Pharmacokinetic evaluation, molecular docking and in vitro biological evaluation of 1, 3, 4-oxadiazole derivatives as potent antioxidants and STAT3 inhibitors.

1, 3, 4-Oxadiazole derivatives (4a-5f) were previously synthesized to investigate their anticancer properties. However, studies relating to their antioxidant potential and signal transducer and activator of transcription (STAT) inhibition have not been performed. We investigated previously synthesized 1, 3, 4-oxadiazole derivatives (4a-5f) for various radical scavenging properties using several in vitro antioxidant assays and also for direct inhibition of STAT3 through molecular docking. The data obtained from various antioxidant assays such as 2, 2,-diphenyl-1-picrylhydrazyl radical (DPPH), nitric oxide, hydrogen peroxide, and superoxide anion radical revealed that among all the derivatives, compound 5e displayed high antioxidant activities than the standard antioxidant L-ascorbic acid. Additionally, the total reduction assay and antioxidant capacity assay further confirmed the antioxidant potential of compound 5e. Furthermore, the molecular docking studies performed for all derivatives along with the standard inhibitor STX-0119 showed that binding energy released in direct binding with the SH2 domain of STAT3 was the highest for compound 5e (-9.91kcal/mol). Through virtual screening, compound 5e was found to exhibit optimum competency in inhibiting STAT3 activity. Compound 5e decreased the activation of STAT3 as observed with Western blot. In brief, compound 5e was identified as a potent antioxidant agent and STAT3 inhibitor and effective agent for cancer treatment.

Introduction

In a previous investigation on pan class="Chemical">5-aryl-2-butylthio-1, 3, 4-oxadiazole derivatives (4a–5f), we obclass="Chemical">pan class="Chemical">served antiproliferative effects of these compounds on MCF-7 breast cancer cells as a result of the induction of apoptosis [1]. In the present study, we further explored the potential of these compounds as antioxidants and STAT3 inhibitors through computational analyses to establish the authenticity of these derivatives as potent anticancer agents because antioxidants and STAT3 inhibitors are known to play prominent roles against the progression of cancer.

pan class="Chemical">Reactive oxygen species (class="Chemical">pan class="Chemical">ROS) such as superoxide, singlet oxygen, hydroxyl radical and hydrogen peroxide produced under oxidative stress damage various biological macromolecules, causing serious health problems such as cancer, inflammation, cardiovascular and neurodegenerative disorders [2], [3]. Thus, effective antioxidants are needed as they inhibit the survival and proliferation of cancer cells by reducing the oxidative stress [4].

Because of the possibility of simple transfer of pan class="Chemical">hydrogen atom and resonance stability of the resulting class="Chemical">pan class="Chemical">carboxylate radical, the substituted benzoic acid could inhibit the formation of free radical species by reducing oxidation [5]. The derivatives of benzoic acid are found in various natural and synthetic antioxidants such as chalcones, flavones, salicylic acid and parabens [6]. 2, 5-disubstituted-1, 3, 4-oxadiazole exhibits a wide spectrum of biological activities [7]. As reported earlier, many derivatives containing the 1, 3, 4-oxadiazole moiety including sulfonamidomethane [8], benzoxazole [9], pyrimidines [10] and methoxyphenyl [11] display significant antioxidant properties. Thus taking into consideration the antioxidant properties of benzoic acid and 1, 3, 4-oxadiazoles scaffold, new derivatives (4a–5f) containing the 1, 3, 4-oxadiazole ring formed through cyclization of derivatives of benzoic acid synthesized in our previous study could serve as potent anticancer agents by effectively scavenging the free radical formation because of extended conjugation and stability.

Signal transducer and activator of transcription (STAT) comprises a group of seven proteins, namely, n class="Gene">STAT1, 2, 3, 4, 5a, 5b and 6. class="Chemical">pan class="Gene">STAT3 is closely related to the occurrence of cancers [12]. Many cancers, such as those of breast, prostate, lung and ovaries, as well as leukemia and lymphoma, occur due to the abnormal activation of STAT3 [13], [14]. STAT3 acts as a prime factor in the regulation of many cellular events involving cell proliferation, differentiation, apoptosis and angiogenesis [13], [15], [16], [17] and, therefore, an inhibitor of STAT3 activation could be an interesting lead for cancer drug discovery [18], [19], [20], [21].

The pan class="Chemical">1, 3, 4-oxadiazole scaffold is extensively used in the inhibition of class="Chemical">pan class="Gene">STAT3 in various cancer cells. Dell'Orto et al. [22] showed that an oxadiazole based compound effectively inhibited the activation of STAT3 by targeting the STAT3-SH2 domain in a dose-dependent manner. Shin et al. [23] reported that oxadiazoles containing ureido derivatives inhibited STAT3 signaling by blocking the upstream tyrosine kinases involved in the activation of STAT3. Matsuno et al. [24] reported the inhibition of dimerization of STAT3 through STX-0119 (an oxadiazole derivative) in several cancer cell lines. Using the virtual screening approach, the ability of the synthesized compounds (4a–5f) as effective STAT3 inhibitors through direct binding to the STAT3-SH2 domain, and thereby blocking the dimerization and activation of STAT3, was investigated.

