Genus Staphylococcus contains gram-positive, bacitracin-resistant, catalase-positive, non-spore-forming coccus bacteria (Cheung et al., 2021). It is a coagulase-positive bacterium, which is the common colonizer of the human body. It is a highly virulent pathogen of “Risk Group Level 2” because of its ability to invade, persist, replicate, and infect human tissues (Wang et al., 2016). It is an opportunistic pathogen responsible for several human infections, from localized skin and soft tissue infections to life-threatening severe bacteremia (Matuszewska et al., 2020), endocarditis, osteomyelitis, atopic dermatitis, staphylococcal food poisoning, staphylococcal toxic shock syndrome (TSS) (Turner et al., 2019), prosthetic joint infections, medical device-related infections, and pneumonia (Senobar Tahaei et al., 2021). Due to its toxin-producing abilities (i.e., staphylococcal enterotoxin), it is a significant cause of staphylococcal foodborne disease (Fri et al., 2020). S. aureus can also cause infections in livestock, like mastitis (in ruminants) and bumblefoot (in poultry), as well as diseases in domestic animals (dogs and cats), resulting in substantial morbidity rates in animals and a significant economic loss to the farmers.

Methicillin-resistant S. aureus (MRSA) was first isolated in 1961 in a hospital in the UK (Ullah et al., 2016). MRSA is resistant to almost all β-lactams (Craft et al., 2019), including penicillin, trimethoprim, erythromycin, clindamycin, and tetracyclines (Turner et al., 2019), hence minimizing the safe and effective treatment options for MRSA. These strains also resist other antibiotic groups, e.g., fluoroquinolones, macrolides, and aminoglycosides, and are, therefore, known as “Superbug” (Senobar Tahaei et al., 2021). The success of this pathogen is related to the presence of its extensive virulence factors such as enzymes (hyaluronic acid, lipases, proteases, coagulase, and staphylokinase), toxins (hemolysins and leukocidins), antigens, and evasive immune factors (protein A and capsule) coupled with its remarkable ability to quickly acquire resistance to different antibiotics used in clinical practice over the years (Fri et al., 2020).

Methicillin resistance is associated with the modifications in penicillin-binding proteins (PBP2a/2c/2ʹ), due to the presence of mecA, mecB, or mecC genes in S. aureus. In addition to PBP1-4, MRSA strains encode a high-molecular-weight fifth penicillin-binding protein, called penicillin-binding protein 2a (PBP2a), responsible for their resistance (Fergestad et al., 2020). PBP2a is encoded by the mecA gene, which is present inside a mobile genetic element known as the Staphylococcal cassette chromosome mec (SCCmec) (Miragaia, 2018; Craft et al., 2019). PBP2a enables the synthesis of the bacterial cell wall in the presence of antibiotics, as it has a low affinity for all beta-lactam antibiotics, thus allowing cross-linking to proceed (Craft et al., 2019; Fergestad et al., 2020). Like other PBPs, the active site of PBP2a contains the active site serine (Ser403) located at the N-terminus. Still, it lies deep in the pocket and is inaccessible to beta-lactam antibiotics. This closed active site indicates the resistance offered by PBP2a (Bien et al., 2011). Moreover, some additional genes essential for methicillin resistance, such as factors necessary for the expression of methicillin resistance (fem) and auxiliary factors (aux), have also been identified (Bien et al., 2011).

