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.

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.

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.

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.

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).

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.

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.

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).

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.