Materials and methods

Chemicals and software

All the chemicals were of the highest quality available. The reagents used in experiments including pan class="Chemical">L-Ascorbic acid, class="Chemical">pan class="Chemical">2, 2-diphenyl-1-picrylhydrazyl (DPPH), Griess reagent, sodium nitroprusside, Tris-HCl buffer, dimethyl sulfoxide (DMSO), and hydrogen peroxide, were purchased from Sigma Aldrich (India). The human cell line MCF-7 (breast cancer cell line) was procured from National Centre for Cell Science (Pune, India). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum along with 1% penicillin–streptomycin–neomycin (GIBCO Grand Island, New York, USA) in a humidified atmosphere having 5% CO2 maintained at 37 °C. All the computational studies were performed using Windows 10 Professional on a personal computer with Intel Core i3microprocessor, 4 GB RAM and a 64-Bit operating system. We used biological databanks such as PubChem, and Protein Data Bank (PDB) and online tools such as Online Pass Server along with softwares like Chemdraw Ultra 8.0, AutoDock 4.2, and Discovery Studio version 3.5.

In vitro antioxidant assays

DPPH radical scavenging assay

The antioxidant property of the synthesized compounds was evaluated pan class="Chemical">through class="Chemical">pan class="Chemical">free radical scavenging assay by observing the changes in optical density of DPPH radicals. One milliliter of synthesized compounds in the concentration range of 0–100 μg/mL dissolved in methanol were added to 5 mL of 0.005% (w/v) methanol solution containing DPPH. After incubating for 30 min at room temperature, the absorbance was measured at 519 nm. Ascorbic acid was taken as a standard. All the experiments were repeated thrice.

Nitric oxide radical scavenging activity

The pan class="Chemical">nitric oxide (NO) radical scavenging ability was evaluated using slight changes in the methods employed by Green et al. [25] and Marcocci et al. [26]. The tested compounds were taken in a concentration range of 0–100 μg/mL, and added to the solution containing 1.2 mL of class="Chemical">pan class="Chemical">sodium nitroprusside (10 mM) and 1.7 mL of phosphate buffer saline (0.2 M, pH 7.6); the mixture was left for incubation for 152 min at 25 °C. Following incubation, 1 mL of Griess reagent was used to treat 1 mL of reaction mixture and the absorbance was measured at 545 nm. Ascorbic acid was taken as a standard antioxidant. The NO scavenging activity was calculated using the following equation:where , and represent the absorbance of the control reaction (reagents and ascorbic acid), test compound (reagents and synthesized compounds) and blank (reagents only). All experiments were carried out in triplicates.

H2O2 scavenging activity

Forty millimolar of pan class="Chemical">H2O2 solution was preclass="Chemical">pared in class="Chemical">pan class="Chemical">phosphate buffer (50 mM, pH 7.4) [27]. All the synthesized compounds in the concentration range of 0–100 μg/mL containing 3.5 mL phosphate buffer were added to 0.5 mL of H2O2 solution. The absorbance of the reaction mixture was measured at 232 nm. Ascorbic acid was used a reference. All experiments were repeated thrice.

O2- scavenging activity

In brief, 1.2 mL of test compounds were added pan class="Species">to 7 mL of 5 mM class="Chemical">pan class="Chemical">Tris-HCl buffer (pH 8.3) [28]. Forty-two microliters of 4.4 mM of pyrogallol was added to the reaction mixture. The mixture was stirred and a single drop of ascorbic acid was added after 5 min. The absorbance was measured at 450 nm. All the experiments were repeated thrice.

Total reduction capacity and total antioxidant activity

The method of Oyaizu et al. [29] was used to determine the total reduction capacity with some modifications. In brief, 2.52 mL of 0.2 M pan class="Chemical">phosphate buffer (pH 6.8) along with 1% (w/v) class="Chemical">pan class="Chemical">K3Fe(CN) 6 was added to 1.5 mL of test compounds dissolved in distilled water. After incubating the reaction mixture for 20 min at 50 °C, 2.6 mL of 10% (w/v) trichloro acetic acid was added. The mixture was then centrifuged at 3000 rpm for 15 min to separate and collect the top layer of the solution, which was then mixed with distilled water and 0.76 mL of 0.1% (w/v) FeCl3. All the absorbance values were taken at 695 nm. The method of Prieto et al. [30] with some modifications was employed to calculate the total antioxidant activity. In brief, 0.2 mL of test sample (200 μg) was mixed with 1.5 mL of the reagent (0.5 M H2SO4, 25 mM Na2PO3 and 4.5 mM ammonium molybdate). The tube was capped and incubated in a boiling water bath at 95 °C for 90 min. The samples were cooled to room temperature and absorbance of the solution was read with reference to the blank at 690 nm using UV spectrophotometer.