The common antibiotics used against MRSA currently include daptomycin, gentamicin, vancomycin, linezolid, TMP–SMZ (trimethoprim/sulfamethoxazole), tigecycline, clindamycin, and amikacin, but these antibiotics are losing their efficacy (Okwu et al., 2019). In addition, teicoplanin, daptomycin, and linezolid are expensive (Okwu et al., 2019). As the therapeutic options available at present for the treatment of MRSA infections are limited, there is an urgent need to develop novel drugs effective for treating MRSA infections (Okwu et al., 2019). Cyclic imides and their N-substituted derivatives [-CO-N(R)-CO-] are an important class of organic compounds containing bis-amide linkages. Their neutral structures and hydrophobicity allow them to cross biological membranes easily. The nitrogen and oxygen atoms as donor sites can co-ordinate these molecules with the biological system causing some pharmacological effects (Hassoun et al., 2017). Among the imide-ring-containing heterocyclic compounds, phthalimides and their N-substituted derivatives possess extensive biological and pharmaceutical properties. They have also been used as tumor necrosis factor-alpha (TNF-alpha) inhibitors, which has a crucial role in several physiological immune systems (Kushwaha and Kaushik, 2016). These molecules are reported to possess important biological properties including antifungal, antibacterial, antiviral, anticancer, analgesic, antidepressant, apoptosis induction, anti-inflammatory, androgen receptor antagonist, anxiolytic, anticonvulsant, and muscle relaxant activities (Jafari et al., 2017). The antibacterial properties of different phthalimide derivatives of N-alkyl, N-alkyloxy, N-acyl, butyl, and N-hydroxyl have been reported against both Gram-positive (Streptococcus pneumoniae, Micrococcus luteus, and Listeria monocytogenes) and Gram-negative (Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli) bacterial strains which have shown promising results because of their smaller size and high potency in disease control management (Othman et al., 2019). Similarly, the derivatives of chiral phthalimides with N-alkyloxy and N-alkyl substitution in combination with maleimide have shown antifungal activity against fungi (Candida albicans and Aspergillus fumigatus) (Imran et al., 2019). One of the reasons for inflammatory diseases is the denaturation of the protein present in tissues that leads to the development of autoantigens. Phthalimide derivatives with the combination of N-aryl or aryl amines in the presence of an L-proline catalyst can prevent this denaturation process and, hence, inhibit the development of inflammatory diseases (Perveen and Orfali, 2018). So, these compounds can serve as an excellent therapeutic approach for treating MRSA infections; therefore, our study used three different chiral phthalimides to check their in silico and in vitro inhibitory effects. Regardless of the enormous investments and the time consumed in discovering new drugs, its success rate through clinical trials is only 13%, with a relatively high failure rate. These failures have been reported at a later stage in the majority of cases (40%–60%) because of the lack of optimum pharmacokinetic properties (absorption, distribution, metabolism, excretion, and toxicity) of the drugs (Gurung et al., 2021). So, recently, a decrease in the market has been noted in the number of new drugs because they failed in different phases of the clinical trials. Therefore, it is essential to cope with the limitations in the conventional drug discovery methods with a cost-effective, efficient, and broad-spectrum computational drug development process. Such computational techniques will aid the drug discovery and development process by minimizing the costs and final stage failure chances when used by various pharmaceutical companies and research groups in the preliminary studies (Batool et al., 2019; Gurung et al., 2021).

Molecular docking is a structure-based drug-designing approach (SBDD). It was first developed for predicting the binding action of known active molecules and virtual screening of sizeable digital compound libraries to minimize the cost and speed up drug discovery processes (Torres et al., 2019). It has been widely used as an inexpensive and fast technique in industrial and academic settings. The objective of molecular docking is to get an optimized conformation for both the ligand and the protein and the relative orientation between the ligand and the protein in such a manner that the free energy of the overall system is minimized (Torres et al., 2019). This drug-designing approach is effectively fast and specific for the identification and opsonization of lead molecules, which has helped to understand the molecular level of a disease (Gurung et al., 2021). In the current study, the chiral phthalimides (FIA, FIB, and FIC) used were docked against the PBP2a protein of MRSA before their in vitro antibacterial activity. The reason for selecting these chiral phthalimides was their good antibacterial activity against several Gram-positive and Gram-negative bacteria present in the literature.

Antibiotic resistance has been declared a “Global Public Health Concern” by the Centers for Disease Control and Prevention (CDC), WHO, the World Economic Forum, and the Infectious Diseases Society of America. In the past 2 decades, the acquired MDR infections have increased because of the production of beta-lactamase enzymes (including extended-spectrum β-lactamases (ESBLs), carbapenemases, and metallo-β-lactamases), which leads to 3rd-generation cephalosporin and carbapenem resistance (Aslam et al., 2018; Vestergaard et al., 2019). Currently, a group of MDR pathogens collectively known as “ESKAPE” bacteria also includes MRSA, which is considered the most important pathogen due to its resistance, mortality, and clinical importance. So, being a member of a highly pathogenic group of microorganisms, S. aureus was selected for this project (Senobar Tahaei et al., 2021). Considering the resistance story of S. aureus, it rapidly became MDR over several decades, starting with the production of penicillinase and penicillin resistance early in the 1940s, followed by the macrolide and tetracycline resistance in the 1950s and, finally, the emergence of MRSA (methicillin resistance) in the early 1960s (Morehead and Scarbrough, 2018), showing resistance to β-lactam antibiotics such as methicillin, oxacillin, nafcillin, cloxacillin, and dicloxacillin. This increased resistance rate was attributed to the presence of a “mecA”-resistant gene encoding a “penicillin-binding protein 2a” (PBP2a), which made the MRSA strains more resistant to methicillin (Liu et al., 2016). This is because the active site of PBP2a contains the active site serine (Ser403) located at the N-terminus which lies deep in the pocket and is inaccessible to beta-lactam antibiotics. This closed active site indicates the resistance offered by PBP2a (Bien et al., 2011). Although this multidrug resistance was developed subsequently, it has become a worldwide issue; therefore, the CDC has labeled it a severe threat to the healthcare system (Morehead and Scarbrough, 2018; Senobar Tahaei et al., 2021). Hence, due to the vital role of PBP2a protein in the resistance of MRSA, it was used as a target protein for checking the anti-MRSA activities of the novel chiral phthalimides in the current project.

