TARGET-BASED ANTICANCER GLYCYRRHETINIC DERIVATIVES: DESIGN, SYNTHESIS, BIOLOGICAL ASSESSMENT AND MOLECULAR DOCKING STUDIES

. 18-Glycyrrhetinic acid (GA) is regarded as the principal active component isolated from the Chinese medicinal plant of licorice root, and it has considerable anticancer actions. This work was built on the discovery and design of brand-new 18-glycyrrhetinic acid (GA) amino acid peptides and peptide ester analogs. The cytotoxic evaluation exhibited that despite the promising cytotoxic activity of the tested peptides 2 , 3 , 4 , 6 , and 7 in MCF-7 and HCT-116 cancer cells, with IC 50 values ranging from 5.1-7.4 and 6.6-72.7 µg/mL, respectively. Furthermore, all freshly produced GA-peptides with moderate to high activity on tumor cell lines produced a favorable safety profile versus typical human dermal fibroblasts (BJ-1) cellular lineage. Interestingly, 2 , 4 and 6 demonstrated excellent multitargeting inhibitory profiles against CDK-2, VEGFR-2, and PDGFR-α kinases. Moreover, since peptide 6 was the most active cytotoxic agent, it was chosen as an illustrative candidate to examine its influence on many apoptotic markers in invitro studies, including Bax, caspase-3 and 7, DNA fragmentation, BCl-2, p53, and tubulin polymerization inhibition. All novel analogs were tested for their antimicrobial efficacy versus a panel of microbial strains. The peptide 6 was also subjected to molecular docking simulations in the active sites of the prior kinases.