In silico pharmacokinetics analysis for STAT3 inhibition

Molecular modeling analysis

The molecular docking study was performed using the software AutoDockTools (pan class="Chemical">ADT) version 1.5.6 taken from the Scripps Research Institute [31]. The X-ray 3D crystal structure of class="Chemical">pan class="Gene">STAT3 with a ligand (PDB ID: 1BG1) was retrieved from the RSCB Protein Data Bank (web address: http://www.rscb.org/pdb/) and was used as a docking receptor.

Preparation of ligands and receptors

To propose new inhibitors, the build-and-edit module of Chemdraw ultra 12.0 was used. It was used to draw the structures of ligands, which were then converted into PDB file. The PDB ligand files, thus obtained were converted to PDBQT file using n class="Chemical">ADT version 1.56, which helped in the conversion by detecting the root, choosing the torsion and setting the torsion numbers. The structure was made class="Chemical">pan class="Chemical">free from all the co-crystallized ligands and water molecules. Using ADT, all the polar hydrogen atoms were added. This was followed by the removal of residue structures and replacement of all incomplete side chains [31]. The Gasteiger charges were added to each atom and non-polar hydrogen atoms were dissolved with the structure of protein. All the structures were saved in PDBQT files for further evaluation using ADT.

Molecular docking

The Lamarckian genetic algorithm was employed with the help of AutoDock 4.2 software to perform the docking study of the ligand molecules with pan class="Gene">STAT3 (class="Chemical">pan class="Disease">PDB ID: 1BG1) . Auto grid was used to calculate the center of the active site pocket for the ligand which is represented by the grid maps [33]. The dimensions of the grid for STAT3 protein were 80 × 80 × 80 grid points with spacing of 0.750 Å between the grid points but centered on the ligand for receptor (101.36, 87.332 and 31.445 coordinates). Based on the values of binding energy (kcal/mol) and inhibition constant (ki) value (µM), the best docked conformation was selected. The final results, thus obtained featuring hydrogen and hydrophobic interactions of the docked compounds with the modeled structures, were interpreted using PyMOL software.

Evaluation of the ADMET and TOPKAT properties

The absorption, distribution, metabolism, excretion and pan class="Disease">toxicity (ADMET) properties for the synthesized compounds were evaluated using Discovery Studio 3.5 (Accelrys San Diego, USA) as evaluation of these properties before designing a drug plays a crucial role in clinical phases [34]. The after effects of drug intake were assessed using TOPKAT, which helps in determining the toxicological final points using quantitative structure-class="Chemical">pan class="Disease">toxicity relationship (QSTR).

Biological activity spectrum (BAS)

To evaluate the pharmacological properties of the best docked compounds (4e, 4f, 5e and 5f), the biological activity spectrum (BAS) was determined. It is useful software for determining the pharmacological properties as these properties are related to the structural properties. The pharmacological properties of a compound and its association with the biological system were assessed using PASS by uploading the SMILES string of the respective compounds online.

Alpha screen based assay

It is a bead-based nonradioactive assay carried out in a microtiter plate and is used to elucidate the biomolecular interactions [35]. Forty millimeters of the reaction mixture was taken in a 96-well plate containing 13 mM n class="Chemical">HEPES-class="Chemical">pan class="Chemical">NaOH (pH 7.2), 65 mM NaCl , 1 mM EDTA (pH 7.6), 0.1% NP- 40 and 10 mg/mL BSA at 25 °C. The Phospho-Tyr (p-Tyr) peptide fragment used in this study was 5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3. The SH2 and 80 µM protein were incubated with the compound for 20 min. The protein and the FITC-pTyr peptide were incubated for 90 min. The streptavidin coated donor beads and anti-FITC acceptor beads were mixed and reading was taken at 575 nm using EnVison Xcite (Perkin Elmer).

Western blot analysis

The Western blot anapan class="Chemical">lysis was carried out using a previously described protocol after some modifications [36]. The class="Chemical">pan class="CellLine">MCF-7 cells were cultured in RPMI-1640 medium supplemented with 10% FBS purchased from GIBCO Grand Island, New York, USA. The cells were seeded in six-well plates and cultured overnight to allow them to become confluent. The medium in the plate was changed with fresh medium containing 10% FBS (control) and compound 5e at different concentrations after 24 h. The cell lysate was prepared using the lysis buffer containing the protease and phosphatase inhibitors. Total protein was extracted and protein estimation was done using Bradford reagent. The total protein (70 µg) was loaded on a 12% SDS polyacrylamide gel and blotted onto a PVDF membrane after electrophoresis. The blocking of membrane was done, using the 5% BSA, and then the membrane was incubated overnight in STAT3 (Cat no. SC-482) primary antibody at a dilution of 1:1000 at 4 °C. The blot was subsequently washed thrice using TBS-T and incubated for 1 h at room temperature with the secondary antibody (Cat no. SC-2030). Images were visualized with the LAS-4000 using the ECL reagent. All the important reagents were purchased from Sigma Aldrich (India) while primary STAT3 and secondary antibodies were brought from Santa Cruz Biotechnology, Texas, USA.

Statistical analysis

All the experimental data are presented as means ± SD of triplicate results. The data were analyzed using unpaired Student's t-test to calculate the P values (value of significance) with the GraphPad Prism version 7.0 software (San Diego, CA, USA). The P values < 0.05 were considered statistically significant.

