Potentiating the intracellular killing of Staphylococcus aureus by dihydroquinazoline analogues as NorA efflux pump inhibitor.

Staphylococcus aureus is an emerging human pathogen that has become difficult to treat due to its high resistance against wide range of drugs. Emergence of drug resistant isolates has further convoluted the treatment process. Among different resistance mechanisms, efflux pump proteins play a central role and has made itself a direct approach for therapeutic exploration. To demarcate the role of dihydroquinazoline analogues as NorA efflux pump inhibitor in S. aureus1199B (NorA over producing) strain total seventeen analogues were synthesized and tested for their modulatory effects on norfloxacin and Etbr resistance. Further accumulation assays, bacterial time kill kinetics, cytotoxicity assay were also carried out. The intracellular killing ability of analogues, as EPI was determined using THP-1 monocytes. The binding interaction of analogues with NorA was also predicted. Dihydroquinazoline analogues notably reduced the MIC of norfloxacin and Etbr in S. aureus1199B. In addition to their very low toxicity, they showed high Etbr and norfloxacin accumulation respectively. Further effective over time log reduction in bacterial kill kinetics in presence of these analogues confirmed their role as NorA efflux pump inhibitor. FESEM analysis clearly depicted their effect on the cell surface morphology owing to its lyses. The most significant finding of this study was the ability of analogues to significantly reduce the intracellular S. aureus1199B in human THP-1 monocytes in presence of norfloxacin. Our study has shown for the first time the possibility of developing the dihydroquinazoline analogues as NorA efflux pump inhibitors for S. aureus and control its infection.

Inhibition of the NorA efflux pump of S. aureus by (Z)-5-(4-Fluorobenzylidene)-Imidazolidines.

Searching for new alternatives to antibiotic treatments is crucial to surmount the multidrug-resistant bacteria. In this work, the antimicrobial activity of synthetic imidazolidines was evaluated as well as their modulating effect on the resistance to fluoroquinolones in a S. aureus strain (SA-1199B), which overexpresses the norA gene that encodes the NorA efflux pump. Results showed weak antimicrobial activity (512 µg mL-1) for two fluorobenzylidene derivatives against this bacterial strain, while the other benzylidene derivatives were inactive. Despite this fact, both fluorinated compounds were able to enhance the activity of norfloxacin and ciprofloxacin against SA-1199B up to 6.4- and 3.2-fold, respectively. In addition, both derivatives potentiated the action of ethidium bromide against this strain, suggesting that the modulating effect probably involves the inhibition of the NorA efflux pump, which is in concordance with the fluorimetic assays and molecular docking analyses performed in this work.

1,3,4-oxadiazole conjugates of capsaicin as potent NorA efflux pump inhibitors of Staphylococcus aureus.

NorA efflux pump pertaining to the major facilitator superfamily (MFS) is known to play a key role in antibiotic and biocide resistance in Staphylococcus aureus (S. aureus). It accounts for the extrusion of antibiotics like fluoroquinolones (e.g. ciprofloxacin). Several compounds including synthetic and natural products have been identified as potential NorA efflux pump inhibitors (EPIs) and found to restore the antibacterial activity of antibiotics. However, none of the reported EPIs have reached to clinical approval probably due to their high toxicity profiles. Considering the NorA efflux pump inhibitory potential of capsaicin, a series of capsaicin-based 1,3,4 oxadiazole conjugates were prepared and evaluated for ciprofloxacin activity potentiating effect. Among the new capsaicinoids tested, 17i displayed a minimum effective concentration (MEC) of 12.5 µg/mL against NorA overexpressing S. aureus strain (SA1199B), whereas capsaicin showed MEC of 50 µg/mL. The kill kinetics curve for the combination showed that ciprofloxacin at a sub-inhibitory concentration (0.25 × MIC) was equipotent in effect, to its MIC. 17i has significantly decreased the ethidium bromide efflux confirming NorA inhibition as the mode of action. Mutation prevention concentration of the ciprofloxacin was reduced in combination with 17i.In silico studies revealed the binding efficiency and binding affinity of 17i with NorA. This compound may serve as a template for the further drug discovery.

Synthesis, biological evaluation and computational studies of acrylohydrazide derivatives as potential Staphylococcus aureus NorA efflux pump inhibitors.

The NorA efflux pump decreases the intracellular concentration of fluoroquinolones (ciprofloxacin, norfloxacin) by effluxing them from Staphylococcus aureus cells. The synthesis of novel acrylohydrazide derivatives was achieved using well-known reactions and were characterized by various spectroscopy techniques. The synthesized 50 compounds were evaluated for the NorA efflux pump inhibition activity against S. aureus SA-1199B (norA++) and K1758 (norA-) strains. The study provided two most active compounds viz. 19 and 52. Compound 19 was found to be most active in potentiating effect of norfloxacin and also it showed enhanced uptake, efflux inhibition in ethidium bromide assay. Further compound 19 also enhanced post antibiotic effect and reduced mutation prevention concentration of norfloxacin. The homology modeling study was performed to elucidate three-dimensional structure of NorA. Docking studies of potent molecules were done to find the binding affinity and interaction with active site residues. Further, all the tested compounds exhibited good ADME and drug-likeness properties in- silico. Based on the in-silico studies and detailed in vitro studies, acrylohydrazides derivatives may be considered as potential NorA EPI candidates.

