Chemical agents used in the present study included (DMEM and DMSO; S.D. Fine chemicals Ltd., Bengaluru, India), (Trypsin-EDTA Solution; SRL Chemicals, Mumbai, India), (FBS; Sigma, Bengaluru, India), (Camptothecin; Sigma, Bengaluru, India), (Antibodies; Abcam, Waltham, Boston, United States), (37°C incubator with humidified atmosphere of CO₂; Healforce, China), (Cell line: K562—procured from NCCS, Pune, India). HT microplate spectrophotometer reader was purchased from BioTek (Gujarat, India).

The structural analogues of MMs02943764 and MMs03738126 consisting of 1, 2, 4 triazole scaffold derived from molecular docking were synthesized as per the Scheme-1 and molecular physicochemical properties of 1, 2, 4-triazole derivatives are presented in (Table 1).

Perkin Elmer FT-IR type 1650 spectrophotometer was used to record the infrared spectra of the synthesized compounds within the range of 4000–400 cm⁻¹ considering the Potassium bromide pellets. The 1H-NMR was analyzed using a Bruker AV-500 spectrometer and DMSO-d₆ was used as a solvent and tetramethylsilane was used as internal standard. The mass spectroscopy of synthesized compounds was analyzed using Agilent technologies (HP) 5973 mass spectrometer with an ionization potential of 70 eV.

The K562 cells (1 × 10⁶ cells/well) were procured from ATCC. The stock cells were cultured in DMEM which is supplemented with inactivated FBS (10%), penicillin (1%; 100 IU/mL), and streptomycin (100 μg/mL) was added in a humidified atmosphere containing CO₂ (5%) at 37°C until confluent. The cells are dissociated with TPVG solution having a composition of trypsin (0.2%), EDTA (0.02%), and glucose (0.05%) dissolved in PBS. The viability of the cells is then checked using trypan blue and centrifuged. Further, 5.0 × 10⁴ cells/well were seeded in a 96 well plate and finally incubated for 24 h in CO₂ (5%) incubator at 37°C.

The monolayered cell culture was trypsinised. The cell count was brought to 5.0 × 10⁵ cells/mL by using the medium containing 10% FBS. Next, 100 µL of diluted cell suspension was added to each well of the microtiter plates. After 24 h, when the monolayer is formed, the supernatant is removed. 100 μL of different experimental compounds added to the wells containing the monolayer. These plates were kept for incubation in CO₂ (5%) incubator for 24 h at 37°C and cells will be periodically checked for physical changes such as granularity, shrinkage, and swelling. After 24 h of incubation, the sample solution was removed from the wells. 100 μL of MTT (5 mg/10 mL of MTT in PBS) was added to the wells. The plates were gently shaken and incubated for 4 h at 37°C under the CO₂ (5%) environment. The supernatant was removed and DMSO (100 µL) was added. The plates were again gently shaken to solubilize the formazan produced in the viable cells. The absorbance values were taken from the microplate reader at a wavelength of 590 nm. The percentage growth inhibition has been calculated as per the protocol (Tian et al., 2022).

To analyze the cell cycle phase distribution, K562 cells (1 × 106 cells/well) were seeded in a 6- well plate for 48 h and then exposed to the experimental compounds (PAC (68.44 μM) and standard drug, Calprotectin (25 μM)) and control. After incubation for 48 h, the untreated and treated cells were rinsed 2x with PBS. The cells are then fixed in 70% ethanol at −20°C for 30 min. The fixed cells were again washed with PBS and 50 μL of RNase solution was added. 400 μL of PI solution/million cells added directly to the cell’s RNase A suspension. Mixed well and incubated for 20–30 min at room temperature in the absence of light. The cell cycle was measured with BD FACSCalibur™ and the percentages of cells (10,000 cells in total) in the different phases (G1, S, and G2) were calculated by the Cell Quest software (BD Biosciences) (Bunney et al., 2017).