Results and discussion

In vitro antioxidant activity

The synthesized compounds (Scheme 1) (4a–5f) reported by us previously [1] were tested for their antioxidant potential using the n class="Chemical">DPPH [37], NO [38], class="Chemical">pan class="Chemical">H2O2 [2], O2- [39], total reducing capacity [2] and total antioxidant assays [40]. The results for the in-vitro antioxidant assays for the synthesized compounds and the standard antioxidant were compared.

Scheme 1

Schematic representation for synthesis protocol for compounds (4a–5f).

DPPH radical scavenging activity

The pan class="Chemical">DPPH radical scavenging activity of all the synthesized compounds (4a–5f) was evaluated and is presented in Table 1. It was obclass="Chemical">pan class="Chemical">served that among all the compounds 4e, 4f, 5e and 5f had effective antioxidant activities. In particular, the compound 5e showed an IC50 values that was very close to the IC50 value of the standard antioxidant, L-ascorbic acid. The higher scavenging ability of the compound 5e relative to those of other compounds was a result of the high resonance stability of radical being formed after heterolytic cleavage delocalizing through the aromatic and 1, 3, 4-oxadiazole rings (Scheme 2). The compounds substituted with butyl hydrocarbon chain (5a–f) exhibited higher antioxidant activities than the compounds with a free mercapto group (4a–f), suggesting that the presence of an alkyl chain at C-2 further enhanced their activities [41]. The compound 5e showed 89.90% inhibition of the DPPH radical at a concentration of 80 μg/mL. The inhibition percentage of the DPPH radical was found to be 35.24%, 48.54% and 77.38% at ½ × IC50, IC50 and 2 × IC50 concentrations of the compound 5e, respectively. Moreover, the radical scavenging activity of the compound 5e and L-ascorbic acid was not statistically significant (P > 0.05) in the concentration range of 50–80 μg/mL (Fig. 1A).

Table 1

The IC50 values (μM) of synthesized compounds (4a–5f) calculated from various in vitro antioxidant assaysa.

Compound No.	R	DPPH	NO	O2–	H2O2
4a	H, H	89.96 ± 3.4	112.10 ± 2.3	165.01 ± 3.2	121.03 ± 4.4
4b	Cl; H	74.07 ± 3.3	79.31 ± 3.1	142.04 ± 1.1	105.48 ± 3.4
4c	Cl, Cl; H	83.57 ± 1.1	101.03 ± 4.1	141.92 ± 1.2	107.06 ± 1.1
4d	tertbutyl; H	56.90 ± 3.3	94.39 ± 2.2	83.23 ± 3.2	117.01 ± 3.3
4e	NH2; H	47.04 ± 2.2	44.05 ± 3.0	50.81 ± 2.2	47.72 ± 3.3
4f	Cl, NO2; H	54.07 ± 3.9	47.72 ± 1.1	48.07 ± 1.9	46.58 ± 1.5
5a	H; n-butyl	62.11 ± 3.8	110.30 ± 5.1	164.47 ± 3.4	90.09 ± 2.2
5b	Cl; n-butyl	68.18 ± 5.1	63.85 ± 4.4	134.77 ± 4.4	61.85 ± 1.3
5c	Cl, Cl; n-butyl	64.18 ± 4.4	89.92 ± 1.3	84.88 ± 2.2	50.34 ± 3.2
5d	tert-butyl;n-butyl	71.63 ± 2.8	76.45 ± 1.1	107.75 ± 3.3	50.01 ± 3.1
5e	NH2; n-butyl	30.65 ± 4.2	38.49 ± 6.1	40.98 ± 1.9	40.65 ± 2.2
5f	Cl, NO2; n-butyl	44.62 ± 5.5	39.16 ± 2.2	45.52 ± 1.2	44.64 ± 1.1
L-Ascorbic acidb	–	31.26 ± 2.3	38.93 ± 2.5	39.96 ± 1.2	41.04 ± 3.1

IC50: concentration of compound required to inhibit radical formation by 50%. Values (in µM) are expressed as mean ± SD for a set of three experiments.

L-Ascorbic acid: standard antioxidant drug used for comparison.Bold values represent the active compounds.

Scheme 2

Resonance stabilization of radical formed from compound 5e after heterolytic cleavage.

Fig. 1

Percentage inhibition of radicals of compounds (4a–5f) using different in-vitro antioxidant assays. (A) DPPH, (B) nitric oxide radical, (C) hydrogen peroxide radical, (D) superoxide anion radical, (E) total reduction capacity assay, and (F) total antioxidant capacity assay. L-ascorbic acid is taken as the standard antioxidant. Values are expressed as mean ± SD for a set of three experiments.

IC50: concentration of compound required to inhibit radical formation by 50%. Values (in µM) are expressed as mean ± SD for a set of pan class="Chemical">three experiments.

pan class="Chemical">L-Ascorbic acid: standard antioxidant drug used for comclass="Chemical">parison.Bold values represent the active compounds.

