Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H₄ receptor.
OBJECTIVE
The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H₄ receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182(5.46) in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98(3.36) in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98(3.36) by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research.


METHODS
We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and β-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically.


RESULTS
VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH₄ receptor with submicromolar affinity. Serine substitution of C98(3.36) prevented this covalent interaction.


CONCLUSIONS
VUF14480 is thought to bind covalently to the hH₄ receptor-C98(3.36) residue and partially induce hH₄ receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH₄ receptor.
Faculty of Sciences, Amsterdam Institute for Molecules, Medicines and Systems, VU University Amsterdam, The Netherlands.
S
S
Nijmeijer
H
H
Engelhardt
S
S
Schultes
A C
AC
van de Stolpe
V
V
Lusink
C
C
de Graaf
M
M
Wijtmans
E E J
EE
Haaksma
I J P
IJ
de Esch
K
K
Stachurski
H F
HF
Vischer
R
R
Leurs
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019390:Arrestins / Q000378:metabolism*; D015195:Drug Design; D054333:Drug Partial Agonism; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D017442:Histamine Agonists / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D011487:Protein Conformation; D011743:Pyrimidines / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D011968:Receptors, Histamine / Q000737:chemistry / Q000378:metabolism; D000074040:Receptors, Histamine H4; D015398:Signal Transduction / Q000187:drug effects; D014753:Vinyl Compounds / Q000494:pharmacology*; D000071557:beta-Arrestins
C000591333:4-(4-methylpiperazin-1-yl)-6-phenyl-2-vinylpyrimidine; D019390:Arrestins; C418607:HRH4 protein, human; D017442:Histamine Agonists; D008024:Ligands; D011743:Pyrimidines; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D000074040:Receptors, Histamine H4; D014753:Vinyl Compounds; D000071557:beta-Arrestins; D019204:GTP-Binding Proteins
GPCR; covalent binder; histamine H4 receptor; pyrimidine