1. Introduction
Gossypol (
Figure 1), an orally active polyphenolic compound, is widely distributed in cottonseed (
Gossypium spp.) and has also been detected in
Hampea integerrima Schltdl, a tree belonging to the family
Malvaceae [
1,
2]. This compound is formed in the plant by the dimerization of two molecules of hemigossypol and is best classified as a dimeric-sesquiterpenoid [
3]. Gossypol has previously been studied in various preclinical and clinical experiments and showed various therapeutic properties, including antioxidant, anticancer, antiviral, antiparasitic, and antimicrobial properties along with lower plasma cholesterol properties. This compound was also observed to act as a male contraceptive [
4,
5,
6,
7,
8]. Gossypol remains a little-examined molecule and, therefore, further studies need to be performed [
9]. Consequently, our study aims to draw attention to the novel biological properties of this compound.
Helicobacter pylori (HP), the predominant species of the human gastric microbiome, is the most frequent bacterial infection in the world and poses a significant threat to public health, as an estimated 50% of the world’s population is infected by this pathogen [
10]. HP is a Gram-negative bacterium known to cause peptic ulcer diseases (gastric and duodenal ulcers) [
11]. Several critical complications were also described to be associated with HP infection, including chronic gastritis, gastric cancer, and gastric-mucosa-associated lymphoid tissue (MALT) lymphoma [
12]. Currently, the most effective eradication regimen for HP is mainly based on the use of a proton pump inhibitor (PPI) and a gastric mucosal protective agent coupled with one or two antibiotics (such as clarithromycin, metronidazole, levofloxacin, and amoxicillin), resulting in triple or quadruple therapy [
13,
14]. As with all antibiotics, extended or recurrent use has increased the risk of developing antibiotic resistance, leading to an increase in the failure rate of standard therapy for HP infection [
15]. Therefore, to overcome this global problem, new sources such as natural products [
16,
17,
18] that deliver successful anti-HP drugs with less resistance, reduced undesirable effects, and diverse mechanisms of action are urgently required [
19].
Studies on enzyme inhibition continue to play a vital role in drug discovery since these studies have led to the discovery of novel drugs useful in the therapy of numerous diseases [
20,
21]. Urease is a nickel-dependent metalloenzyme that is naturally synthesized by plants, bacteria, fungi, and algae. It catalyzes the hydrolysis of urea, producing ammonia and carbon dioxide [
22,
23]. Urease is an essential enzyme produced by HP, where the amount of ammonia released by the urease-catalyzed reaction neutralizes the gastric acid, leading to optimal conditions for the survival and colonization of HP. Therefore, inhibition of HP urease (HPU) represents a valuable method for designing novel anti-HP drugs [
24,
25].
By using in vitro systems, this study was designed to investigate the antibacterial effect of gossypol against HP and the inhibitory properties against the urease that plays a critical role in its pathogenesis. The cytotoxicity of gossypol was also tested to evaluate its safety. In addition to in vitro tests, molecular docking studies were performed to predict the binding mode and molecular interaction of gossypol with HPU.
2. Materials and Methods
Gossypol (from cottonseed with a purity of ≥95% and a molecular weight of 518.6 g/mol) used in all experiments was obtained from Sigma-Aldrich, Berlin, Germany.
2.1. Antimicrobial Assay
2.1.1. Bacterial Strains and Culture Requirements
The HP standard strain 43504 (American Type Culture Collection (ATCC); Manassas, VA, USA) and ten HP clinical isolates (HP1–HP10; hospital-acquired from Motol University Hospital (MUH), Prague, Czech Republic) were used for the antimicrobial assay. All bacterial strains were cultured and grown in Mueller–Hinton agar (MHA) supplemented with 7% horse blood (Sigma-Aldrich, Prague, Czech Republic). Further, the strains were incubated at 37 °C for three days under microaerobic conditions with 5% O
2, 10% CO
2, and 85% N
2 [
14,
26]. All used clinical strains had previously been identified based on the microaerophilic growth condition, Gram’s stain, morphology, and the presence of virulence factors along with verified enzyme activities (urease, catalase, and oxidase). Moreover, the impacts of temperature, aging, aerobiosis, starvation, and antibiotics on the morphologic conversion rate to coccoid forms along with the culturability of bacterial strains were ascertained as previously recommended by the Clinical and Laboratory Standards Institute (CLSI) [
26]. All used strains were susceptible to gossypol and standard antibiotics (clarithromycin, metronidazole, and levofloxacin; purchased from Sigma-Aldrich, Berlin, Germany).