The K562 cells (1 × 10⁶ cells/well) was seeded in a 6-well plate and incubated under CO₂ atmosphere at 37°C for 24 h. After 24 h, the cells were cultured in a medium containing required concentrations of experimental compounds and controls for 24 h. Cells were then washed twice with PBS and 200 μL of trypsin-EDTA solution was added. The mixture was then incubated at 37°C for 3–4 min. To this, 2 mL culture medium was added, and the cells were harvested directly into the 12 × 75 mm polystyrene tubes followed by centrifugation and 1xPBS wash. PBS was decanted completely, and the cells were fixed with 70% chilled ethanol followed by incubation at −20°C for 30 min 20 μL of primary antibody (p53/Mdm2/Pirh2) was added (Parrales et al., 2016). The cells were washed with 2xPBS and treated with 20 µL of secondary fluorescent antibody Phycoerythrin (PE) and incubated for 30 min at room temperature in dark condition. The cells were then rinsed with 1xPBS to remove unbound secondary antibody and re-suspended in the 0.5 mL of PBS. The cells were analyzed for p53, Mdm2, and Pirh2 proteins expression by using BD FACSCalibur™. At least ten thousand cells were counted for each sample (Bunney et al., 2017).

All experiments were conducted in triplicate, data were presented as mean ± standard error of the mean (SEM). Student’s t-test was employed for comparing between two groups in the in vitro assay. To assess the statistical significance of the data, one-way analysis of variance (ANOVA) was utilized, with a significance level set at p < 0.05. Statistical analysis was performed using GraphPad Prism 8.0 (Aly et al., 2020).

The free mercapto group present in the 1,2,4-triazole intermediate readily reacts with appropriate chloroacetamide under dry conditions to yield the final compounds of PAA and PAC. The synthesized compounds of PAA and PAC were characterized by determining physical constants (Rf values and melting points) and different spectroscopic methods such as IR, ¹H NMR, 13C NMR, Mass spectroscopy and single crystal XRD. The experimental physical data of the synthesized compounds of PAA and PAC is provided in Table 2 and the calculated values of molecular physicochemical properties are shown in Table 1 (see materials and methods section).

IR spectra of all the synthesized compounds of PAA and PAC showed stretching absorption bands around regions 1550–1570 cm⁻¹ due to N=N and 1560–1640 cm⁻¹ due to C=N functions confirming the presence of 1,2,4-triazole ring. The absence of absorption band around 2576 cm⁻¹ which corresponds to–SH group and retaining of absorption band −750 cm⁻¹ corresponded to C-S function confirms the coupling of triazole ring with acetamides via. reactive -SH group. The spectral characterization by ¹H NMR spectra of all the synthesized compounds of PAA and PAC showed the chemical shift signals in the range of δ 6.77–6.9 ppm (Ar-H) resonated as multiplet for aromatic protons. The NH proton corresponded to the amide group resonated as a singlet and showed the chemical shift signal around 10.3 ppm. Also, the chemical shift signals at δ 4.1 ppm and δ 5.0 ppm resonated singlet peaks corresponded to that of α-proteins S-CH₂- C=O and O-CH₂-C=N respectively. Mass spectrum of the compounds of PAA and PAC showed intense molecular ion peak [M]⁺ and [M+1]⁺ peak for chloro-substituted compounds which are consistent to with respective molecular weights. All the screenshots of the spectral data collected for the compounds of PAA and PAC are provided as Figures 2–7. Analysis of overall spectroscopic data for the compounds of PAA and PAC suggests the formation of desired chemical scaffolds (Table 2) (Volkov et al., 2022).

