Small molecule inhibitor of orphan GPCR dimerization improves host defense and blood pressure control in mice
Jeonghyeon Kwon, Margherita Persechino, Jingchen Shao, Jamal Shamsara, Birgit Spitznagel, Isabelle Salwig, Miloslav Sanda, Stefan Offermanns, Peter Kolb, Nina Wettschureck
Jeonghyeon Kwon, Margherita Persechino, Jingchen Shao, Jamal Shamsara, Birgit Spitznagel, Isabelle Salwig, Miloslav Sanda, Stefan Offermanns, Peter Kolb, Nina Wettschureck
View: Text | PDF
Research Article Immunology Vascular biology

Small molecule inhibitor of orphan GPCR dimerization improves host defense and blood pressure control in mice

Abstract

Orphan GPCRs of the GPRC5 family regulate macrophage activity and vascular contractility by dimerizing with other GPCRs, but pharmacological modulation of this process has not been explored. We previously identified the dimerization interface of receptor GPRC5B and show here that both its mutation and inhibition by a decoy peptide disturbed the interaction with the prostaglandin E2 receptor EP2 in macrophages, resulting in reduced EP2 signaling, enhanced migration and phagocytosis, and protection from bacterial peritonitis in mice. Furthermore, we show that a similar interface exists in related receptor GPRC5C, and, the same as in GPRC5B, mutation or inhibition by decoy peptide improved host defense. Through a virtual docking screen, we identified a small molecule inhibitor of both GPRC5B and GPRC5C dimerization, K303MP20, and showed that it reduced EP2 signaling, enhanced macrophage activity, and improved host defense in bacterial peritonitis and influenza A infection. Interestingly, K303MP20 not only blocked dimerization between GPRC5B/C and EP2, but also with prostacyclin receptor IP and angiotensin II receptor AT1, resulting in reduced AT1-dependent contraction and enhanced IP-dependent relaxation in human and murine smooth muscle cells. In vivo, K303MP20 did not affect basal blood pressure, but protected mice from angiotensin II–induced hypertension. Taken together, inhibition of orphan GPCR dimerization by small molecules is feasible and improves infection control and arterial hypertension.

Authors

Jeonghyeon Kwon, Margherita Persechino, Jingchen Shao, Jamal Shamsara, Birgit Spitznagel, Isabelle Salwig, Miloslav Sanda, Stefan Offermanns, Peter Kolb, Nina Wettschureck

×

Figure 4

GPRC5C-mediated effects on cAMP production and macrophage activity are blocked by interface mutation and decoy peptide.

Options: View larger image (or click on image) Download as PowerPoint
GPRC5C-mediated effects on cAMP production and macrophage activity are b...
(A) Amino acid sequences in the putative interaction interfaces. (B) Localization of 3 sets of residues predicted to mediate the interaction. (C and D) Western blot detection of HA and FLAG signals in lysates of HEK cells expressing FLAG-tagged WT (G5C-wt, C) or mutated (D, G5C-mut7) GPRC5C in combination with different HA-tagged prostanoid receptors before (“input”) and after immunoprecipitation of GPRC5B-FLAG (“Pulldown FLAG”). (E) Butaprost-induced cAMP production in HEK cells transfected with cAMP GloSensor plasmid, HA-EP2, and WT or mutant GPRC5C (G5C, middle section) or GPRC5B (G5B, right section). Mutants contain either alanine mutations of all 7 residues (“mut7”) or only of set1 residues (“set1”) as indicated in A and B (n = 4). (F) Principle of decoy peptide–mediated inhibition of GPRC5C dimerization. (G) Effect of GPRC5C decoy peptide and control peptides (1 μM each) on butaprost-induced cAMP production in RPMs (n = 4). (H and I) Peptide effect on phagocytosis of pHrodo E. coli bioparticles in RPMs; exemplary traces (H) and statistical evaluation of AUC (I) (n = 6). (J and K) Fecal peritonitis model: effect of peptides (100 μl of a 100 μM solution) on body weight change (J) and bacterial CFU in peritoneal lavage fluid harvested 24 hours after injection of fecal bacteria (K) (n = 5). Data are means ± SEM; comparisons between treatments were performed using 2-way ANOVA with Dunnett’s (E), Tukey’s (G), or Šídák’s (J) multiple comparisons test, 1-way ANOVA with Tukey’s multiple comparisons test (I), or unpaired, 2-sided t test (K). n, number of independent experiments or mice; RLU, relative luminescence units; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.