2.1.2. Anti-Helicobacter pylori (HP) Activity
For antimicrobial susceptibility testing, the agar dilution method was used to determine the minimum inhibitory concentration (MIC) values as previously advised by the CLSI [
26]. Briefly, the stock solutions of test compounds (gossypol, clarithromycin, metronidazole, and levofloxacin were dissolved in dimethyl sulfoxide (DMSO; 1%) and prepared in serial dilutions with starting concentrations ranging from 0.5 to 10 µg/mL) were added to MHA–7% horse blood and maintained in a microaerobic condition (5% O
2, 10% CO
2, and 85% N
2). All HP strains were subjected to sub-culturing on MHA supplemented with horse blood (7%) under the same microaerobic condition for three days at 37 °C. The bacterial suspensions prepared in MH broth were modified to a final concentration of a McFarland Standard 0.5 (10
8 CFU/mL). Further, 2 µL of the adjusted inocula was transferred to the series of agar plates, including a control plate without test compounds. After 72 h of incubation under the microaerobic condition, the MIC values were assessed as the lowest concentration of test compounds inhibiting visible growth. MIC values were acquired from three individual measurements conducted in triplicate.
2.2. Enzyme Inhibition Assay
2.2.1. Instrumentation and Operational Parameter Settings
The activity of HPU (obtained from MUH, Prague, Czech Republic with a purity of ≥99%) was assessed using a system pump-injector (Agilent 1200, Berlin, Germany) combined with a Sciex-3200QTRAP–hybrid triple quadrupole/linear ion trap mass spectrometer (MS; Toronto, ON, Canada) coupled with Electrospray Ionization (ESI). The instrumental parameter settings were optimized according to the published method [
24] (curtain gas (CUR), 25 psi; nebulizer gas (GS1), 50; auxiliary gas (GS2), 40; declustering potential (DP), 15 V; ion spray voltage, −4000 V; turbo temperature, 450 °C). The analysis was initiated by running ESI-MS without a High-Performance Liquid Chromatography (HPLC) column utilizing the flow injection analysis (FIA) mode. To detect and measure the substrate depletion (urea;
m/z 61→44), MS was used to perform a multiple reaction monitoring (MRM) analysis and was set in positive ion mode using mobile phases (HCOOH (0.1%) and HCOONH
4 (1 mM)). The flow rate was set at 0.5 mL/min with 10 µL of injection volume.
2.2.2. Anti-Helicobacter pylori Urease (HPU) Assay
The HPU-catalyzed reaction was determined using an Electrospray Ionization–Mass Spectrometry (ESI-MS) method as previously detailed [
24], where the assay is based on the monitoring of the decrease in the substrate concentration (urea concentration) in the presence and absence of inhibitors by observing the changes in the concentration of urea. Briefly, gossypol and acetohydroxamic acid (Sigma-Aldrich, Berlin, Germany; purity ≥98%) at a concentration of 16.1 µM were incubated with a solution consisting of HPU (38.2 µg/mL) prepared in HCOONH
4 buffer (1 mM; pH = 7.6) for 20 min to achieve binding equilibrium. Further, the solution mixture was blended with urea (275 µM) and directly injected into the FIA system and the changes in the concentration of urea were detected. The kinetics of urea depletion were analyzed by integrating areas (total counts) under peaks in the FIA system. The repeatability of measurements was confirmed by performing multiple measurements of the same sample. The relative standard deviation (RSD; %) of multiple measured slopes was calculated to determine the precision of time-course analysis. The half-maximal inhibitory concentration (IC
50) values for gossypol and acetohydroxamic acid were assessed following the above-mentioned method [
24].