INTRODUCTION
18-Glycyrrhetinic acid is a pentacyclic triterpene of the oleanane type found mostly in the root of the Chinese plant Licorice (Glycyrrhiza glabra) [1,2].Various findings have reported that GA and its byproducts produce different pharmacological effects, such as bronchodilator, detoxification, anti-inflammatory, antitumor, antiulcer, antimicrobial, and antiviral functions [3,4].Recently, the antiproliferative activity of GA has been extensively investigated in breastovarian cancers, hepatoma, leukemia, and Gastric tumor cells [5,6].
It has been shown that GA exerts its potent anticancer impact by disrupting the actin cytoskeleton, hence inhibiting angiogenesis [7,8] along with impairing the p38 MAPK-AP1 signaling axis [9] as well as an immunomodulatory effect through suppression of COX-2 and its enzymatic product PGE-2, which have been reported to display crucial roles in the development of cancer disease [10].In addition, mechanistic studies represented that the GA-based cytotoxic activity is correlated with the mitochondrial apoptotic pathway via potential depolarization of the mitochondrial membrane, which consequently results in the upregulation of caspase-9, caspase-3 and activation of the production of reactive oxygen species (ROS) as well as causing a balance between pro-proteins and anti-apoptotic proteins, leading to apoptosis [11].Many studies have been carried out in the last decades to modify the GA backbone, aiming to improve its anticancer effectiveness, such as ring A, ring C, and C-30 carboxylic acid modifications [12].In addition, hybridization of GA was also performed with other biological moieties of pronounced cytotoxic activity, including "cinnamamide, triphenylphosphonium, and Rhodamine B" [13].Numerous semi-synthetic oleanolic acid analogs, for example, the CDDO and CDDO-Me with conjugation of 1-en-2-cyano-3-oxo moiety to ring A, were discovered to be the most efficient anticancer drugs and have undergone clinical studies.(Figure 1A) [14].Likewise, GA similar compound CDODA-Me displayed potent antitumor activity (Figure 1A) [15,16].COOTO-Meanalogue displayed a strong suppression of cancer cell proliferation and activation of apoptosis in conjunction with decreased (HDAC) histone deacetylase protein levels [17].
Amino acids are the basic material that sustains biological life and play vital functions.Different pharmacological actions, including anticancer activity, are shown by amino acids, which are extremely water soluble [18].Despite the wide spectrum of the biological activities related to natural products, the low bioavailability and poor solubility are considered their major disadvantages [19].Interestingly, conjugation with amino acid residues might increase the bioavailability features and ADME qualities of the natural product.These characteristics distinguish amino acids as the primary natural structural enhancers [18].
Inspired by the preceding discoveries and in keeping with our prior studies in the area of identification of novel effective anticancer and antibacterial compounds [20][21][22][23][24][25][26][27][28][29], this manuscript represented the development and formulation of novel 18β-glycyrrhetinic amide derivatives of potential anticancer activities.The synthetic strategy was based on coupling GA with different amino acids at its C-30 carboxylic group.In addition, esterification of the hydroxyl group of ring A with substituted glycine derivatives was also carried out, aiming to improve the anticancer potentiality of the new derivatives and to explore the structure-activity relationship (Figure 1B).
The cytotoxic potential of all produced compounds was evaluated versus three human cancer cell lines: (MCF-7) breast cancer, (HCT-116) colon cancer, and (HepG-2) liver cancer.Since different studies related the anticancer activity of various macrocycles to different enzyme inhibitions that have been involved in carcinogenesis cases such as CDK-2, VEGFR-2, PDGFRα, JAK-2, FLT-3 and EGFR, representing them as successful attractive targets for anticancer therapies [30], it was of interest in this study to examine the GA-peptides which exhibited the most promising anticancer activity 2, 4 and 6 as multitargeting kinase inhibitors against various kinases such as CDK-2, VEGFR-2, PDGFR-α.In addition, further evaluation was also carried out for the promising peptide 6 against different oncogenic parameters such as (TubB) tubulin polymerization, p53, Bax, Bcl-2, caspase-3 and 7, and % fragmentation of DNA to find out its cytotoxic mechanism-based Since peptide 6 exhibited the most promising multitargeting kinase inhibitory action, a molecular modeling investigation was conducted to justify its promising pharmacological impact by elucidating its mechanisms of binding with the building blocks of proteins residues of the examined kinases CDK-2, VEGFR-2, and PDGFR.It has been documented that specific infections are primarily responsible for various forms of cancer [31].Many cytotoxic antibiotics, such as "Actinomycin, Adriamycin/Doxorubicin", and certain other compounds, exert their effects by causing damage to DNA and simultaneously triggering the host's existing defense mechanisms.This process is considered one of the key pathways through which such chemotherapeutic agents exert their antimicrobial effects [32].Consequently, there was a keen interest in investigating the antimicrobial effectiveness of the recently synthesized GA-peptides.This research aimed to develop new, safer agents with dual capabilities, serving as both potent anticancer and antimicrobial agents of natural origin.

Cytotoxic assay
Human breast MCF-7, colon HCT-116 and liver HepG-2 carcinoma cell line were taken from Vacsera (Giza, Egypt).The culture was retained in RPMI 1640 medium with 1% antibioticantimycotic mixture (25 μg/mL amphotericin B, 10,000 μg/mL streptomycin sulfate and 10,000U/mL potassium penicillin), 1% L-glutamine, and complemented by 10% heat inactivated fetal bovine serum.The culturing and subculturing were done according to [33].Doxorubicin was used as a positive control and DMSO as a negative control.This experiment was carried out according to the reported MTT assay method and the absorbance was measured at 595 nm.

Kinase assays
The activities of the examined compounds against CDK-2, EGFRand PDGFR-α were in vitro tested usingCDK4 ELISA kit, Cloud-Clone Corp for CDK4, USabcam's Human In cell ELISA Kit (ab 126419) for EGFR and ADP-GloTM Kinase Assay for PDGFR-α, respectively.Theprocedures of the used kits were done according to the manufacturer ' s instructions.

Estimation of Bcl2 level
The samples and standards having Bcl-2 were estimated as previously mentioned [34].Addition of biotin-conjugated antibody was followed by streptavidin-HRP.Then the reaction was terminated by adding acid and the absorbance was measured at 450 nm.

Estimation of Bax level
The levels of Bax protein were evaluated according to the reported method [35].Addition of monoclonal antibody specific to Bax captured on the plate was followed by incubation and addition of Streptavidin conjugated to Horseradish peroxidase.Then the reaction was terminated by adding acid and optical density of the produced color was measured at 450 nm.