Percentage inhibition of radicals of compounds (4a–5f) using different in-vitro antioxidant assays. (A) n class="Chemical">DPPH, (B) class="Chemical">pan class="Chemical">nitric oxide radical, (C) hydrogen peroxide radical, (D) superoxide anion radical, (E) total reduction capacity assay, and (F) total antioxidant capacity assay. L-ascorbic acid is taken as the standard antioxidant. Values are expressed as mean ± SD for a set of three experiments.

NO radical scavenging activity

The NO scavenging activities for all the tested compounds and the reference drug are shown in Table 1. From the data, it is clear that the compounds showing considerable pan class="Chemical">DPPH scavenging activity also exhibited significant NO scavenging activity. Owing to the resonance stability and the inductive effect of the -NH2 group at class="Chemical">para position to the class="Chemical">pan class="Chemical">benzene ring, compound 5e exhibited the most significant antioxidant activity, which was not statistically significant compared with that of the standard antioxidant (P > 0.05) (Fig. 1B).

pan class="Chemical">H2O2 is an oxidizing species present in many animal tissues. It is a biologically important reagent because it diffuses acclass="Chemical">pan class="Chemical">ross many biological membranes, bringing oxidative damage to DNA, lipids and protein molecules. As represented in Table 1, among all the compounds, four compounds, namely, 4e, 4f, 5e, and 5f, exhibited significant H2O2 scavenging that was almost similar to that obtained for L-ascorbic acid at its IC50 in contrast to the low H2O2 activities previously reported for compounds having 1, 3, 4-oxadiazole ring [2]. In particular, the compound 5e exhibited a prominent antioxidant activity because of low bond dissociation energy for the -NH2 group [42] and showed increased scavenging of (OOH.) radicals in a concentration-dependent manner without significant statistical difference (P > 0.05). The H2O2 scavenging activity for the compound 5e at ½ × IC50, IC50 and 2 × IC50 concentrations was found to be 42.6%, 58.04% and 81.90%, respectively (Fig. 1C).

pan class="Chemical">O2- radical is very reactive and acts as a biological cataclass="Chemical">pan class="Chemical">lyst in the reduction of molecular oxygen [39]. As shown in Table 1, the IC50 value for the compound 5e was almost equal to that of L-ascorbic acid. As revealed from Fig. 1D, at higher concentrations the compound 5e inhibited the formation of O2- radical up to 83.9%, thus displaying a significant potential as an antioxidant. Furthermore, it was observed that the percentage of inhibition of radicals by the compound 5e and L-ascorbic acid was not statistically different (P > 0.05) within the concentration range of 10–80 μg/mL.

Total reduction capacity and total antioxidant capacity

The total reduction capacity and total antioxidant capacity of all the synthesized compounds (4a–5f) and pan class="Chemical">L-ascorbic acid are presented in terms of the optical densities in the concentration range of 0–100 μg/mL in Fig. 1E and Fig. 1F, respectively. Both the assays revealed that only four compounds, namely, 4e, 4f, 5e and 5f, showed better results for the total reduction caclass="Chemical">pacity and total antioxidant caclass="Chemical">pacity (P > 0.05) in terms of absorbance in a concentration-dependent manner. Particularly, the compound 5e exhibited the highest activity class="Chemical">pan class="Chemical">throughout.

Structure-activity relationship (SAR)

The data obtained from in vitro antioxidant assays for the synthesized compounds (4a–5f) showed that the presence of various substituents on the phenyl ring at n class="Gene">C-5 and the alkyl group at class="Chemical">pan class="Gene">C-2 of the 1, 3, 4-oxadiazole ring is important in determining the SAR. The possible reason for the increased antioxidant activity for compounds 5a–5f compared to that for 4a–4f was the presence of the hydrocarbon chain. The prominent radical scavenging activity of the compounds 4e and 5e was due to the high inductive (+I effect) and mesomeric effects (+M effect) of a strong electron donating (-NH2) group at para position to the phenyl ring. The plausible reason for the antioxidant activity of compounds 4f and 5f might be the presence of strong electron withdrawing group (-NO2) at the meta position. No significant activity was exhibited by the compounds 4a–4d and 5a–5d, which could presumably be due to the presence of weak electron donating (alkyl) and withdrawing (-chloro) groups.

In silico pharmacokinetic analysis for STAT3 inhibition

The synthesized compounds (4a–5f) and the known inhibitor n class="Gene">STX-0119 were analyzed by molecular docking using the rigid docking method [31]. AutoDock 4.2 was used to determine the orientation of inhibitors bound in the active site of class="Chemical">pan class="Gene">STAT3 (PDB ID: 1BG1) and also to determine the conformation exhibiting the highest binding energy value for each molecule. With the aim of developing new STAT3 inhibitors, the binding modes for STAT3 inhibitors were analyzed using the PyMOL software. The binding site in the SH2 domain of STAT3 as described by Becker et al. [43] was used to elucidate the interactions, contributing to the binding affinity of new inhibitors, which can be used to design potent inhibitors.

