Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling

Abstract

G protein-coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2-mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates' ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation-independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

Keywords: CADD; GPCR; GRK2; RGS; desensitization.

FIGURE 1

Obtention and characterization of probe and control systems. (A) HEK293 control cells or clones transfected with pCDNA3.1 Zeo (+) H2R and resistant to zeocin (H2.1, H2.2, and H2.3) were evaluated for cAMP response after stimulation with 10 μM amthamine (Amtha) in the presence of IBMX (left panel) and by saturation binding assay with [³H]‐tiotidine (right panel) as detailed in the “Materials and methods” section. Data were analyzed by two‐way ANOVA and Dunnett's post‐test. *p < .05; ****p < .0001. (B) H2.1 clone was transfected with pcDNA3‐GRK2K220R (left panel) or pcDNA3‐GRK2K220R/R106A (right panel) and the clones resistant to geneticin were analyzed for GRK2 overexpression by SDS‐PAGE and western blot. Data were analyzed by one‐way ANOVA and Dunnett's post‐test. *p < .05; ****p < .0001. (C) Probe system (left panel) and control system (right panel) were exposed for 10 min to increasing concentrations of Amtha at 37 °C in the presence of 1 mM IBMX. cAMP levels were determined as detailed in the “Materials and methods” section. Data represent the mean ± SD of assay duplicates. Similar results were obtained in three independent experiments

FIGURE 2

Biological activity of hit compounds: positive modulation of cAMP response of H2R. (A) Probe system or (B) Control system cells were pre‐treated with 10 µM or 100 µM of indicated compounds for 40 min, and basal cAMP response or stimulated with 10 µM amthamine (Amtha) in the presence of IBMX was determined as detailed in the “Materials and methods” section. Grey bars correspond to basal and white to the stimulated response. Data were analyzed by two‐way ANOVA and Dunnett's post‐test. **p < .01, ***p < .001; ****p < .0001 with respect to control (DMSO)‐stimulated response. Data represent the mean ± SD of assay duplicates. Similar results were obtained in three independent experiments

FIGURE 3

Preliminary profiling of physicochemical and in vitro ADMET properties of hit compounds. (A) U937 (left panel) and HEPG2 (right panel) cells were treated with increasing concentrations of C2Z858, C3Z392, C4Z102, and C5Z299 or DMSO for 48 h, and cell viability was determined by Trypan Blue exclusion test. Data represent media ± SD of three independent experiments. (B) Lipophilicity of hit compounds was determined by RP‐HPLC as detailed in the “Materials and methods” section. Processing of retention time “RT” and dead time “T0” (solvent front, DMSO) for each compound is shown. Experimental log Kw values correspond to extrapolations at 0% acetonitrile (ACN)

FIGURE 4

Mechanism of action of candidates: inhibition of GRK2 (45‐178) GFP translocation. (A) HELA cells were transiently cotransfected with GRK2 (45‐178) GFP and H2R plasmids or empty vector (MOCK) and fixed after 48 h. Subcellular localization was assessed by confocal microscopy in basal or in 10 µM amthamine (Amtha)‐stimulated condition. (B) HELA cells were transiently cotransfected with GRK2 (45‐178) GFP and H2R plasmids. Forty‐eight hours after transfection cells were treated with 100 nM of C2Z858, C3Z392, C5Z299, or C9Z817 (negative control) or DMSO (vehicle control) for 40 min and fixed. At least 100 cells were examined in three independent experiments. For each cell in a given image, a line intensity profile across the cell was obtained. Representative intensity profiles are shown for each condition

FIGURE 5

Mechanism of action of candidates: inhibition of interaction between GRK2 and Gαs subunit. Co‐immunoprecipitation of Gαs and GRK2 is shown. HEK293 cells co‐transfected with GRK2, H2R, and HA‐tagged Gαs, or Mock were incubated for 10 min with 10 µM amthamine (Amtha) (A) or treated with 10 µM C3Z392 and C5Z299 for 30 min (B), and cross‐linking with 2.5 mM dithiobis(succinimidyl propionate) was done. Cells were lysed, and HA‐Gαs was immunoprecipitated (IP) using agarose beads coupled to anti‐HA antibody. Co‐precipitated GRK2 was detected by western blot using specific antibodies. Total Gαs was detected by western blot using anti‐HA antibodies. A representative image is shown. Densitometry analysis was performed with ImageJ as indicated in the “Materials and methods” section and analyzed by Student's t‐test comparing GRK2 levels in candidate pre‐treated versus control cells (n = 3)

FIGURE 6

In vitro efficacy of active compounds: inhibition of desensitization. (A) U937 cells endogenously expressing H2R and GRK2 were exposed to 10 µM amthamine (Amtha) for 1 h and after washing, rechallenged with 10 µM Amtha for 10 min in the presence of IBMX. cAMP response was determined as detailed in the “Materials and methods” section. Data were calculated as the means ± SD of assay duplicates and are expressed as the ratio of stimulated over basal cAMP with respect to control (without pre‐treatment) which is considered as 100%. (B) HEKT Epac‐SH187 transiently co‐transfected with H2R and GRK2 were pre‐treated during 1 h with either 10 µM Amtha (red) or vehicle (green) in the presence of different concentrations of C3Z392 as indicated. After that, cells were washed and challenged with 10 µM Amtha. (C) Concentration–response curves were constructed with the AUC values of 10‐min R/R0 cAMP response of time course of FRET changes with respect to control (without pre‐treatment) determined in FlexStation^®3 at 37°C as described in the “Materials and methods” section