Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists

The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent <i>in vitro</i> ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1

Emetine disrupts HCMV mediated MDM2-p53 complex.

<p><b>A)</b> MDM2 and p53 expression increases with emetine treatment. Cells were seeded at 0.5 or 2 million cells/plate, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5 μM). Cell lysates were harvested at 24 or 72 hpi for Western blotting. <b>B)</b> Cells were seeded at 2 million cells/plate, infected with HCMV Towne and treated with emetine (75 nM) or GCV (5 μM). RNA was harvested and qRT-PCR was performed for p21. Data represent mean ±SE of triplicate determinations from a representative of three independent experiments <b>C)</b> HFFs were seeded at 1 or 2 million cells/plate and treated with emetine (75 nM) or GCV (5 μM). Cell lysates were harvested at 72 hpi for Western blotting. <b>D)</b> HFFs were seeded at either 0.5 or 2 million/plate in 100 mm dishes, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5μM) for 24h. MG132 (10 μM) was added after 12 h. At 24 hpi, lysates were collected and subjected to IP with anti-MDM2 antibody followed by immunoblotting with anti-p53 antibody or <b>E)</b> IP with anti-p53 antibody followed by immunoblotting with anti-MDM2 antibody. <b>F</b>) A model depicting the mechanism by which emetine disrupts HCMV mediated MDM2-p53 interaction in high-density cells, but not in cycling low-density cells.</p

Model of HCMV inhibition by emetine.

<p>In high-density infected cells (A) emetine induces (1) nuclear translocation of RPS14 (2) followed by RPS14 binding to MDM2 (3 & 4) resulting in disruption of the interaction between MDM2-p53 (6) and MDM2- viral IE2 (5 & 7), and by RPS14 ubiquitination and degradation (8). In low-density infected cells (B) although emetine induces (1) nuclear translocation of RPS14 (2), it is unable to interact with MDM2 (4) which is already bound to p53 to facilitate virus replication (3).</p

Emetine achieves high tissue concentrations and is efficacious against MCMV replication.

<p><b>A)</b><i>In vivo</i> pharmacokinetics of emetine. Plasma, liver, lung and spleen samples were collected at the indicated time points after single oral administration of emetine at 0.1 mg/kg in male BALB/c mice. Concentrations were measured using UPLC-MS/MS methods. For plasma, the concentrations observed were mostly below the level limit of quantification. <b>B)</b> Quantitative real-time PCR of viral gB was performed on DNA extracted from blood at day 14 post infection. Plaque assay was performed from salivary glands <b>C)</b> or liver <b>D)</b> collected at day 14 post infection. Emetine was administered orally starting 24 hpi or 72 hpi at 0.1 or 1.0 mg/kg every 3 days. GCV dose was 10 mg/kg/dose administered intraperitoneally twice daily.</p

Emetine induces an interaction between MDM2 and RPS14 in infected high-density cells.

<p><b>A)</b> Emetine abrogates infection-mediated overexpression of RPS14 at 72 hpi. Cells were seeded at 0.5 or 2 million cells/plate, infected with Towne HCMV and treated with emetine (75 nM) or GCV (5μM). Lysates were collected at 24 or 72 hpi for Western blotting. <b>B)</b> HFFs were seeded at 1 or 2 million cells/plate and treated with emetine or GCV. Lysates were collected at 72 hpi for Western blotting. <b>C)</b> Cells were seeded at 0.5 or 2 million/plate in 100 mm dishes, infected with Towne HCMV followed by treatment with emetine (75 nM) or GCV (5μM) for 24h. MG132 (10 μM) was added after 12 h. At 24 hpi, lysates were collected and subjected to IP with anti-MDM2 followed by immunoblotting with anti-RPS14 antibody or <b>D)</b> In reverse reaction, IP was performed with anti-RPS14 followed by immunoblotting with anti-MDM2 antibody. Densitometry analysis was performed to normalize immunoprecipitated protein to its input level. <b>E</b>) A model showing the interaction of MDM2 and RPS14 in high-density but not in low-density cells.</p

Nuclear localization of RPS14 persists during infection and emetine treatment in low-density cells.