Considering these susceptibility reports for MRSA (Fri et al., 2020; Senobar Tahaei et al., 2021) mentioned in the literature, we conducted AST using the CLSI guidelines 2022 in our project, and it was examined that all the MRSA isolates showed complete resistance to the antibiotics: cefoxitin (30 ug), penicillin (10 ug), ciprofloxacin (5 ug), vancomycin (30 ug), and cefepime (30 ug), and the highest level of susceptibility to the antibiotics was shown by gentamicin (10 ug), amikacin (30 ug), and linezolid (30 ug), whereas some isolates showed variable degree of resistance to two of the antibiotics, erythromycin (15 ug) and clindamycin (2 ug), because of the phenomenon of the D-test. The results of AST obtained followed the previous studies and indicated a high threat. Therefore, current-day researchers need to use alternative treatment options for microbial resistance. In this regard, an interdisciplinary approach to dealing with the resistance issue is gaining interest in the scientific community. So, our project used methods such as bioinformatics, synthetic chemistry, and microbiology. The emphasis on an interdisciplinary approach, involving bioinformatics for compound selection, synthetic chemistry for compounds synthesis and characterization, and microbiology for their antibacterial actions, ensures a comprehensive investigation. This collaboration increases the study’s depth and validity by highlighting the distinct contributions of each discipline to the overall findings. This research project was focused on using the novel compound “chiral phthalimides” by checking its antibacterial activities under in silico and in vitro environments against the PBP2a protein of MRSA. This will provide safe preventive and treatment approaches against S. aureus infections, contributing to the efficient management of infections and reducing the spread of antimicrobial resistance. The in silico part (molecular docking) strengthens the importance of this research project, which was carried out before reporting the in vitro activity of chiral phthalimides. The chiral phthalimides were initially docked against the PBP2a protein of MRSA to report their in silico antibacterial activity. For this purpose, online computational databases such as NCBI and PubChem were used for retrieving the sequences of the target protein and the compounds, respectively. Once the active sites of PBP2a protein were determined using CASTp, docking of all the three chiral phthalimides was performed using PatchDock. Results generated through molecular docking reported that these compounds gave the best results by interacting with the active site of PBP2a through hydrogen bonds. Compound 1 (FIA) interacted with the amino acid ARG-151, compound 2 (FIB) interacted with the amino acid THR 216, and compound 3 (FIC) interacted with the amino acid THR 216 within the active site of PBP2a through hydrogen bonds. FIB also interacted with PBP2a through a 2nd hydrogen bond (LYS 218) which resides outside the active pocket of the target protein. A similar molecular coupling study was conducted in 2019 in the active pocket of the PBP2a protein of MRSA with the monomeric units of “Eudragit E-100 chloride” (EuCl) and “the sodium salt of poly(maleic acid-alt-octadecene)” (PAM18Na) polymers in both neutral and ionized forms as well as with antibiotic ampicillin. Results suggested that the monomeric forms of both the EuCl and PAM18Na polymers, as well as ampicillin antibiotic, showed interactions of different types and intensities [i.e., hydrophobic interactions (HIs), hydrogen bonds (HBs), and electrostatic interactions (EIs)] with the amino acids in the active site of PBP2a which, in turn, suggested antibacterial activities against the resistant strains of S. aureus (Liscano et al., 2021). Another docking study was conducted in 2016 for the synergistic effects of metronidazole–triazole hybrids with oxacillin against PBP2a of MRSA. The results of the docking studies and docking confirmations for these MTZ–triazole hybrids gave the best scores in the active pocket of PBP2a by interaction through hydrogen bonds as well as π–π interactions in the predicted binding pockets (Negi et al., 2016).

