bradykinin B 2 receptor that extends molecularcellular studies the isolated umbilical vein François Marceau Professeur associé Importance of bradykinin BK Derived from ID: 929886
Download Presentation The PPT/PDF document "A robust bioassay of the human" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
A robust bioassay of the human
bradykinin
B
2
receptor that extends molecular/cellular studies: the isolated umbilical vein François MarceauProfesseur associé
Slide2Importance of bradykinin (BK)Derived from kininogens via the action of kallikreins
A small and unstable peptide
Target cell types: Endothelial
cells: edema, hyperemia
Sensory nerve terminals: pain, etc.Epithelial cells: various inflammatory consequencesSmooth muscle cells
Slide3abnormal surfaces
BK
Lys-BK
plasma-
kallikrein
tissue
kallikrein
=
KLK-1
LK
N
C
N
C
HK
FXIIa
inactive
fragment
s
plasmin
tPA
,
uPA
plasminogen
B
2
R
C1-INH
ACE
ACE
inhibitors
N
C
N
C
prekallikrein
FXII
C1-INH
C1-INH
N
C
B
1
R
Lys-des-Arg
9
-BK
contact system
Slide4BDKRB1
→
BDKRB2
→
B
1
R
↑
B
2
R
leaky
microvessels
G
q
G
q
IP
3
ER
Ca
2+
calmodulin
MLCK
myosin
actin
PLC-
β
eNOS
NO
cytosol
nucleus
cPLA
2
PGI
2
vasodilation
MEK/ERK
c-Fos
DAG
PKC
BK
Lys-BK
Lys-des-Arg
9
-BK
Signaling
in
endothelial
cells
Slide5Need for a human bioassayIn vitro screening of new ligands based on radioligand
binding competition, simple signaling
(e.g., intracellular calcium)BK B2R has a notoriously
species-specific pharmacological profile
Naturally expressed BK B2R in the umbilical vein (not overexpressed)
Slide6vein
B2R antagonists exert species-specific effect: need for a bioassay based on human tissues
Stable in a time scale of hours
Slide7vein
α
-actin
monoclonal
ACE (C28)
polyclonal
vWF
polyclonal
Obtained after uncomplicated elective caesarean sections (with informed consent)
SMCs dominate → contractile response mediated by BK B
2
Rs
HUVECs: this is where they
reside
Koumbadinga
et al.
Peptides
2010;
31
: 1546
95% O
2
+ 5% CO
2
drain
Krebs solution reservoir
Force transducer
Slide9icatibant (Hoe 140; Firazyr; D-
Arg[Hyp3, Thi5, D-Tic7, Oic
8]-bradykinin)reported by Hock et al., Br J Pharmacol 1991; 102, 769
c
linical use: auto-injected s.c., hereditary angioedema1304.5 g/mol
Slide101 g
10 min
0.94
nM
icatibant1
μ
M
3.3
1074
10508
36.3
131
12.7
0.94
nM
3.3
12.7
36.3
131
1074
10508
34093
Lesage et al.,
Front.
Pharmacol
.
2020;
11
: 916
pA
2 = 8.06 ± 0.37
Lesage et al., Front.
Pharmacol. 2020; 11
: 916
Slide12Small molecule antagonists of the BK B2R
Slide13WIN 64338anatibant = LF16-0687
bradyzide
19c
47a
Pharvaris
Compound 3
Slide14WIN 64338First non-peptide BK B2R antagonist
Slide15Marceau et al. J Pharmacol Exp Ther 1994;
269: 1136
pA
2 = 5.99
Slide16pA2 = 7.53The rodent-specific antagonist bradyzide is modified to improve potency at human B2R
bradyzide
19c
Marceau et al.,
Int.
Immunopharmacol
.
2003;
3
: 1529
Slide17Anatibant (LF 16-0687)Bawolak et al., Regul.Peptides 2007; 140: 125
pA2 = 8.3
Slide18Compound 47a, a partial agonist identified by Fujisawa scientistsBawolak et al., Br. J. Pharmacol. 2009; 158: 1375
Slide19Pharvaris Compound 3pA2
= 9.67 ± 0.42
Lesage et al., Front.
Pharmacol. 2020; 11
: 916
Slide20Lesage et al.,
Front. Pharmacol.
2020; 11: 916
Lesage et al.,
Front.
Pharmacol
. 2020; 11
: 916
Slide22Verifying the properties of special peptide ligands, such as latent agonists activated by peptidases
Arg
-carboxypeptidases
Plummer’s
inh. H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Arg-OH BK-Arg
H-
Arg
-Pro-Pro-
Gly
-
Phe
-
Ser-Pro-Phe-Arg-OH bradykinin (BK)
Charest-Morin et al., Front. Pharmacol.
2014; 5: 32
B
2
R
Slide23Verifying the claim that tissue kallikrein (KLK-1) is a direct BK B2R agonist
KLK-1 1 nM at 1-hr intervals
KLK-1 10 nM at 1-hr intervals
10 min
BK 10 nM
BK 10 nM
1 g
Tachyphylaxis
, as well as the inhibitory effect of
aprotinin
or
icatibant
,
indicated that KLK-1 releases
a
kinin
from residual
kininogen
(s) adherent to the freshly isolated vein
Jean et al.,
Life
Sci
.
2016;
155
: 180
Slide24Gera et al., BMC Res. Notes 2016; 9: 452
HEK 293
cells expressing B2R-GFP
Verifying the properties
of special peptide ligands, such as fluorescent probesGFPCy7
Slide25Antagonists have variable affinities for B2Rs from various mammalian species; the human umbilical vein smooth muscle naturally expresses the BK B2RNot a “low tech” approach: remarkably quantitative and complementary to molecular/cellular pharmacology. In a time scale of several hours, this assay allows determining potency, surmountability, residual agonist activity, specificity and reversibility This assay has an intermediate level of complexity between cellular and molecular pharmacology on one hand, and in vivo studies in subhuman primates on the other
conclusions
Slide26acknowledgements
Ms. Johanne Bouthillier
Dr. Xavier Charest-Morin
pharvaris.com
funding