Estimation of human p53 level
The samples or standard having human p53 binds to antibodies adsorbed to the microwells.Addition of biotin-conjugated was followed by incubation and addition of dispense of unbound biotin-conjugated streptavidin HRP.Then the reaction was terminated by adding acid and the absorbance was measured at 450 nm [36].

Calculation of IC50 values
The IC50 values were estimated for the promising active compounds using the SPSS computer program (SPSS for windows, statistical analysis software package /version 9/ 1989 SPSS Inc., Chicago, USA) and utilizing probit analysis.

Human CASP7 (Caspase 7) Estimation
The micro ELISA plate provided in this kit pre-coated with CASP7 specific antibody.A biotinylated CASP7 antibody and Avidin-Horseradish Peroxidase (HRP) conjugate was added.Aspire the excess components.The substrate solution was added.Wells that contain CASP7, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color.The color turns yellow followed the addition of sulphuric acid solution.The optical density (OD) was measured at a wavelength of 450 nm ± 2 nm [37].

Enzyme-linked Immunosorbent Assay Kit for Tubulin Beta (TubB)
The MCF-7 cells were inoculated using RPMI 1640 (supplemented with 1% penicillinstreptomycin and 10% FBS) at concentration 10,000 cells/well.For 24 hours before the enzyme assay of Tubulin, the tested compounds were added, followed by addition of Avidin conjugated to Horseradish Peroxidase (HRP) to each microplate well and incubated.After addition of TMB substrate solution, the wells containing TUBb, enzyme-conjugated Avidin and biotin-conjugated antibody revealed a change in color.Addition of sulphuric acid solution terminated the enzymatic reaction.The color change was measured spectrophotometrically at a wavelength of 450 nm ± 10 nm [38].

Estimation of DNA Fragmentation through DPA assay
Assessment of DNA fragmentation of the cells was done, as described by the reported method [39].Briefly, the cells were lysed for 15 min on ice with 0.5% (v/v) Triton X-100, 20 mM EDTA and 5 µM Tris (pH 8.0).Then, the cells were centrifuged to separate intact chromatin from DNA fragments for 20 min, at 27,000 Xg.Measurement of the amount of DNA was done using a diphenylamine reagent and the optical density was measured at 600 nm.

In-vitro antimicrobial activity
Antimicrobial activity of the tested chemical peptides was determined by well diffusion method for pathogenic bacteria having Gram-positive bacteria (Staphylococcus aureus ATCC25923, Streptococcus pneumonia and Micrococcus luteus) and Gram-negative bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC7853 and Proteus sp.) and Candida albicans as human pathogen yeast using well diffusion method [40,41].

Molecular docking study
The 2D structure of the newly synthesized derivatives 6 was drawn through ChemDraw.The protonated 3D was employed using standard bond lengths and angles, using molecular operating environment (MOE-Dock) software version 2014.0901[42,43].Then, the geometry optimization and energy minimization were applied to get the Conf Search module in MOE, followed by saving of the MOE file for upcoming docking process.The co-crystallized structures of CDK-2, VEGFR-2 and PDGFR-α kinases with their ligands PY8, sorafenib and imatinib were downloaded (PDB codes: 2J9M, 4ASD and 6JOL, respectively) [42,43] from protein data bank.All minimizations were performed using MOE until an RMSD gradient of 0.05 kcal•mol −1 Å −1 with MMFF94x force field and the partial charges were automatically calculated.Preparation of the enzyme structures was done for molecular docking using Protonate 3D protocol with the default options in MOE.London dG scoring function and Triangle Matcher placement method were used in the docking protocol.At the first, validation of the docking processes were established by docking of the native ligands, followed by docking of the derivative 6 within the ATP-binding sites after elimination of the co-crystallized ligands.

RESULTS AND DISCUSSION Chemistry
New target peptides (1-8) have been synthesized according to Schemes 1 and 2. GA has been conjugated with many amino acids.These coupling reactions were carried out using conventional synthetic peptide methods (in solution) [6,21,24].

