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

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.

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 E182^5.46 (Schultes et al., 2013b). In addition, substituents at this position are also in close proximity to C98^3.36 as indicated by the green dotted line (distance ring carbon to thiol group of C98^3.36 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

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

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

[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

Functional characterization of VUF14480 and VUF14481 on hH₄-C98^3.36S receptor. (A) GTPγS-binding assay. Membranes (10 μg per well) from cells expressing hH₄ receptor-C98^3.36S 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-C98^3.36S-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

[³H]-histamine binding to hH₄-C98^3.36S receptor expressing HEK293T cell membranes pre-incubated with VUF14480 or VUF14481. Crude membrane fractions of hH₄-C98^3.36S 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

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.