2.3. Cytotoxicity Assessment
2.3.1. Cell Lines Preparation
The cytotoxicity of gossypol and cisplatin (a standard cytotoxic drug obtained from Sigma-Aldrich, Prague, Czech Republic; European Pharmacopoeia reference standard) was evaluated using human gastric epithelial cells (GES-1; MUH, Prague, Czech Republic). The GES-1 cells were prepared as previously described [
27]. Concisely, GES-1 cells were cultivated in a culture medium (Roswell Park Memorial Institute RPMI-1640; Sigma Chemicals Co., Saint Louis, MO, USA) enhanced by a mixture consisting of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (25 mM), 10% of heat-inactivated fetal calf serum (FCS; pH = 7.2),
L-glutamine (3 mM), streptomycin (200 mg/mL), and penicillin (192 U/mL). The prepared cells were cultivated in a humidified condition with 5% CO
2 at 37 °C, and then sub-cultivated two times for 7 days at the same experimental conditions.
2.3.2. Cytotoxicity Test
The cytotoxicity of gossypol was assessed by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide; Sigma-Aldrich, Berlin, Germany) with GES-1 cells following the detailed method [
27]. Briefly, dimethyl sulfoxide (DMSO; 1%) was used to prepare the stock solutions of gossypol, while NaCl (0.9%) was employed to prepare the stock solutions of cisplatin. The stock solutions of gossypol and cisplatin were then diluted with nutrient medium to final concentrations of up to 200 µg/mL and 10 µg/mL, respectively, on GES-1 cells. The absorbance of test samples (λ 570 nm) was detected by a microplate reader (Infinite M200, Tecan, Salzburg, Austria). The IC
50 values of test compounds were determined by cell survival diagrams and require the inhibition of 50% of the GES-1 cells’ survival.
2.4. Molecular Docking Studies
For the preparation of investigated ligands and proteins, the SDF file of the three-dimensional (3D) structure of gossypol (CID: 3503) was recovered from the PubChem database, while the 3D crystal structure of HPU in complex with acetohydroxamic acid (PDB ID: 1E9Y) was retrieved from the RCSB Protein Data Bank (
www.rcsb.org; accessed on 5 October 2021).
The molecular docking analyses were completed using a PyRx virtual screening tool combined with Autodock VINA software (version 0.8, The Scripps Research Institute, La Jolla, CA, USA). The docking analyses and settings were performed according to the previously described protocol with an exhaustiveness of 8 [
21,
28], where all docking settings, including the preparation of PDBQT files for the receptor and ligands, energy minimization, determination of binding sites, calculations (performed five times and the best-scored result was chosen), the protonation state, and the overall charges, were established and optimized. The cubic grid box with a size of 60 Å (x, y, and z) and a spacing of 0.375 Å along with the grid maps were optimized. The center of the grid was adjusted to the average coordinates of the two Ni
2+ ions.
The binding affinity values (kcal/mol) of ligand–receptor complexes were utilized to assess the docking results. The binding affinity values are based on hydrogen bonds, hydrophobic interactions, and electrostatic interactions. The validation processes of docking results were confirmed by removing the co-crystallized ligands and re-docking them back into their receptors. The best-scored docking poses were selected, and the docking results were further graphically processed by Discovery Studio Visualizer version v19.1.0.18287 (BIOVIA, San Diego, CA, USA).
4. Conclusions
Although numerous studies have disclosed the pharmacological actions of gossypol against several biological and molecular targets, there are still many activities that have yet to be investigated. The results of this study reveal the pronounced therapeutic utilities of gossypol via its ability to effectively suppress the growth of all HP strains tested along with a remarkable capacity to inhibit urease, a potent virulence factor for HP. Moreover, no remarkable cytotoxicity of gossypol was detected against human gastric epithelial cells evaluated by an MTT assay. The therapeutic efficacy was determined by in vitro microbiological, biochemical, and in silico molecular docking studies. The binding modes and molecular interactions were predicted by molecular docking analyses. Despite the therapeutic value induced by this compound against the investigated targets, additional in-depth in vivo studies are needed to authenticate the findings obtained by in vitro investigations. Moreover, combined pharmacokinetic and pharmacodynamic experiments need to be performed.