In vitro anticancer action
In this study, all the new target peptides 1-8 were initially screened for their cytotoxic action versus three human cancer cell lines, namely MCF-7, HCT-116, colon carcinoma, and HepG2 at a starting concentration of 10 µg/mL via using MTT assay.The resulting data are depicted in Figure 2A.It has been found that the tested peptides 2, 3, 6, 7, and 4 possessed the highest cytotoxic activity against the breast tumor cells (MCF-7) with cytotoxicity percentages of 98.6, 98.1, 97.7, 95.2 and 86.4 %, correspondingly.On the contrary, compound 1 showed a moderate cytotoxic effect, and derivatives 5 and 8 presented low activity.Similarly, compounds 6, 2, 3, 4, and 7 represented the most promising cytotoxic effect against the colon tumor cells (HCT-116) with growth inhibition percentages 99.1, 97.7, 97.3, 95.3 and 89.2 %, respectively.Conversely, compounds 1, 5, and 8 showed weak activity.Furthermore, with respect to the examined hepatocellular carcinoma cells (HepG-2), compound 6 was the only promising derivative showing tumor growth inhibition of 97%.Compounds 2 and 4 showed moderate activity, with growth inhibition percentages of 58.9 and 56.4 %, respectively, while the rest of the compounds possessed weak cytotoxic potency (Figure 2A).Afterward, all the compounds that showed moderate to high cytotoxic capabilities were further screened versus the typical human dermal cell lineage (Bj-1) to identify the safety of these tested peptides.As shown in Figure 2B, all the tested peptides represented a promising safety profile against the tested normal cells.GA-peptides 1, 2, 3, 4, 6, and 7 exhibited cytotoxic activity ranging from 0.1-22.9%.
In order to investigate their median growth inhibitory concentrations (IC50s), all the compounds that represented cytotoxic activity over 70% inhibition of the tested cancer cells were subjected to the dose-dependent MTT assay at varying concentrations (0.625, 1.25, 2.5, 5 and 10 µg/mL), taking doxorubicin as a standard drug.Thus, GA-peptides 2, 3, 4, 6, and 7 were investigated as anticancer agents against their respective human cancer cell lines "breast MCF-7, colon HCT-116, and liver HepG-2 cell lines" at different concentrations (0.625, 1.25, 2.5, 5 and 10 µg/mL).The results were conveyed as IC50 values (μg/mL) and depicted in Table 1 (Figure 2C).According to the biological results in Table 1, it could be noted that the conjugation of glycylglycine peptide 2 with the glycylglycyl-S-tritylcysteinate moiety as the peptides 3, 4, and 6 retained the potent cytotoxic activity.The glycylglycine peptide 6 exhibited two-fold higher growth inhibition activity against MCF-7 than the standard drug doxorubicin, with IC50 values of 6.2 and 13±1 µg/mL, respectively.On the other hand, compound 6 was slightly less active than doxorubicin against HCT-116, with IC50 values of 6.60±0.11and 2.00±3 µg/mL, respectively.A detectable drop in the activity of compound 6 was recorded against HepG-2 compared to the reference drug, with IC50 values of 63.73±0.21 and 1.00 µg/mL, correspondingly.Also, the cytotoxicity of compounds 3 and 4 was approximately two-fold higher than that obtained by doxorubicin against MCF-7, with IC50 values of 6.32±0.15and 7.40±0.05µg/mL, respectively.Compound 4 exhibited a small drop in potency against HCT-116 with an IC50 value of 7.11±0.10µg/mL.In contrast, compound 3 produced a significant drop in potency compared to the reference drug with an IC50 value of 53.82±0.20 µg/mL.Although the glycylglycyl-S-tritylcysteinate-OMe peptide 7 has maintained its activity against MCF-7 cancer cells with an IC50 value of 6.22 µg/mL, its activity remarkably decreased against HCT-116 with an IC50 value of 72.73 µg/mL.Unfortunately, all the tested peptides were inactive against HepG-2 cancer cells, while compound 6 was moderately active with an IC50 value of 63.73 µg/mL (Table 1).It could be noted that all the assessed peptides, the glycyl-glycine peptide 2 and its S-trityl-Lcysteine analogs 3, 4, 6, and 7, appeared to be potent cytotoxic candidates against the human breast cancer cells (MCF-7) as related to the standard drug as doxorubicin HCL.Alternatively, the O-methylated derivatives (3, 7) observed a detectable decrease in potency, which were moderately active against HCT-116 cancer cells.In contrast, the other analogs (2, 4, and 6) retained their potent activity against HCT-116 cancer cells.This result could be explained by the fact that the presence of free hydroxyl groups is preferable for cytotoxic activity because they act as H donors to bind with the active sites of the ligands' sites.It could be observed that the conjugation of the tert-butoxycarbonyl)leucine moiety at ring A as compound 7 did not enhance the potency against MCF-7 and might be a reason for the detectable decrease in the activity towards HCT-116 cancer cells due to the steric hindrance that might occur, preventing the molecular structure from having a good fit with the target protein on which they act.The hepatic carcinoma cells (HepG-2) were insensitive towards the evaluated GA-peptides except peptide 6, which produced moderate activity.13.00 ± 0.10 2.00 ± 0.31 1.00 ± 0.10 IC50: The concentration of a substance that suppresses cellular survival by half; every value is the average of 3 repeats ± SD.