After getting the results for molecular docking, the in vitro antibacterial activity of the selected chiral phthalimides was checked against MRSA using standard protocols. Different zones of inhibition were obtained for FIA, FIB, and FIC, indicating positive outcomes for their antibacterial activity. A related study carried out in Egypt by Lamie et al. has reported the antibacterial and antifungal activities of the novel phthalimide derivative against the fungus (C. albicans), as well as Gram-positive and Gram-negative bacteria, i.e., Bacillus subtilis and P. aeruginosa, respectively, by performing the agar-well diffusion assay. Similarly, those compounds were characterized to be non-cytotoxic after completing the cytotoxicity assay in cancer and normal human cell lines (Lamie et al., 2015). From this study, it may be assumed that our synthesized chiral phthalimides (FIA, FIB, and FIC) may be non-toxic in normal therapeutic concentration ranges. Still, the cytotoxicity of these chiral phthalimides is recommended for further confirmation.

To further evaluate the antibacterial activity of these compounds, we performed the micro-broth dilution method (96-well plate method) and visual plate assay to identify the minimum inhibitory and minimum bactericidal concentrations of these chiral phthalimides. All the compounds showed excellent anti-MRSA activities, with FIB being the strongest anti-staphylococcal agent having an MIC of 0.022 ug (probably because of the structural differences in FIB and two covalent bonds formed with the PBP2a of MRSA in contrast to only one covalent bond in case of FIA and FIC) compared to FIA and FIC, which have MICs of 0.73 ug and 93 ug/mL of DMSO, respectively. In our study, the MIC and MBC values obtained for these chiral phthalimides were the same, meaning our compounds were bactericidal at the lowest concentration. This strengthens our research because bactericidal compounds are usually preferred over bacteriostatic compounds due to the complete elimination of the pathogenic agent from a particular site rather than just inhibiting its growth. A related study conducted in Bangladesh by Nayab et al. (2015) reported the antibacterial, antioxidant, and DNA-binding activities of the novel phthalimide derivatives, where significant antibacterial activity was shown by the novel N-substituted phthalimides against E. coli and S. mutans. Similarly, the chiral phthalimides used in our project along with some additional derivatives, i.e., FIB and FIC, as well as FIF: 2-(1,3-dioxoisoindolin-2yl)-3 mercaptopropionic acid, FIH: 2-(1,3-dioxoisoindolin-2yl)-3 (4-hydroxyphenyl prophanic acid, and FII: 3-(1,3-dioxoisoindolin-2yl) prophanic acid, have been reported to have good antibacterial activities against the MDR hypervirulent strain of Klebsiella pneumonia (hvKp) when the agar-well diffusion method and micro-broth dilution assay were performed in a similar study conducted at IPDM, Khyber Medical University. Similarly, these chiral phthalimides were reported to have no cytotoxic effects (non-cytotoxic) when their cytotoxicity assay was performed against human red blood cells (RBCs) (Khan et al., 2022).

As the results obtained during our research project align with the findings of similar studies conducted in the past, it confirms that, yes, the chiral phthalimides used against MRSA possess good antibacterial activities and could be a possible treatment option for the drug-resistant staphylococcal infections in future. Moreover, to our understanding, this is a novel study because no such studies have been explicitly conducted against S. aureus previously. These findings provide useful insights within the defined area of the investigation, despite the fact that the current study has a limited sample size of ten MRSA due to resource limitations. The findings of this study add to the existing body of information and have the potential to serve as a basis for additional research.

In conclusion, the chiral phthalimides exhibited antibacterial activities against multidrug-resistant MRSA and have the potential to be considered as alternative chemotherapeutics to treat the infections caused by MRSA after the confirmation of their cytotoxic and pharmacokinetic studies. The observed activities highlight their importance in the fight against antibiotic-resistant strains. Recognizing the need for a comprehensive assessment, future research will concentrate on cytotoxicity and pharmacokinetics. Confirming these aspects will help us better understand the safety and efficacy profiles of chiral phthalimides, positioning them as potential candidates for treating MRSA infections. This study lays the groundwork for further investigation into the full therapeutic potential of these compounds, providing a potential solution to the problems caused by MRSA infections.