N-(4-chloro phenyl)-2-((5-(phenoxy methyl)-4-phenyl-4H-1, 2, 4-triazol-3 yl)thio) acetamide (8a; PAC): IR (νmax, cm^-1): 1666 (C=O), 2924 (Ali C-H), 3032 (Ar C-H), 3232 (N-H), 1172 (C-N), 756 str (C-Cl), 1550 (N=N, triazole), 1597 (C=N, triazole); ¹H NMR (400 MHz, CDCl3) δ 10.482 (s, -NH-, ¹H), 7.561-6.844 (m, Ar-CH-, 14H), 5.053 (s, O-CH2-, 2H) 3.962 (s, S-CH2-, 2H); 13C NMR (100 MHz, CDCl3) δ 166.43, 157.34, 136.89, 132.12, 130.68, 130.03, 129.59, 129.07, 128.85, 126.74, 122.01,120.97, 114.84, 59.71, 36.26; Mass: Exact mass calculated for C₂₃H₁₉C_lN₄O₂S, 450.09; found (m/z), 451.13 [M + H] ⁺ (Figures 1–4).

2-((5-(phenoxy methyl)-4-phenyl-4H-1, 2, 4-triazol-3-yl)thio)-N-phenyl acetamide (8e; PAA): IR (νmax, cm⁻¹): 1666 (C=O), 1550 (Ar C=C), 2931 (Ali C-H), 3055 (Ar C-H), 3240 (N-H), 1172 (C-N),763 str (C-S), 1550 (N=N, triazole), 1597 (C=N, triazole); ¹H-NMR (400 MHz, DMSO-d6) δ 10.323 (s,-NH-, ¹H), 7.533–6.816 (m, Ar-CH-, 15H), 5.048 (s, O-CH2-, 2H), 4.162 (s, S-CH2-, 2H); Mass: Exact mass calculated for C₂₃H₂₀N₄O₂S, 416.50; found (m/z), 417.05 [M+] (Figures 5, 6).

Since PAC exhibited relatively better anti-proliferation activity in all the tested cancer cell lines, therefore, it was further selected for cell cycle studies. The cell cycle study was conducted on K562 cells in order to decipher the growth phase which is prone to the PAC treatment. The results indicated that the higher percentage of K562 cells are arrested at SubG0/G1 and S phases as evident. A marked increase in the percentage of gated cell populations which are in SubG0/G1, S and G2 phases was detected as compared to that in control K562 p53 null cells. This implies that PAC induces cell cycle arrest predominantly at the SubG0/G1, S and G2 phases. This also suggests an increased level of p53 due to inhibition of Mdm2/Pirh2 by PAC in K562 p53 null cells has occurred thus blocking cell cycle progression since p53 also acts at the G1 checkpoint during G1/S transition phase of the cell cycle and induces the expression of cyclin-dependent kinase (cdk) inhibitor. The cdk inhibitor inactivates the Cdk-G1/S cyclin complex, which later blocks cell cycle progression. These facts suggest that PAC rescues p53 by inhibiting ubiquitination by Mdm2/Pirh2 in K562 p53 null cells (Sane and Rezvani, 2017).

Whereas, in case of cells treated with Camptothecin (CPT; 25 µM), the highest percentage of cells got arrested during G2/M phase (30%) which suggests CPT induces arrest G2/M phase of the cell cycle by a distinct mechanism.

Since PAC and its analogues have been designed in silico to modulate the ubiquitination of p53 by both Mdm2 and Pirh2 E3 enzymes, hence its impact on the expression levels of p53, Mdm2 and Pirh2 proteins have been studied by flow cytometry. The expression levels of p53 and Mdm2 and Pirh2 proteins were measured in K562 cells under control and treated conditions. In the histogram graphs Figures 5C–H, the M1 region represents lower expression levels of p53, Mdm2, and Pirh2 protein while the M2 region represents normal to high expression levels.

Generally, K562 cells express a negligible amount of p53, and the results obtained from the flow cytometric analysis has also indicated the lower expression of the p53 tumour suppressor protein under control conditions. Interestingly, exposure of the K562 cells to the indicated concentrations of CPT (reference) and PAC has caused a dramatic increase in the p53 levels. Quantitatively, the exposure of K562 cells to CPT, which is a topoisomerase inhibitor derived from natural source, has triggered ∼10 folds rise in the p53 level, while the PAC treatment has caused ∼4-fold rise in the p53 levels (Figure 7).