In vitro enzymatic assays
It has been reported that the enzymes (CDK-2) cyclin-dependent kinase-2, (VEGFR-2) vascular endothelial growth factor receptor, and platelet-derived growth factor receptor -α (PDGFR-α) play significant roles in tumor cell proliferation, survival, differentiation, angiogenesis, and apoptosis.Accordingly, they are considered successful targets for anticancer therapies, particularly for gaining multitargeting anticancer drugs of improved activity [44].Thus, it was of interest to determine the enzymatic inhibitory efficacy of the most promising cytotoxic GA-peptides with the safest profile against the normal cells (BJj-1) 2, 4, and 6 against a panel of three different kinases: CDK-2, VEGFR-2, and PDGFR-α using the multitarget inhibitor staurosporine as a standard drug.The resultant data were tabulated in Table 2.It could be observed that glycylglycine peptide 6 represented the most promising inhibitory effect against the three examined kinases.It represented a suppressing activity against CDK-2 that was about 2-fold higher than the reference drug, with IC50 values of 0.074 μM and 0.150 μM, respectively.Furthermore, peptide 6 appeared to be approximately equipotent to staurosporine against both VEGFR-2 and PDGFR-α with IC50 values of 0.024 and 0.051 μM and IC50 staurosporine; 0.030 and 0.040 μM, respectively.Although the other two tested peptides 2 and 4 exhibited promising suppression impact against the three examined enzymes with IC50 values ranging from 2.640 to 6.125 μM, they appeared much less potent than the standard drug staurosporine.The data obtained increased understanding of the pharmacological mechanisms of the glycylglycine peptide 6, indicating that the suppression impact of CDK-2, VEGFR-2, and PDGFR-α could be considered one of the anticancer mechanisms that it relied on.In contrast, the anticancer activity of the other two peptides, 2 and 4, was primarily based on other modes of action.

