Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep;170(1):89-100.
doi: 10.1111/bph.12113.

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H₄ receptor

S Nijmeijer  1 , H Engelhardt, S Schultes, A C van de Stolpe, V Lusink, C de Graaf, M Wijtmans, E E J Haaksma, I J P de Esch, K Stachurski, H F Vischer, R Leurs
Affiliations

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H₄ receptor

S Nijmeijer et al. Br J Pharmacol. 2013 Sep.

Abstract

Background and purpose: 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.

Experimental approach: 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.

Key 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.

Conclusion and implications: 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.

Keywords: GPCR; covalent binder; histamine H4 receptor; pyrimidine.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Rational design of a covalent hH4 receptor ligand. (A) Chemical structures of hH4 receptor antagonists. JNJ 7777120, Compound 10 from Janssen Pharmaceutica (Chavez et al., 2008), 2-amino pyrimidine VUF10460, VUF14480 and VUF14481. (B) Binding mode of VUF10460 in the previously validated hH4 receptor homology model (Schultes et al., 2013b) based on the hH1 receptor crystal structure template (Shimamura et al., 2011). The amino group at position 2 of 2-aminopyrimidines interacts with E1825.46 (Schultes et al., 2013b). In addition, substituents at this position are also in close proximity to C983.36 as indicated by the green dotted line (distance ring carbon to thiol group of C983.36 is 4.2 Å). The backbone of TM helices 5, 6 and 7 (right to left) are presented as yellow helices, TM3 is presented as a yellow ribbon in the front. Important binding residues are depicted as ball-and-stick models with grey carbon atoms. Oxygen, nitrogen sulfur and polar hydrogen atoms are coloured red, blue, yellow and light blue respectively. H-bonds between VUF10460 and hH4 receptors are depicted by black dotted lines.
Figure 2
Figure 2
LCMS analysis of glutathione addition to VUF14480 and VUF14481. VUF14480 (top panel) or VUF14481 (bottom panel) were mixed with glutathione in a 1:1 molar ratio and incubated overnight at 22°C. The incubated mixtures were subsequently separated and analysed by LCMS. (Top panel) Reaction product (588 g·mol−1; m+1) was formed that corresponded to the mass of VUF14480 (280 g·mol−1) and glutathione (307 g·mol−1). Peak with mass 295 g·mol−1 (m+2/2) corresponded to a double protonated product. (Bottom panel) VUF14481 (269 g·mol−1; m+1) and glutathione (307 g·mol−1; GSSG m+2/2) could only be detected separately and no product was formed.
Figure 3
Figure 3
Functional characterization of VUF14480 and VUF14481. (A) GTPγS-binding assay. Membranes (10 μg per well) from cells expressing hH4 receptors were incubated with increasing amounts (1 nM–10 μM) of histamine, VUF14480 or VUF14481 for 1 h at 22°C. (B) BRET-based β-arrestin2 recruitment assay. Cells expressing the hH4 receptor-Rluc8 and β-arrestin2-mVenus were incubated with 10 μM histamine (HA), VUF14480 or VUF14481 in absence (open columns) and presence (hatched columns) of thioperamide (10 μM) for 20 min at 37°C. Results shown are from at least three pooled experiments performed in triplicate. Data were normalized to the histamine response (100%) and fitted to a three-parameter response model. Error bars indicate SEM values.
Figure 4
Figure 4
[3H]-histamine binding to hH4 receptor-expressing HEK293T cell membranes pre-incubated with compounds VUF14481, VUF14480 or JNJ7777120. Crude membrane fractions of hH4 receptor-transfected cells were pre-incubated with increasing concentrations of VUF14480, VUF14481 or JNJ7777120, at 22°C for 1 h. Pretreated membranes fractions were washed at least three times very extensively with 50 mM Tris-HCl before further incubation at 22°C for 1 h with ∼10 nM [3H]-histamine and increasing amounts (10 pM–10 μM) of non-labelled histamine. Curves are fitted to a one-site competition-binding model. Data shown are pooled data from three experiments performed in triplicate, error bars indicate SEM values.
Figure 5
Figure 5
Functional characterization of VUF14480 and VUF14481 on hH4-C983.36S receptor. (A) GTPγS-binding assay. Membranes (10 μg per well) from cells expressing hH4 receptor-C983.36S were incubated with increasing amounts (1 nM–10 μM) of histamine, VUF14480 or VUF14481 for 1 h at 22°C. (B) BRET-based β-arrestin2 recruitment assay. Cells expressing the hH4 receptor-C983.36S-Rluc8 and β-arrestin2-mVenus were incubated with 10 μM histamine (HA), VUF14480 or VUF14481 in the absence (open columns) and presence (hatched columns) of thioperamide (10 μM) for 20 min at 37°C. Results shown are from at least three pooled experiments performed in triplicate. Data were normalized to the histamine response (100%) and fitted to a three-parameter response model. Error bars indicate SEM values.
Figure 6
Figure 6
[3H]-histamine binding to hH4-C983.36S receptor expressing HEK293T cell membranes pre-incubated with VUF14480 or VUF14481. Crude membrane fractions of hH4-C983.36S receptor transfected cells were pre-incubated with increasing concentrations of VUF14480 or VUF14481 at 22°C for 1 h. Pretreated membranes were subsequently washed extensively and at least three times with 50 mM Tris-HCl before further incubation at 22°C for 1 h with ∼10 nM [3H]-histamine and increasing amounts (10 pM–10 μM) of non-labelled histamine. Curves are fitted to a one-site competition-binding model. Data shown are pooled data from three experiments performed in triplicate, error bars indicate SEM values.
Figure 7
Figure 7
Proposed binding modes of VUF14481 (A, green) and VUF14480 (B, magenta) in previously validated hH4 receptor homology model (Schultes et al., 2013b). Rendering and colour coding is the same as in Figure 1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ballesteros JW, Weinstein H. Integrated methods for the construction of three dimensional models and computational probing of structure funciton relations in G protein-coupled receptors. Methods Neurosci. 1995;25:366–428.
    1. Buck E, Bourne H, Wells JA. Site-specific disulfide capture of agonist and antagonist peptides on the C5a receptor. J Biol Chem. 2005;280:4009–4012. - PubMed
    1. Chapman KL, Kinsella GK, Cox A, Donnelly D, Findlay JB. Interactions of the melanocortin-4 receptor with the peptide agonist NDP-MSH. J Mol Biol. 2010;401:433–450. - PMC - PubMed
    1. Chavez FCM, Edwards JP, Gomez L, Grice CA, Kearney AM, Savall BM, et al. 2008. Benzofuro- and benzothienopyrimidine modulators of the histamine H4 receptor, (Janssen Pharmaceutica NV). WO-2008008359.
    1. Chen C, Xue JC, Zhu J, Chen YW, Kunapuli S, Kim de Riel J, et al. Characterization of irreversible binding of beta-funaltrexamine to the cloned rat mu opioid receptor. J Biol Chem. 1995;270:17866–17870. - PubMed

Publication types

MeSH terms

LinkOut - more resources