The active residue sites for SH2 domain of pan class="Gene">STAT3 were class="Chemical">pan class="Chemical">Leu 438, Asp 369, Arg 382, Gly 442, Arg 423, His 457, Lys 244, Glu 444, Thr 443, Thr 456 and Lys 244 as analyzed by DoGsiteScorer server. We designed the STAT3 inhibitors having 1, 3, 4-oxadiazole ring with different phenyl substituents having free mercapto group (4a–f) and butyl chain attached to a sulfur atom forming butyl-mercapto linker (5a–f) (Scheme 1). These compounds sufficiently act as H-bond acceptors as well as donors due to the presence of various polar linkages such as C-O-C, N=C-O and C=S, acting as hydrophobic and hydrophilic sites in the form of these dipoles.

Docking results showed that all compounds (4a–f) with a pan class="Chemical">free -SH group exhibited similar orientations in the binding pocket of the SH2 domain of class="Chemical">pan class="Gene">STAT3. However, compounds substituted with the butyl mercapto linkage exhibited different conformational behaviors because of their diverse atomic compositions and chemical properties. To explore the SAR profile, the role of alkyl linkage (5a–f) and free –SH group (4a–f) at C-2 of 1, 3, 4-oxadiazole ring with substituted phenyl ring at C-5 was analyzed. The compound 4a bearing an unsubstituted phenyl ring at C-5 formed three H-bonds: the first H-bond formed between the –C-O-C-linkage of oxadiazole ring and Ser 372 residue with a bond distance of 3.5 Å, while the other two H-bonds formed between the -N=C-O- linkage of oxadiazole moiety and Thr 440, and Leu 438 residues with a bond distance of 1.1 Å and 1.9 Å, respectively. For compound 5a, the –N=C-O- linkage of oxadiazole ring formed two H-bonds with Arg 379 residues with a bond distance of (3.2 Å, 3.4 Å) and one pi-interaction. The compound 4b, having o-Cl atom, also formed 2 H-bonds but no pi-interaction with His 457 or Lys 244 was observed. Compound 5b showed 2 H-bonds and one pi-interaction with Arg 335 with bond distance of 3.1 Å and 3.3 Å, respectively. The 2, 4-dichloro and para- tert-butyl group of compounds 4c and 4d, respectively formed no hydrogen bond and no pi-interaction with amino acid residues. Compounds 5c and 5d also interacted in a similar way as compounds 4c and 4d respectively did not show any interaction. The compound 4e having a p-amino group formed 6 H-bonds with His 457 (2.3 Å, 3.3 Å), Thr 456 (2.9 Å, 3.1 Å) and Lys 244 (2.6 Å, 1.7 Å) residues and two pi-interaction with Thr 456 and His 457 residues respectively. Compound 5e with a p-amino group and butyl mercapto linkage at C-2 showed 6 H-bond interaction with Thr 456 (3.0 Å, 2.6 Å), His 457 (3.4 Å, 3.0 Å), Lys 244 (3.4 Å) and Gln 247 (2.0 Å) residues and one pi-interaction with Thr 456 residue. Compound 4f with nitro and chloro group formed 3 H-bonds with two amino acid residues, Asp 566 (3.5 Å) and Asp 334 (3.3 Å, 3.1 Å), while compound 5fformed 3 H-bonds with Gly 380 (2.9Å), Gly 442 (3.0 Å) and Arg 423 (3.0 Å) residues (Table 2).

Table 2

Binding energy and specific interaction of STAT3 with the synthesized compounds.

Compound no.	Binding energy (kcal/mol)	Inhibitory constant (μM)	Protein ligands interaction
			No. of H bonds	Amino acid residues	Distance (Å)
4a	− 4.58	442.52	3	Ser372	3.5
				Leu438	1.1
				Thr440	1.9
4b	− 5.48	110.16	2	His457	2.9
				Lys244	3.4
4c	− 5.18	249.97	–	–	–
4d	− 5.25	239.70	–	–	–
4e	− 8.04	16.50	6	His457	2.9, 3.1
				Thr456	2.6, 3.1
				Lys244	2.6, 1.7
4f	− 7.82	19.19	3	Asp566	3.5
				Asp334	3.3, 3.1
5a	− 5.68	98.59	2	Arg379	3.2, 3.4
5b	− 5.89	91.79	2	Arg335	3.1, 3.3
5c	− 5.20	210.36	0	–	–
5d	− 5.30	241.44	0	–	–
5e	− 9.91	10.36	6	Thr456	3.0, 2.6
				His457	3.4, 3.0
				Lys244	3.4
				Gln247	2.0
5f	− 8.81	14.21	3	Gly380	2.9
				Gly442	3.0
				Arg423	3.0
STX-0119	− 6.37	21.30	3	Glu415	2.0
				Gln416	3.5, 3.2

Binding energy and specific interaction of pan class="Gene">STAT3 with the synthesized compounds.