The effect of the GA-peptide 6 on Bax, Bcl-2, Bax/Bcl-2 ratio and p53 levels
The Bcl-2 protein is essential in tumor growth and suppressed cell apoptotic intrinsic process [45].Unlike, the pro-apoptotic Bax induces apoptosis inside the cell [46].It has been reported that cell survival through apoptosis inhibition is enhanced by raising the amount of the anti-apoptotic Bcl-2 in the cancer cells, leading to apoptosis [44].The fate of the cell is determined by the balance among Bax and Bcl-2 proteins [44].Additionally, p53 is a tumor suppressor transcription factor protein.This protein is commonly mutated in human cancers and expressed in many biological stressors, including DNA damage [47] and [48].The p53 pathway was reported to either arrest the cell cycle or apoptotic cascades through the mRNA copies of Bcl-2 and Bax genes [48].The concentration of a substance that suppresses cellular survival by half; every value is the average of 3 repeats ± SEM.
Based on the promising results referring to the anticancer activity of peptide 6, it was of interest to study its various pathways, such as its influence on the apoptotic-induced pathway, including P53 and Bax apoptotic proteins, BCL-2 anti-apoptotic proteins, the BAX/Bcl-2 proportion, tubulin polymerization inhibition, fragmentation of DNA, and caspase-7 induction.
Using peptide 6 at its IC50 concentration of 6.2 g/mL for 24 hours reduced the anti-apoptotic protein Bcl-2 from 7.2 to 4.1 ng/mL compared to the typical MCF-7 cells, but less potently than doxorubicin, which decreased Bcl-2 concentrations to 0.40 ng/mL.In addition, compound 6 increased the amount of Bax protein from 65.5 to 259.4 pg/mL compared to typical MCF-7 cells, which was comparable to the level attained by doxorubicin, which was 259.2.Accordingly, peptide 6 led to a predictable elevation in the Bax/BCL-2 ratio, supporting its efficacy in elevating the therapeutic response in MCF-7 cells (Figures 3-A and 3-B).In addition, compound 6 increased the amount of p53 in treated MCF-7 cells by roughly 9.8-fold, from 111. pg/mL to 1090 compared to the typical untreated cells.

Impact of GA-peptide 6 on the quantities of caspase-3 and 7, the assembly of tubulin (TubB), and the percentage of fragmentation of the DNA
All apoptosis events involve shrinking cell size, consolidation of chromatin, and fragmentation of the DNA, requiring the association of caspase-7, which is considered a marker of cell apoptosis.Caspases, or cysteine-dependent aspartate-specific proteases, constitute a family of enzymes that are essential for initiating and executing the apoptosis process within a cell.Treating the MCF-7 cells with an IC50 value of 6.2 µg/mL of peptide 6 for 24 hours increased the level of caspase-7 as compared to unsubjected cells from 0.3 to 1.4 ng/mL, similar to doxorubicin, which elevated the caspase-7 level to 1.9 ng/mL (Figure 3C).In addition, peptide 6 has raised the level of caspase-3 from about 0.3 ng/mL in the control MCF-7 cells to 0.90 ng/mL in the cells subjected, while the reference drug doxorubicin raised the caspase-3 level to a higher level of 1.90 ng/mL (Figure 3D).Furthermore, peptide 6 has also had a profound influence on DNA fragmentation levels in the treated MCF-7 cells.It increased the ratio of fragmentation of the DNA from 7.8% in the control untreated MCF-7 cells to reach to approximately 25.4% in the peptide 6-treated MCF-7 cells.The reference drug colchicine appeared to be more effective, producing a DNA fragmentation percentage of 40.7 %, as shown in (Figure 3E).In addition, peptide 6 has had a profound influence on the DNA fragmentation rate of treated MCF-7 cells.It increased the percentage level of DNA fragmentation from 7.8% in the control, untreated MCF-7 cells to approximately 25.4% in the peptide 6-treated MCF-7 cells.The reference drug colchicine appeared to be more effective, producing a DNA fragmentation percentage of 40.7 percent, as shown in (Figure 3E).Furthermore, α-and β-tubulins polymerize to generate polarized filaments, which are known as microtubules, which are essential for a multiple of cellular activities, such as cell division, morphology, and intracellular transport [49].Accordingly, the identification of new inhibitors is still an active area of anticancer research [50].Accordingly, the effect of peptide 6 was also studied against tubulin B. The resultant data showed that IC50 of peptide 6 on MCF-7 is 14886.5 ng/mL for 24 h while the reference drug colchicine reaches 487.7 ng/mL, which implicates that peptide 6 was not potent enough to suppress tubulin B as the reference drug colchicine.GApeptide 6 appears to be a powerful pro-apoptotic candidate by activating the intrinsic mitochondrial route of apoptosis.

