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. 2019 Dec 11;24(24):4541.
doi: 10.3390/molecules24244541.

Covalent Inhibition of the Histamine H3 Receptor

Gábor Wágner  1 , Tamara A M Mocking  1 , Albert J Kooistra  1 , Inna Slynko  1 , Péter Ábrányi-Balogh  2 , György M Keserű  2 , Maikel Wijtmans  1 , Henry F Vischer  1 , Iwan J P de Esch  1 , Rob Leurs  1
Affiliations

Covalent Inhibition of the Histamine H3 Receptor

Gábor Wágner et al. Molecules. .

Abstract

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H3 receptor (H3R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H3R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H3R was validated with washout experiments and leads to inverse agonism on H3R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H3R conformation and to study the consequences of prolonged inhibition of the H3R.

Keywords: G protein-coupled receptor (GPCR); Histamine H3 receptor; covalent binder; isothiocyanate.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The three design cycles of the potential H3R covalent ligands and their non-covalent reference compounds. Red compounds proved too unstable to be isolated in pure form.
Figure 2
Figure 2
Proposed binding mode of a representative compound from the third design cycle (Figure 1 top right, R2 = -NCS) covalently bound to C1183.36. The ligand is shown in cyan sticks and the protein is shown in light yellow sticks and ribbons. Only polar hydrogens are shown in white with protein-ligand hydrogen bonding indicated in black dashed lines. For clarity purposes, parts of the protein are not shown. Residue numbers are shown as UniProt and as GPCR-specific Ballesteros–Weinstein numbers [30].
Scheme 1
Scheme 1
Synthesis of pyrimidine-based covalent ligands. (a) POCl3, reflux, 3 h, 26–33%; (b) N,N-dimethylazetidin-3-amine or 1-cyclobutylpiperazine, N,N-Diisopropylethylamine (DIPEA), dioxane, 150 °C, 30 min, μW, 59–65%; (c) t-butyl azetidin-3-yl(methyl)carbamate or t-butyl piperazine-1-carbamate, DIPEA, dioxane, 150 °C, 30 min, μW, 50–85%, 8: used without full purification; (d) 1. HCl, dioxane, rt, 2 h, 2. cyclobutanone, NaBH(OAc)3, dichloromethane (DCM), rt, overnight, 33% (two steps); (e) tBuONO or iPeONO, SbCl3, DCM, rt, 3 h, 23–40%; (f) 1. HCl, dioxane, rt, 2 h-overnight, 2. H2CO or cyclobutanone, NaBH(OAc)3, DCM, rt, overnight, 56–73% (two steps); (g) ethynyltrimethylsilane, CuI, Pd(dppf)Cl2, TEA, DME, 100 °C, 1 h, μW, 54%; (h) K2CO3, MeOH, rt, 1 h, 72%; (i) vinyl boronic acid MIDA ester, Pd(PPh3)4, Na2CO3, DME, H2O, 120 °C, 1 h, μW, 21–62%.
Scheme 2
Scheme 2
Synthesis of pyrimidine-based reference ligands. (a) methyl 4-methyl-3-oxopentanoate, NaOMe, MeOH, rt, overnight, 91–97%; (b) POCl3, reflux, 3 h, 86–87%; (c) t-butyl azetidin-3-yl(methyl)carbamate, DIPEA, dioxane, 150 °C, 30 min, μW, 34–38%; (d) HCl, dioxane, rt, 3 h-overnight, 77–90%; (e) H2CO, NaBH(OAc)3, DCM, rt, overnight, 82%; (f) 1-cyclobutylpiperazine, DIPEA, dioxane, 150 °C, 30 min, μW, 36–51%.
Scheme 3
Scheme 3
Synthetic of phenylpiperazine derivatives. (a) t-butyl piperazine-1-carboxylate, NaOtBu, Pd2(dba)3, Xantphos, dioxane, 80 °C, 1 h, μW, 57%; (b) HCl, dioxane, rt, 1 h, 78%; (c) cyclobutanone, NaBH(OAc)3, DCM, rt, overnight, 76%; (d) HCOONH4, Pd/C, MeOH, H2O, rt, overnight, 25%; (e) 2-chloroacetyl chloride, TEA, DCM, rt, 1 h, 68%; (f) CSCl2, NaHCO3, DCM, H2O, rt, 1 h, 64%.
Figure 3
Figure 3
LC-MS analysis of 44 (0.40 mM) in 50 mM Tris-HCl pH 7.4 without (A,B) and with (C) GSH (1.2 mM). The mixtures were incubated for 10 min (A,C) or 100 min (B) at rt. The incubated mixtures were analysed by LC-MS. A signal from the DMSO used for the stock solution of 44 is visible at 1.2 min. (D) Postulated identified products.
Figure 4
Figure 4
MS/MS spectrum of NP modified by 44 (m/z 676.5, see Figure S1A). Two-fold charged species were examined.
Figure 5
Figure 5
(A) Binding of [3H]NAMH to hH3R-expressing HEK293T cell homogenates that were pre-incubated with vehicle, 10 µM 44, 10 µM 42 or 10 µM of non-covalent reference H3R ligands thioperamide, pitolisant and histamine for 1 h at 25 °C, followed by washout. (B,C) Radioligand displacement curves of [3H]NAMH with histamine on cell homogenates preincubated for 1 h at 25 °C with increasing concentrations of (B) 44 or (C) 42. After thorough washing pretreated cell homogenates were subjected to displacement of [3H]NAMH with increasing histamine concentrations. Data are normalized to specific binding of vehicle treated cell homogenates. Data shown are pooled data mean ± S.E.M. of at least three experiments performed in triplicate. * p < 0.05 (one-way ANOVA with Fisher’s LSD test).
Figure 6
Figure 6
Functional characterization of 44 as measured by [35S]GTPγS binding to hH3R expressing HEK293T cell homogenates at 25 °C for 1 h. (A) Inhibition of basal hH3R activity by 44. (B) Inhibition of 31.6 nM immepip-induced hH3R activity by 44 or pitolisant.
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