SUPPLEMENTAL DATA

Manuscript title: Detailed analysis of biased histamine H4 receptor signalling by

JNJ7777120 analogues

Authors: S Nijmeijer, H F Vischer, F Sirci, S Schultes, H Engelhardt, C de Graaf, E

M Rosethorne, S J Charlton, R Leurs

Supplemental Table 1. Overview of potency and intrinsic activity values in a β-

arrestin2 recruitment and CRE-luciferase assay. Intrinsic activity is calculated as

percentage of maximal histamine (HA) response. Data shown are pooled data from at

least three experiments performed in duplicate. Error bars indicate SEM values.

Supplemental Figure 1. Effect of different basic side chains influence compound

intrinsic activity in β-arrestin2 recruitment. U2OS-H4R cells were stimulated with

indicated amount of indolecarboxamides. A) R5-Cl, R7-CH3 B) R7-F C) R7-CH3.

Intrinsic activity is plotted as percentage of maximal histamine (HA) response. Data

shown are pooled data from at least three experiments performed in duplicate. Error

bars indicate SEM values.

 
 Compound β-arrestin2 recruitment Compound β-arrestin2 recruitment
# Structure pEC50 Intrinsic act. (%HA) # Structure pEC50 Intrinsic act. (%HA)
CH3 CH3
N N

75* 6.0(0.09) 95 (4.1) 26 5.4(0.08) 44 (6.1)

Cl N
O N
N
H3 C
O
+
H
N N O
O O
H

CH3 CH3
N
N

35 6.4(0.12) 75 (4.8) 41 7.8(0.10) 44 (4.1)

N
N

N O
H2 N N O H
H NH2

CH3 CH3
N NH2 N
Cl

48 Cl N
8.0(0.11) 63 (4.5) 22 N
6.1(0.06) 42 (4.0)
N O N O
H H

CH3
CH3
N N

50 7.8(0.10) 63 (4.0) 59 5.8(0.10) 41 (3.9)

Cl
F
N
N
N O
H
CH3 N O
H
CH3 CH3
N O + O N
O N

28 7.0(0.03) 62 (4.3) 21 5.6(0.02) 39 (3.5)

+
N N N
O

N O N O
H H

CH3
N N

1 Cl N
8.0(0.12) 62 (4.2) 46 N
6.8(0.13) 38 (2.2)
N O
N O H
H CH3

CH3 H3C
N NH
F

47 Cl N 8.3(0.13) 59 (1.1) 72 Cl N
6.2(0.09) 37 (2.5)
N O
N O H
H F
CH3 CH3
N N
OH
Cl
51 8.2(0.20) 58 (4.2) 20 6.2(0.00) 37 (1.3)
N
N
N O
H N O
NH2 H
CH3
N
N

29 H2N N 7.4(0.07) 57 (2.8) 67 Cl N 7.5(0.19) 36 (3.3)

N O N O
H H

CH3 CH3
N N

49 Cl N
7.9(0.12) 57 (2.4) 69 Cl

N
N
6.2(0.07) 36 (2.2)
N O O
H H
F
F

CH3 H
N N
F

76 6.9(0.12) 57 (1.5) 64 6.4(0.06) 36 (1.3)

N N

N O N O
H H
CH3 F

H CH3
N CH3
N
O

2 7.5(0.12) 56 (3.6) 19 5.3(0.08) 36 (0.9)

Cl N N

N O N O
H
H
CH3
N N

70 7.3(0.06) 55 (4.0) 71 7.2(0.12) 35 (0.8)

F
Cl N
Cl N

N O
N O H
H F

CH3
Cl N N

18 N
7.0(0.12) 53 (0.8) 63 N
7.1(0.09) 33 (4.7)
N O N O
H
H F
CH3
N

12 Cl N 6.5(0.12) 52 (2.8) 54 6.2(0.12) 33 (1.6)

O O
CH3 CH3
N N
F

17 N 6.9(0.07) 52 (1.3) 57 N 5.6(0.07) 32 (3.5)

N O N O
H H
CH3 CH3
N N

56 H
N N 6.5(0.09) 51 (3.8) 31 N
6.8(0.09) 30 (1.4)
Cl N O
Cl O H
H
N
Cl
74 7.4(0.06) 50 (0.4) 45 5.9(0.09) 29 (2.5)
N

N O
H
F
CH3 CH3
N N

36 N
7.2(0.13) 49 (3.9) 61 N
5.4(0.07) 28 (3.2)
N O N O
H H
F F
H H3C
N NH

43 7.0(0.10) 48 (4.8) 66 5.6(0.08) 24 (2.3)

N
N
N O
H N O
CH3 H
F

Chiral
N Cl H
N N

55 Cl

N
N

O
7.1(0.03) 48 (4.8) 73 N
H
O
CH3
6.6(0.11) 19 (1.9)
H
CH3 F

CH3 Chiral
N
Cl H
N N

10 N 7.2(0.07) 47 (4.2) 53 N O
CH3
6.6(0.07) 18 (2.4)
H
N O CH3
H
CH3 Chiral
N H
F N N

77 N
N

O
6.7(0.09) 46 (1.9) 44 N O
CH3
6.0(0.07) 14 (3.4)
H H
F CH3

H Chiral
N H
N N

68 Cl N
6.7(0.13) 45 (2.3) 65 N O
CH3
6.0(0.12) 13 (2.5)
N O H
H F

*
NB. 75 is the only compound that displays agonism in CRE luciferase assay:
Eff=105±5%, pEC50=6.4±0.1
Supplemental Figure 1

A) Basic Chain

100 Structure

-arrestin2 recruitment
(Intrinsic activity %HA)
N
Cl R
Cl N #
N O

75
H
CH3
N O
H
CH3 50 N

50 55 N

25 54
NH

NH
53
0
N

-10 -9 -8 -7 -6 -5 -4
log [compound] M
B) Basic Chain Structure

100
-arrestin2 recruitment
(Intrinsic activity %HA)

N #
N O
H
F
N
75 36 N

N O N
H
NH
F
64
50 N

63
N

25 N

61 N

NH

0 66 N

NH

65 N

-10 -9 -8 -7 -6 -5 -4
log [compound] M
C) Basic Chain

100
-arrestin2 recruitment
(Intrinsic activity %HA)

Structure
N
R

N
#
75
N O
H
CH3
N O
H NH
CH3
43
N

50
46 N

25 45
NH

NH
44
0 N

-10 -9 -8 -7 -6 -5 -4
log [compound] M