Antimicrobial activity
The recently synthesized peptides 1-4, 6, and 7 (disregarding compounds 5 and 8) underwent assessment as potential antimicrobial candidates against a set of pathogenic gram-positive bacteria, namely "Staphylococcus aureus ATCC25923, Streptococcus pneumonia RCMB 010010, and Micrococcus luteus, as well as three pathogenic gram-negative bacteria: Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC7853, and Proteus vulgaris RCMB 010085, in addition to the yeast Candida albicans".This evaluation was conducted using the well diffusion method [40].Gentamycin and ketoconazole were employed as reference substances for assessing antibacterial and antifungal activities, respectively.The data obtained for each analog was recorded as the mean size of the inhibitory zones (IZ) in millimeters, representing the area where microorganism Inhibition of growth occurred close to the discs (see Table 3).
It is evident that all the tested analogs of GA displayed stronger inhibitory activity against Micrococcus luteus compared to gentamicin, with inhibition zones ranging from 25 to 30 mm, while gentamicin had an IZ of 24.4 ± 0.72 mm.While ordinarily innocuous, it may become an opportunity-based pathogen and has been related with a number of illnesses, including but not limited to meningitis, septic arthritis, and endocarditis [41].In contrast, all of the chemicals studied showed very mild action versus the fungal and microbial strains used in the study, with inhibition zone ranges of 12-17 mm, in contrast to ketoconazole, which had an IZ of 22.8 ± 0.10 mm, and gentamicin, which exhibited IZ ranges of 12-19 mm, compared to its reference range of 20-27 mm.

Molecular docking study on CDK-2, VEGFR-2, and PDGFR-α
The objective of the docking study was to gain a deeper understanding of how the promising derivative 6 binds to the ATP binding sites of CDK-2, VEGFR-2, and PDGFR-α kinases, including their binding orientations and modes.In order to conduct this docking experiment, we integrated the "Molecular Operating Environment (MOE-Dock) software, specifically version 2014.0901"[42,43].To ensure the reliability of our docking process, we validated it by performing redocking experiments of co-crystallized ligands PY8, sorafenib, and imatinib within the active sites of CDK-2, VEGFR-2, and PDGFR-α.These co-crystallized ligands are associated with the respective protein structures with PDB codes 2J9M, 4ASD, and 6JOL [42,43].The results of the redocking revealed energy scores of -10.25, -12.44, and -11.20 kcal/mol for CDK-2, VEGFR-2, and PDGFR-α, respectively, along with small RMSD values of 0.68 Å, 0.81 Å, and 1.12 Å, indicating a close alignment among docked ligands and their locations in co-crystals.
The native ligand, PY8 was depicted in the vicinity of CDK-2 through five H-bond forces; two were formed between sulfonamide moiety the backbones of Ile10 and Asp86 (distance: 3.10 and 2.74 Å, respectively) and the other H-bonds was between the aminopyrimidine scaffold and the backbone of Glu81 and Leu83 (distance: 3.33, 1.94 and 2.90 Å, respectively) (Figure 4A).Within the binding site VEGFR-2, the ligand sorafenib illustrated two H-bonds between the urea group and the essential amino acids Asp1046 and Glu885 (distance: 2.79 and 2.58 Å, respectively).On the other hand, the pyridine nitrogen and the carbamoyl proton exhibited two hydrogen bonds with the backbone of Cys919 (distance: 3.10 and 2.88 Å, respectively) (Figure 4B).Moreover, there were arene-cation interactions with Asp1046 and Phe1047.Regarding the PDGFR-α receptor, imatinib formed several H-bonds with Met648, Thr674, Cys677, Val815, His816, and Asp836 (distance: 3.45, 3.00, 2.88, 3.14, 2.98 and 2.94 Å, respectively), and arenecation interactions with Leu599, Val607 and Phe837 (Figure 4C).The sulfur atom of S-tritylcysteinylglycylglycine potentiates fitting within ATP binding sites of VEGFR-2 and PDGFR-α through the formation of H-bonds with Asn923 and Leu599 (distance: 3.90 and 2.45 Å, respectively).The terminal carboxylic group showed an H-bond acceptor with the cornerstone of the essential amino acid Cys919 within the VEGFR-2 receptor (distance: 2.27 Å).Additionally, within PDGFR-α, there were H-bond and arene-cation interactions with Asp681 (distance: 3.08 Å) and Arg597, respectively.Finally and by focusing upon the superimposition views in Figure 7, the compound 6 bearing 18β-glycyrrhetinic amide scaffold was well fitted and embedded nicely within the active sites of CDK-2, VEGFR-2, and PDGFR-α through different H-bond formation afforded from the hydroxyl group at p-10, sulfur atom of S-tritylcysteinylglycylglycine fragment and the terminal carboxylic group with the essential amino acids sharing the native ligands within the same vicinity of the screened enzymes.This study involved creating and synthesizing novel peptides based on 18-glycyrrhetinic acid (GA).The synthesis process followed conventional methods of peptide coupling in a liquid phase, combining GA with various amino acids to yield eight novel peptides labeled as 1-8.All these new compounds were subjected to cytotoxicity testing to assess their potential using the MTT assay against MCF-7, HCT-116, and HepG-2 cell lines.Among the tested peptides, namely 2, 3, 4, 6, and 7, it was observed that they displayed significant cytotoxic effects against MCF-7 and HCT-116 cancer cells.In contrast, these peptides did not exhibit cytotoxicity against HepG-2 liver cancer cells, with the exception of peptide 6.Importantly, all the newly synthesized GA-peptides were found to be non-toxic to normal skin fibroblast cells (BJ-1).Additionally, when compared to the reference drug staurosporine, enzymatic assays demonstrated that peptides 2, 4, and 6 exhibited promising potential as multitargeting kinase inhibitors against CDK-2, VEGFR-2, and PDGFR-α kinases.Because target peptide 6 produced the most significant activities, it was chosen as a representative example to investigate its influence on the following cancer-related variables: Bax, BCl-2, p53, caspase-7, caspase-3, tubulin-B, and DNA fragmentation percentage.Furthermore, all of the new analogs appeared to be moderately active when tested versus a panel of gram-negative and positive, as well as Candida albicans.To explain peptide 6's pharmacological efficacy, molecular docking experiments were also performed in order to determine how peptide 6 binds to the active sites of the three examined kinases, CDK-2, VEGFR-2, and PDGFR.

