4).

Inhibitory activity against the Substance P induced muscle contraction in the presence of 1 μM naloxone, Ke: concentration of antagonist needed to inhibit Substance P to half its activity (n = 4). See reference 3 for detailed procedures.

Reference 3a.

Reference 3b.

ⁿ

Reference 7.