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Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

ABSTRACT: G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs in vitro can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery systems. Glycerosomes have greater morphological stability than liposomes. Proteoglycerosomes are defined as glycerosomes that contain membrane proteins. Human histamine H1 receptor (HRH1) is one of the most studied GPCRs. In this study, we synthesized wild-type HRH1 (WT-HRH1) proteoglycerosomes and D107A-HRH1, (in which Asp107 was replaced by Ala) in a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. The mutant HRH1 has been reported to have low affinity for the H1 antagonist. In this study, the amount of synthesized WT-HRH1 in one synthesis reaction was 434 ± 66.6 ?g (7.75 ± 1.19 × 103pmol). The specific binding of [3H]pyrilamine to the WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased. The dissociation constant (Kd) and maximum density (Bmax) of the synthesized WT-HRH1 were 9.76 ± 1.25 nM and 21.4 ± 0.936 pmol/mg protein, respectively. However, specific binding to D107A-HRH1 was reduced compared with WT-HRH1 and the binding did not become saturated. The findings of this study highlight that HRH1 synthesized using a wheat germ cell-free protein synthesis system combined with glycerosomes has the ability to bind to H1 antagonists.

SUBMITTER: Suzuki Y

PROVIDER: S-EPMC5808195 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

Suzuki Yasuyuki Y Ogasawara Tomio T Tanaka Yuki Y Takeda Hiroyuki H Sawasaki Tatsuya T Mogi Masaki M Liu Shuang S Maeyama Kazutaka K

Frontiers in pharmacology 20180206

G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs <i>in vitro</i> can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery syste ...[more]

PMID: 29467651

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Project description:Numerous host factors, in addition to human angiotensin-converting enzyme 2 (hACE2), have been identified as coreceptors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrating broad viral tropism and diversified druggable potential. We and others have found that antihistamine drugs, particularly histamine receptor H1 (HRH1) antagonists, potently inhibit SARS-CoV-2 infection. In this study, we provided compelling evidence that HRH1 acts as an alternative receptor for SARS-CoV-2 by directly binding to the viral spike protein. HRH1 also synergistically enhanced hACE2-dependent viral entry by interacting with hACE2. Antihistamine drugs effectively prevent viral infection by competitively binding to HRH1, thereby disrupting the interaction between the spike protein and its receptor. Multiple inhibition assays revealed that antihistamine drugs broadly inhibited the infection of various SARS-CoV-2 mutants with an average IC50 of 2.4 µM. The prophylactic function of these drugs was further confirmed by authentic SARS-CoV-2 infection assays and humanized mouse challenge experiments, demonstrating the therapeutic potential of antihistamine drugs for combating coronavirus disease 19.IMPORTANCEIn addition to human angiotensin-converting enzyme 2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can utilize alternative cofactors to facilitate viral entry. In this study, we discovered that histamine receptor H1 (HRH1) not only functions as an independent receptor for SARS-CoV-2 but also synergistically enhances ACE2-dependent viral entry by directly interacting with ACE2. Further studies have demonstrated that HRH1 facilitates the entry of SARS-CoV-2 by directly binding to the N-terminal domain of the spike protein. Conversely, antihistamine drugs, primarily HRH1 antagonists, can competitively bind to HRH1 and thereby prevent viral entry. These findings revealed that the administration of repurposable antihistamine drugs could be a therapeutic intervention to combat coronavirus disease 19.

Dataset Information

Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

Publications

Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

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