Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo

Abstract

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue-alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure-activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate S_P and R_P diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist-AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the S_P-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial-mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the S_P-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

Keywords: ATP analogue; P2Y2 receptor; Purinergic signaling; Skin regeneration; Wound healing.

Figure 1

ATP derivatives used in current studies: ATP (1), α-thio-ATP (2a,b), β,γ-methylene-ATP (3) and α-thio-β,γ-methylene-ATP (4a,b), respectively.

Figure 2

The schematic procedure for synthesis and purification of diastereomerically pure α-thio-modified ATP analogues. The single-modified 2a and 2b derivatives are presented in the left panel, double-modified 4a and 4b compounds in the right panel. The general synthesis scheme (upper part) and HPLC profiles below including: post-synthetic mixture of P-diastereoisomers 2a and 2b, 4a and 4b, respectively (top), followed by profiles after separation of compound 2 and 4 into individual P-isomers: fast (middle) and slow (lower), respectively, according to their chromatographic mobility on a C18 column.

Figure 3

The viability of HaCaT cells after 72 h incubation with tested compounds at the concentration of 100 μM. Data represent mean percentage viability ± standard error of measurement (SEM) estimated for the control (untreated cells, assumed as 100%) from at least 3 independent experiments performed in triplicate. ***p < 0.0001 compared to the control.

Figure 4

Impact of diastereomerically pure ATP analogues on intracellular calcium mobilization. Intracellular calcium mobilization measurements. The following designations were adopted for compounds: ATP (orange) and tested ATP analogues: α-thio-ATP (2a and 2b blue), β,γ-methylene-ATP (3 violet) and α-thio-β,γ-methylene-ATP (4a and 4b green). Data represent the means ± SEM from at least 3 independent experiments. ****p < 0.0001 vs control ^####p < 0.0001 vs ATP. C—untreated control cells, RFU—relative fluorescence units.

Figure 5

Migration of human keratinocytes after treatment with α-thio-modified ATP analogues. The rate of wound healing after 24 h treatment of HaCaT cells with 100 μM of α-thio-modified ATP derivatives. Upper panel: relative migration rate of HaCaT cells quantified based on the size of the uncovered area after 24 h incubation with tested compounds compared to the untreated cells (taken as control sample 100%). Data represent the means ± SEM from at least 3 independent experiments. Lower panel: the microscopy images of the wound area after 24 h incubation with tested compounds. *p < 0.05 compared to the control, Scale bars 50 μm. The initial sizes of the scratches are available in Supplementary Materials (Fig. S16).

Figure 6

Impact of α-thio-β,γ-methylene-ATP on the epithelial–mesenchymal transition- related changes in the model MDA-MB-231 cells. (A) Transwell migration rate of cells through 8 μm pore sizes. Data represent the means ± SEM from at least 3 independent experiments, (B) Crossing the collagen barrier in the presence of tested compounds. (C) The impact of tested analogues on in vivo cells migration in zebrafish xenograft model (D) QPCR analysis of EMT markers after treatment with 4a and 4b. Data represent the means ± SEM from at least 3 independent experiments ***p < 0.0005, **p < 0.001, *p < 0.05 compared to the control.

Figure 7

The in vivo impact of α-thio-β,γ-methylene-ATP on the tissue regeneration in Danio rerio model organism. (a) The images of tail zebrafish embryo were recorded by stereo microscope, dpi (day post incubation) represent the day after drug treatment. (b) The 4a and 4b promote the fin regrowth (n = 22, *** p < 0,001, **** p < 0,001). The regenerated parts of the tails have been marked with lines for better visibility.

Figure 8

In silico studies on the interaction between ATP analogues and P2Y2 receptor. (A) LigPlot graph presents the 2D plot of P2Y2 receptor-ligand interactions with ATP (1) as a natural ligand. (B) The binding energy and number of hydrogen bonds calculated using molecular docking for ATP (1) and ATP analogues (2a-4b).

Figure 9

In vitro verification of ATP analogues activity in the presence of P2Y2 receptor antagonist. (A) The impact of tested ATP analogues on Ca²⁺ mobilization in HaCaT cells in the presence and absence of P2Y2 antagonist (B) The influence of 4a derivative on transwell migration of MDA-MB-231 cells in the presence and absence of P2Y2 antagonist. Data represent the means ± SEM from at least 3 independent experiments. ****p < 0.0001 vs control. C—untreated control cells, RFU—relative fluorescence units. Scale bars 50 μm.