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
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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

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Figure 1

Enhanced macrophage activity in mice expressing a dimerization-deficient GPRC5B mutant (G5b-mut).

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Enhanced macrophage activity in mice expressing a dimerization-deficient...
(A) Generation of G5b-mut mice: (left) GPRC5B amino acids F97, L101, and L104 were mutated to alanine, indicated by the letter A; (right) localization of guide RNAs (gRNA) and repair DNA within the Gprc5b allele; resulting founder mice were used to produce Gprc5bmut/mut mice (G5b-mut) and Gprc5bwt/wt mice (G5b-wt). (B) Coimmunoprecipitation of endogenous GPRC5B (G5B) with EP2 in RPMs from G5b-wt or G5b-mut mice; IgG-mediated precipitation in G5b-wt RPMs as negative control. (C) Butaprost-induced cAMP production was determined by ELISA in RPMs from G5b-wt and G5b-mut mice (cells from control mice and M-G5b-KOs as reference) (n = 8). (D) Basal and chemokine-induced transwell migration of G5b-wt and G5b-mut RPMs; cells from control mice and M-G5b-KOs as reference (n = 6). (E and F) Phagocytosis of E. coli bioparticles in G5b-wt and G5b-mut RPMs. Exemplary traces (E) and statistical evaluation of area under the curve (AUC, F); cells from control and M-G5b-KOs as reference (n = 6). (G–J) Fecal peritonitis in G5b-wt and G5b-mut mice: body weight change (G), bacterial colony-forming units (CFU) in peritoneal lavage fluid 24 hours after injection of fecal bacteria (H), numbers of CD11b+, F4/80lo, MHCII+, CCR2+ macrophages (I), and CD11b+, Ly6G–, Ly6C+ monocytes (J) before and 24 hours after injection of fecal bacteria (n = 5–6). For gating strategy, see section 4 of Supplemental Materials. Data are means ± SEM; comparisons between treatment groups were performed using 2-way ANOVA with Šídák’s (C, G, I, and J) or Tukey’s (D) multiple comparisons test or unpaired, 2-tailed t test (F and H). n, number of independent experiments or mice; *P < 0.05; **P < 0.01; ****P < 0.0001.