Thus, on the basis of SAR studies it can be inferred that an amino group at para position of the phenyl ring of compound 4e and a n class="Chemical">butyl mercapto linkage at class="Chemical">pan class="Gene">C-2 present in compound 5e in comparison to other compounds were more interactive and competent in forming stable protein-ligand complex. In general, compound 5e interacted with residues Thr 456 (3.0 Å, 2.6 Å), His 457 (3.4 Å, 3.0 Å), Lys 244 (3.4 Å) and Gln 247 (2.6 Å) through H-bonds and with Thr 456 residue through pi-interaction (Fig. 2 C and D) in comparison to the standard drug (STX-0119)Fig. 2 A and B), where the pi-interactions were absent and only H-bonds could be formed with Glu 415 (2.0 Å) and Gln 416 (3.5 Å and 3.2 Å) residues.

Fig. 2

Docked conformations of the known inhibitor STX-0119 (A and B) and compound 5e (C and D) into the active site of STAT3 binding pocket.

Docked conformations of the known inhibitor pan class="Gene">STX-0119 (A and B) and compound 5e (C and D) into the active site of class="Chemical">pan class="Gene">STAT3 binding pocket.

ADMET and TOPKAT prediction

The in-silico profile of the selected compounds (4e, 4f, 5e and 5d) was evaluated to determine their putative bioavailability as pan class="Gene">STAT3 inhibitors. Physicochemical properties, primarily aqueous solubility (logS), lipophilicity (clogP), molecular weight (MW) and polar surface area, linearly correspond to the bioavailability and absorption of a drug molecule. CYPs (cytochrome P450) play an important role in drug metabolism and also determine the nature of drugs inside the body. AlogP value (lipophilicity) is a significant property for calculating oral bioavailability of a drug. Here, we employed ADMET (DS3.5) to evaluate the predicted pharmacokinetic profile of compounds by using the standard inhibitor class="Chemical">pan class="Gene">STX-0119. The results showed that all the synthesized compounds 4e, 4f, 5e and 5f had standard AlogP value ≤ 5. Similarly, the compounds 4e, 4f, 5e and 5f showed moderate to good solubility (solubility level = 3). The observed value of human intestinal absorption (HIA) was excellent for entire molecules. The penetration ability of compounds across the blood-brain-barrier (BBB) was high when prediction value was zero and low when the value was 4. All four compounds showed better BBB penetration ability; however, only two compounds 5e and 5f exhibited probability score for hepatotoxicity ≤ 0.5. All four compounds had good HIA value. Except for compounds 5e and 5f, the other two compounds were active against CYP2D6 enzyme. For real drug ability, the ideal plasma protein binding (PPB) level is 0. Except for compound 5e, all the compounds showed poor PPB. Polar surface area (PSA) depends on the conformation and hydrogen bonding. For a drug to be active, the optimum value of PSA is ≤ 140 Å. All predicted compounds showed significant PSA.

With the help of the computer-aided pan class="Disease">toxicity predictor, TOPKAT, the cellular class="Chemical">pan class="Disease">toxicity of compounds 4e, 4f, 5e and 5f was determined after comparison with a known STAT3 inhibitor. The carcinogenic and mutagenic effects of compounds determined using weight of evidence (WOE Prediction) and Ames Prediction were prime factors. These techniques consist of various toxic endpoints and models (irritation, teratogenicity, sensitization, neurotoxicity and immunotoxicology) that are useful in drug development. All the selected compounds showed Ames probability score ≤ 7 and were found to be mutagenic except for compounds 5e and 5f. WOE, another toxicity predictor, is used to evaluate the relative level of a compound that causes cancer in humans. All compounds expect 5e and 5f were carcinogenic (Table 3).

Table 3

Pharmacokinetic profiles of known inhibitors and synthesized compoundsa.

Pharmacokinetic profiles			Synthesized compounds				STX-0119
			4e	4f	5e	5f
ADMET
	BBB		3	3	2	2	2
	AlogP		1.284	2.589	2.794	4.100	3.983
	Sol.		3	3	3	2	2
	HIA		0	0	0	0	0
	HTL		1	1	1	0	1
	HT_Prob		0.94	0.68	0.50	0.45	0.98
	PPB		1	0	2	1	2
		CYP2D6	0	0	1	1	1
		PSA	61.61	77.89	61.61	77.89	89.00
TOPKAT
	Ames Mut.		M	M	NM	NM	M
	Prob		0.76	0.77	0.70	0.71	0.78
	Enrichment		1.37	1.38	1.26	1.27	1.39
	WOE		C	C	NC	NC	C

BBB: blood brain barrier level value: 0 (high penetration); 1 (no penetration), HIA: human intestinal absorption level value: 0 (good); 1(moderate), Sol. (solubility level): 3, HTL: hepatotoxicity level value: 0 (good); 1 (moderate), HT-Prob.: hepatotoxicity probability < 0.5 is ideal, CYP2D6 < 0.5 is good and denoted as level 0, PPB: plasma protein binding value ≤ 0, AlogP value ≤ 5.0 and Polar surface area ≤ 140. Ames Mut.: Ames mutagen prediction, Prob.: Ames probability; Enrichment: Ames enrichment; WOE-Prediction (weight of evidence); M: (mutagen); NM: (non-mutagen); C: (carcinogen); NC: (non-carcinogen).

Pharmacokinetic profiles of known inhibitors and synthesized pan class="Chemical">compoundsa.

