Novel antagonists for proteinase-activated receptor 2: inhibition of cellular and vascular responses in vitro and in vivo.
OBJECTIVE
Proteinase-activated receptor 2 (PAR(2)) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR(2) antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR(2) antagonists and demonstrate inhibitory effects on PAR(2)-mediated intracellular signalling pathways and vascular responses.


METHODS
The peptide-mimetic compound library based on the structures of PAR(2) agonist peptides were screened for inhibition of PAR(2)-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkappaB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models.


RESULTS
Two compounds, K-12940 and K-14585, significantly reduced SLIGKV-induced Ca(2+) mobilisation in primary human keratinocytes. Both K-12940 and K-14585 exhibited competitive inhibition for the binding of a high-affinity radiolabelled PAR(2)-ligand, [(3)H]-2-furoyl-LIGRL-NH(2), to human PAR(2) with K(i) values of 1.94 and 0.627 microM respectively. NFkappaB reporter activity and IL-8 production were also significantly reduced. Furthermore, relaxation of rat-isolated aorta induced by SLIGRL-NH(2) was inhibited competitively by K-14585. K-14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice.


CONCLUSIONS
K-12940 and K-14585 antagonized PAR(2) competitively, resulting in inhibition of PAR(2)-mediated signalling and physiological responses both in vitro and in vivo. These peptide-mimetic PAR(2) antagonists could be useful in evaluating PAR(2)-mediated biological events and might lead to a new generation of therapeutically useful antagonists.
Kanke
T
T
Tokyo New Drug Research Laboratories, Kowa Company Limited, Noguchicho, Higashimurayama, Tokyo, Japan.
Kabeya
M
M
Kubo
S
S
Kondo
S
S
Yasuoka
K
K
Tagashira
J
J
Ishiwata
H
H
Saka
M
M
Furuyama
T
T
Nishiyama
T
T
Doi
T
T
Hattori
Y
Y
Kawabata
A
A
Cunningham
M R
MR
Plevin
R
R
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008297:Male; D051379:Mice; D018716:Molecular Mimicry; D009842:Oligopeptides / Q000494:pharmacology*; D010455:Peptides / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D044464:Receptor, PAR-2 / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000502:physiology*; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology
C549501:K 12940; C547641:K-14585; D009842:Oligopeptides; D010455:Peptides; D044464:Receptor, PAR-2; D014508:Urea