Anti-inflammatory potency of novel ecto-5'-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation

Abstract

Three novel cytosine-derived α,β-methylene diphosphonates designated MRS4598, MRS4552, and MRS4602 were tested in the range of 1 × 10^-9 to 1 × 10^-3 M for their efficacy and potency in inhibiting membrane-bound ecto-5'-nucleotidase/CD73 activity in primary astrocytes in vitro. The compounds were also tested for their ability to attenuate the reactive astrocyte phenotype induced by proinflammatory cytokines. The main findings are as follows: A) The tested compounds induced concentration-dependent inhibition of CD73 activity, with maximal inhibition achieved at ∼1 × 10^-3M; B) All compounds showed high inhibitory potency, as reflected by IC₅₀ values in the submicromolar range; C) All compounds showed high binding capacity, as reflected by Ki values in the low nanomolar range; D) Among the tested compounds, MRS4598 showed the highest inhibitory efficacy and potency, as reflected by IC₅₀ and Ki values of 0.11 μM and 18.2 nM; E) Neither compound affected astrocyte proliferation and cell metabolic activity at concentrations near to IC₅₀; E) MRS4598 was able to inhibit CD73 activity in reactive astrocytes stimulated with TNF-α and to induce concentration-dependent inhibition of CD73 in reactive astrocytes stimulated with IL-1β, with an order of magnitude higher IC₅₀ value; F) MRS4598 was the only compound tested that was able to induce shedding of the CD73 from astrocyte membranes and to enhance astrocyte migration in the scratch wound migration assay, albeit at concentration well above its IC₅₀ value. Given the role of CD73 in neurodegenerative diseases, MRS4598, MRS4552, and MRS4602 are promising pharmacological tools for the treatment of neurodegeneration and neuroinflammation.

Keywords: Astrocytes; Cell migration; Cytosine-based nucleoside 5'-α,β-methylene diphosphates; Ecto-5′-nucleotidase/CD73; Shedding.

Figure 1.. Evaluation of the inhibition of CD73 activity by cytosine-based 5’-α,β-methylene diphosphonates MRS4598, MRS4552, and MRS4602.

Structural and chemical formulas (A, E, C) and concentration-response curve of MRS4598 (B), MRS4552 (D), and MRS4602 (F). Concentration-response curves were obtained by fitting raw data (percent enzyme inhibition vs [inhibitor concentration]) to the sigmoid curve using a four-parameter logistic regression model. Black symbols represent individual measurements, colored symbols represent mean inhibition (E) at each inhibitor concentration ± SEM from n ≥ 3 independent measurements.

Figure 2.. Impact of compounds on metabolic activity and cellular state of astrocytes.

A) Cell metabolic activity was determined by MTT assay in the range of inhibitor concentrations and concentration-response curves for MRS4598 (blue line), MRS4552 (pink line), and MRS4602 (green line) are presented. Dotted line represents basal metabolic activity of control cells, defined as 100%. Significance inside graph: *p < 0.05. B) Bright field images of astrocytes treated with the compounds at the indicated concentrations, taken 24 hours after treatment. Scale bar = 100 μm. C) The proliferation rate was determined by double Ki67/DAPI fluorescence, in the presence of inhibitors for 24 hours at the indicated concentrations. Proliferation rate (%) was expressed as the proportion of Ki67+ cells out of total number of cells (DAPI+) cells. Dotted line represents basal proliferation rate in control culture. D) Representative images of Ki67/DAPI staining in the presence of compounds at indicated concentrations. Scale bar: 100 μm.

Figure 3.. The influence of compounds on astrocyte morphology.

Astrocytes treated with the indicated concentration of compound were prefixed after 24 hours and immunostained for GFAP, whereas nuclei were counterstained with DAPI. Images of the microscopic fields were acquired using confocal laser scanning microscopy. Images were used to determine cell surface area and shape descriptors (Table 1) in ImageJ. Scale bar in a): 20 μm.

Figure 4.. The inhibitory potency of MRS4598 on CD73 activity in astrocytes treated with inflammatory cytokines.

A) Representative immunoblot of astrocyte membrane samples isolated from control cultures and cultures treated with TNF-α and IL −1β. Equivalent amounts of native samples or PNGaseF-treated samples (dControl, dTNF-α, and dIL-1β) were resolved on SDS-PAGE, transffered onto PVDF, and immunoblotted with antiCD73 antibodies. B) CD73 activity in control culture and in cultures treated with TNF-α or IL-1β in the presence (blue bars) or absence (gray bars) of 50 μM MRS4598. Significance inside the graph: # p < 0.05 between cytokine-treated culture and control; * p < 0.05 between cultures exposed to MRS4598 and the corresponding control. C) Concentration-response curve to MRS4598 ranging from 1–250 μM in astrocytes treated with 0.1 μg/ml IL −1β. Data were fitted to a sigmoid curve (red lines) with four-parameter sigmoid function nonlinear regression analysis using Origin 7.0.

Figure 5.. The impact of MRS4598 on astrocyte migration in scratch wound assay.

A) Representative images of astrocytes in the presesnce of MRS4598 (1–100 μM), taken immediately after creating a wound and after 8 and 24 hours. Scale bar = 200 μm. B) The impact of MRS4598 is assessed by determining wound closure (%) as uncovered area at 8 and 24 hours, expressed as a percentage of the initial wound area at 0 hours.

Figure 6.. The impact of MRS4598 on CD73 release from astrocyte membranes.

A) Representative dot-blot membrane of soluble CD73 detected in culture media, 24 hours after addition of APCP or MRS4598 (both in the range of 1–100 μM). The presence of soluble CD73 was detected by anti-CD73 antibody and chemiluminescence detection. B) Integrated density values determined in ImageJ.