BBB: blood brain barrier level value: 0 (high penetration); 1 (no penetration), HIA: pan class="Species">human intestinal absorption level value: 0 (good); 1(moderate), Sol. (solubility level): 3, HTL: class="Chemical">pan class="Disease">hepatotoxicity level value: 0 (good); 1 (moderate), HT-Prob.: hepatotoxicity probability < 0.5 is ideal, CYP2D6 < 0.5 is good and denoted as level 0, PPB: plasma protein binding value ≤ 0, AlogP value ≤ 5.0 and Polar surface area ≤ 140. Ames Mut.: Ames mutagen prediction, Prob.: Ames probability; Enrichment: Ames enrichment; WOE-Prediction (weight of evidence); M: (mutagen); NM: (non-mutagen); C: (carcinogen); NC: (non-carcinogen).

ADMET and TOPKAT properties for the selected compounds 4e, 4f, 5e and 5f along with the known inhibitor suggested that among the four compounds, compound 5e emerged as the lead for pan class="Gene">STAT3 inhibition.

pan class="Chemical">PASS online class="Chemical">pan class="Chemical">server was used to evaluate the related biological activities for selected compounds. As these probabilities can be independently evaluated, the Pa and Pi values ranged from 0 to 1 and Pa + Pi was < 1. The Pa represents the active compounds while Pi represents inactive compounds [44]. The data from PASS prediction showed that the values for Pa were found to be higher than Pi values, suggesting inhibition of transcription factor involved in activation of STAT3 protein. All the four compounds 4e, 4f, 5e and 5f inhibited the transcription factor involved in the activation of STAT3 within the range of 0.607–0.746. Compound 5e, in particular, exhibited significant Pa values in comparison to other compounds (Table 4).

Table 4

Biological activity spectrum of compounds (Pa – Active; Pi – Inactive).

Compounds	Pa	Pi	Activity
4e	0.676	0.004	Transcription factor STAT3 inhibitor
4f	0.607	0.006	Transcription factor STAT3 inhibitor
5e	0.746	0.004	Transcription factor STAT3 inhibitor
5f	0.609	0.006	Transcription factor STAT3 inhibitor

Compound 5e inhibited STAT3 activation in Alpha Screen-based assay

The molecular docking results showed that compound 5e effectively bound with the SH2 domain of pan class="Gene">STAT3 protein. Based on that, we further investigated the direct interaction of the compounds (4a–5f) with class="Chemical">pan class="Gene">STAT3, for which the compounds were subjected to the Alpha Screen based assay [35] (an in vitro competitive binding assay used to recognize the ability of compounds to directly inhibit the binding of SH2 containing proteins to their respective phosphopeptides). Among all, compound 5e considerably interacted with the SH2 domain and exhibited significant activity against STAT3 (74.12% of inhibition at 60 μg/mL concentration) (Fig. 3).

Fig. 3

Concentration-dependent decrease in the percentage activity of STAT3 after treatment with compounds (4a–5f) in the Alpha screen based assay.

Concentration-dependent decrease in the percentage activity of pan class="Gene">STAT3 after treatment with compounds (4a–5f) in the Alpha screen based assay.

Compound 5e inhibited expression of STAT3 via Western blot assay

From previous cytotoxic studies, the IC50 value for compound 5e was found to be 2.45 ± 1.03 µg/mL for pan class="Disease">MCF-7 cancer cells [1]. Thus, we investigated the effect of compound 5e on class="Chemical">pan class="Gene">STAT3 in MCF-7 cancer cells at the same concentrations via Western blot assay. The results showed that compound 5e considerably decreased the expression of STAT3 in a concentration-dependent manner, thus inhibiting its activation (Fig. 4A). The decrease in the folds of protein expression was measured using Image J software (Fig. 4B) [45]. The data collectively suggested that compound 5e could inhibit STAT3 activation.

Fig. 4

(A) Western blotting analysis of STAT3 expression in the total protein extract from MCF-7 cells exposed to different concentrations of 5e for 24 h. (B) Fold changes in the protein expression compared to the loading control GAPDH. Values are expressed as mean ± SD for a set of three experiments. **P < 0.01, ***P < 0.001, compared to control.

(A) Western blotting anapan class="Chemical">lysis of class="Chemical">pan class="Gene">STAT3 expression in the total protein extract from MCF-7 cells exposed to different concentrations of 5e for 24 h. (B) Fold changes in the protein expression compared to the loading control GAPDH. Values are expressed as mean ± SD for a set of three experiments. **P < 0.01, ***P < 0.001, compared to control.

Conclusion

In tpan class="Chemical">his study, we showed that class="Chemical">pan class="Chemical">2, 5-disubstituted-1, 3, 4-oxadiazole derivatives, particularly compound 5e (5-(aminophenyl)-2-(butylthio)-1,3,4oxadiazole), displayed significant antioxidant activities by potentially scavenging the formed radicals. Additionally, computational studies suggested that the compound 5e emerged as a lead for inhibition of STAT3, thus providing a new therapeutic approach to cancer drug development. Further optimization of the derivatives needs to be explored.