Figure 1 .
Figure 1.A: The chemical makeup of GA and many semi-artificial pentacyclic triterpenoids.B: Design of new GA-peptides 1-8 of potential anticancer activity.

Figure 2 .
Figure 2. A: The cytotoxicity percentages of the new eight GA-peptides against (MCF-7) human breast carcinoma, (HCT-116) human colon carcinoma, and (HepG-2) hepatocellular carcinoma at 10 µg/mL.Each result is an average of triplicate, ± standard deviation.B: The cytotoxicity percentages of the active compounds against the human normal skin cell line (BJ-1) at 10 µg/mL.Each result is an average of triplicate ± standard deviation.C: IC50 values of GA-peptides 2-4, 6, and 7 against various human cancer cell lines ± = standard deviation.

Figure 3 .
Figure 3. A: Bax protein levels in breast cells treated with compound 6, typical cells, and cells subjected with the standard drug doxorubicin ± standard deviation.B: Protein levels of Bcl2 and Bax/Bcl2 proportion in MCF-7 treated cells with compound 6, typical MCF-7 cells (as control), and doxorubicin treated MCF-7 cells.C: Caspase-7 protein levels of treated cells with compound 6 compared with untreated cells and doxorubicin treated cells ± standard deviation.D: Caspase 7 protein levels of treated cells with compound 6 compared with untreated cells and doxorubicin treated cells ± standard deviation.E: % of DNA fragmentation in compound 6-treated cells compared to untreated and colchicine-treated cells ± standard deviation.

Table 1 .
The IC50 estimates for GA-based peptides vs. several cancer cell lines.

Table 3 .
Displays the results of the antimicrobial effectiveness assessment for the recently created compounds, employing the well diffusion assay technique.