Generally, the expression level of Mdm2 is not affected in K562 cancer cells (Table 4), and the status Pirh2 expression in K562 cancer cell lines has not been documented till date. However, Mdm2 serves as a primary negative regulator of p53 expression whereas Pirh2 serves as secondary. Hence, it may be presumed that the expression level of Mdm2 is slightly high as compared to the Pirh2. In an agreement, the flow cytometric analysis indicated that Mdm2 expression was slightly high when compared to Pirh2 in control group of K562 cell population (see Figures 11E–H, 13), which reinforces the notion that Mdm2 is a primary negative regulator of p53. On the other hand, CPT, a reference molecule which is a topoisomerase inhibitor, has less influence on Mdm2 and Pirh2 expressions in K562 cells. Strikingly, the PAC treatment has caused a dramatic fall in the expression levels of both Mdm2 and Pirh2 oncoproteins as compared to the untreated K562 cells (see Figure 7). These data partly suggest that PAC inhibits Mdm2 and Pirh2 oncoproteins which resulted in the rise in p53 tumour suppressor protein in K562 cells. On the other hand, the induction of p53 levels by CPT in K562 p53 null cells may be due to distinct mechanisms. Results of the overall in-vitro analysis suggest that PAC exhibited good anticancer activity against all the tested cancer cell lines. Enzyme binding studies are required to further validate the Mdm2 and Pirh2 promiscuous binding nature of PAC and its analogs. (Figures 11–13) (Astalakshmi et al., 2022).

Through our molecular docking experiment, we discovered that PAC is efficient. As a result, PAC had the highest ratings for Glide (−8.312 kcal/mol) and binding affinity −49.601 kcal/mol). The docked complex analysis revealed that the residues HIE96, TYR100 bonded with hydrogen bonding. Lys 173 was interacting with the ligand at the hydration site The Glide ratings for PAA were −7.216 kcal/mol, and the binding affinity was recorded as −42.371 kcal/mol. Analysis of the docked complex indicated the occurrence of hydrogen bonding between PAA and the residues TYR100 exhibited superior docking scores and binding affinity compared to PAA. Consequently, additional investigations and studies were conducted specifically focusing on PAC. (Table 5) (Vettoretti et al., 2016).

A simulation study was conducted using molecular dynamics (MD) to confirm the stability of the receptor-ligand complex, validate the predicted binding mode, and examine potential interactions. These aspects had been previously investigated through Glide XP docking. In the present investigation, we ran a 200 ns MD simulation of the PAC—3LBK complex. This extended simulation duration enabled the examination of molecular and atomic-level changes crucial for understanding the stability of the protein-ligand complex and the dynamic behavior of the ligand. Using the Desmond tool from the Schrödinger suite, a 200 ns simulation was conducted, and resulting trajectories were analyzed with the simulation interaction diagram panel to comprehend deviation fluctuation and intermolecular interactions. The root mean square deviation (RMSD) value was calculated to assess the deviation in the protein’s backbone throughout the 200 ns simulation period. The RMSD plot of the PAC—3LBK complexes are shown in Figure 12A, demonstrating slight conformational fluctuations in the complexes between 135 and 160 ns. RMSD levels range from 1.8 Å to 2.7 Å Around 8 Å. Similarly, there was a modest fluctuation in the ligand RMSD between 0 ns and 30 ns, with RMSD ranging from 0.6A to 2.7 around 2.1 Å. Despite these variations, the PAC—3LBK complex remained stable. The Root Mean Square Fluctuations (RMSF) aid in characterizing local protein alterations. These variations were used to identify the residue in the complex that contributes to structural fluctuations. The region of proteins was represented by peaks that vary throughout the process of simulation. The N and C terminal of protein tails changes more when compared to any other regions of protein. The alpha helices and beat chain are stronger than the unstructured regions of the protein and hence they alter less when compared to loop regions. The alpha and beat regions are represented in red and blue color. These regions were specified by helices or strands that continue 70% of simulation time. The proteins that are interacted with ligand are represented in green color with slight fluctuations, conforms the protein ligand complex are stable. (Figures 14A, B) (Li and Zhang, 2022).