<p><b>A)</b> Cells were seeded at 0.5 million/4-well chamber slide, infected and treated with either emetine (75 nM) or GCV (5μM) for 24 h. Cells were stained with IE1/2 (Alexa 555:Red) for evidence of infection and RPS14 (FITC: Green) and nuclear DAPI. Stained slides were subjected to confocal microscopy and nuclear localization was quantified using NIS elements software (Nikon). <b>B)</b> The same procedure was repeated at 72 hpi. The images shown are a representative of two independent experiments. Percent nuclear localization is represented as mean ± SD of at least 25 cells per condition from two different fields in the slide.</p

The anti-HCMV activity of emetine is reduced in low-density HFFs.

<p><b>A)</b> Cells were seeded at indicated densities in a 96-well plate, infected with pp28-luciferase HCMV Towne (MOI = 1) and treated with emetine (75 nM) or GCV (5 μM). Luciferase activity was measured at 72 hpi and normalized to the activity of infected untreated cells. Data represent mean ± SE of triplicate determinations from a representative of two independent experiments. <b>B)</b> Same cell lysates as in A were collected for Western blotting at 72 hpi and expression of viral proteins UL44 and pp65 was determined. <b>C)</b> Supernatants from each well as in A were used to infect cells (1 million cells in a 96-well plate) in the corresponding well and luciferase activity was measured at 72 hpi. <b>D)</b> Cells were seeded into 12-well plates at 0.5 and 2 million cells/plate, infected with 100 PFU/well of HCMV Towne or MCMV, and treated with emetine (75 nM) or GCV (5 μM). After 10 days (for HCMV) or 3 days (for MCMV) plaques were stained with crystal violet and the number of plaques enumerated. Data shown are average of 2 wells (±SD) for a representative experiment from two different experiments.</p

Anti-HCMV activity of emetine and synergy with GCV.

<p><b>A)</b> Cells were infected with pp28-luciferase HCMV Towne and treated with indicated concentrations of emetine or GCV. Luciferase activity was measured at 72 hpi. Data represent mean ± SE of triplicate determinations from a representative of three independent experiments. <b>B)</b> Cells were treated with the indicated concentrations of emetine and cell viability was determined after 72 h. Data represent mean values ± SE of triplicate determinations from a representative of three independent experiments. <b>C)</b> Inhibition of HCMV protein expression by emetine (75 nM) or GCV (5 μM) at 72 hpi. Cells were infected with HCMV Towne and treated with emetine. Western blot of viral proteins IE1/2, UL44 and pp65 was performed at 72 hpi. <b>D)</b> Cells were infected with pp28-luciferase HCMV Towne, and treated with combination of GCV and emetine. The antiviral activity was evaluated by luciferase assay. Data represent mean values ± the SE of triplicate determinations from a representative of three independent experiments.</p

Emetine induces nuclear translocation of RPS14 in infected high-density cells at 24 hpi followed by cytoplasmic relocalization.

<p><b>A)</b> Cells were seeded at 2 million/4-well chamber slide, infected and treated with either emetine (75 nM) or GCV (5μM) for 24 h. Cells were stained with IE1/2 (Alexa 555:Red) for evidence of infection and RPS14 (FITC: Green) and nuclear DAPI. Stained slides were subjected to confocal microscopy and colocalization was studied and quantified using NIS elements software (Nikon). <b>B)</b> The same procedure was repeated at 72 hpi. The images shown are a representative of two independent experiments. Percent nuclear localization is represented as Mean ± SD from two different fields of at least 40 cells per condition.</p

RPS14 is ubiquitinated and degraded in infected high-density cells.

<p><b>A)</b> Cells were seeded at 0.5 or 2 million/plate in 100 mm dishes, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5μM) for 72h. MG132 (10 μM) was added 12 h before harvest. Lysates were collected at 72 hpi and subjected to IP with anti-Ubiquitin antibody followed by immunoblotting with anti-RPS14 antibody or IP with anti-RPS14 antibody followed by immunoblotting with anti-Ubiquitin antibody. The mono- and poly- ubiquitinated forms of RPS14 are depicted as (Ub)-RPS14 and n(Ub)-RPS14, respectively. <b>B)</b> HCMV-infected cells (left: high-density right: low-density) were treated with emetine or GCV. At 24 hpi cycloheximide (CHX, 100 μg/mL) was added. Cells were harvested at the indicated time intervals up to 8 h following CHX for SDS-PAGE analysis.</p