Synthesis of amides from (E)-3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid and substituted amino acid esters as NorA efflux pump inhibitors of Staphylococcus aureus.

Inhibitors for NorA efflux pump of Staphylococcus aureus have attracted the attention of many researchers towards the discovery and development of novel efflux pump inhibitors (EPIs). In an attempt to find specific potent inhibitors of NorA efflux pump of S. aureus, a total of 15 amino acid conjugates of 3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid (4-18) were synthesized using a simple convenient synthetic approach and bioevaluated against NorA efflux pump. Two compounds 7 and 8 (each having MEC of 1.56 µg/mL) were found to restore the activity of ciprofloxacin through reduction of the MIC elucidated by comparing the ethidium bromide efflux in dose dependent manner in addition to ethidium bromide efflux inhibition and accumulation study using NorA overexpressing strain SA-1199B. Most potent compounds among these were able to restore the antibacterial activity of ciprofloxacin completely against SA-1199B. Structure activity relationship (SAR) studies and docking study of potent compounds 7 and 8 could elucidate the structural requirements necessary for interaction with the NorA efflux pumps. On the whole, compounds 7 and 8 have ability to reverse the NorA efflux mediated resistance and could be further optimized for development of potent efflux pump inhibitors.

Deciphering the Molecular Recognition Mechanism of Multidrug Resistance Staphylococcus aureus NorA Efflux Pump Using a Supervised Molecular Dynamics Approach.

The use and misuse of antibiotics has resulted in critical conditions for drug-resistant bacteria emergency, accelerating the development of antimicrobial resistance (AMR). In this context, the co-administration of an antibiotic with a compound able to restore sufficient antibacterial activity may be a successful strategy. In particular, the identification of efflux pump inhibitors (EPIs) holds promise for new antibiotic resistance breakers (ARBs). Indeed, bacterial efflux pumps have a key role in AMR development; for instance, NorA efflux pump contributes to Staphylococcus aureus (S. aureus) resistance against fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from the cells. Even though NorA efflux pump is known to be a potential target for EPIs development, the absence of structural information about this protein and the little knowledge available on its mechanism of action have strongly hampered rational drug discovery efforts in this area. In the present work, we investigated at the molecular level the substrate recognition pathway of NorA through a Supervised Molecular Dynamics (SuMD) approach, using a NorA homology model. Specific amino acids were identified as playing a key role in the efflux pump-mediated extrusion of its substrate, paving the way for a deeper understanding of both the mechanisms of action and the inhibition of such efflux pumps.

Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA efflux pump inhibitors of Staphylococcus aureus.

A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1-C18) with α-, ß- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors.

Efflux pump inhibitory potential of indole derivatives as an arsenal against norA over-expressing Staphylococcus aureus.

NorA, an extensively studied efflux pump in Staphylococcus aureus, has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus. The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus. Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE The NorA efflux pump is the most effective resistance mechanism in S. aureus. The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus.

From Quinoline to Quinazoline-Based S. aureus NorA Efflux Pump Inhibitors by Coupling a Focused Scaffold Hopping Approach and a Pharmacophore Search.

Antibiotic resistance breakers, such as efflux pump inhibitors (EPIs), represent a powerful alternative to the development of new antimicrobials. Recently, by using previously described EPIs, we developed pharmacophore models able to identify inhibitors of NorA, the most studied efflux pump of Staphylococcus aureus. Herein we report the pharmacophore-based virtual screening of a library of new potential NorA EPIs generated by an in-silico scaffold hopping approach of the quinoline core. After chemical synthesis and biological evaluation of the best virtual hits, we found the quinazoline core as the best performing scaffold. Accordingly, we designed and synthesized a series of functionalized 2-arylquinazolines, which were further evaluated as NorA EPIs. Four of them exhibited a strong synergism with ciprofloxacin and a good inhibition of ethidium bromide efflux on resistant S. aureus strains coupled with low cytotoxicity against human cell lines, thus highlighting a promising safety profile.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Cuminum cyminum L. Essential Oil: A Promising Antibacterial and Antivirulence Agent Against Multidrug-Resistant Staphylococcus aureus.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 µL ⋅ mL-1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell-cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (-3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography-mass spectrometry analysis, cuminic aldehyde (38.26%), α,ß-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains.

The 1,8-naphthyridines sulfonamides are NorA efflux pump inhibitors.

OBJECTIVE: Efflux pumps are transmembrane proteins associated with bacterial resistance mechanisms. Bacteria use these proteins to actively transport antibiotics to the extracellular medium, preventing the pharmacological action of these drugs. This study aimed to evaluate in vitro the antibacterial activity of 1,8-naphthyridines sulfonamides, as well as their ability to inhibit efflux systems of Staphylococcus aureus strains expressing different levels of the NorA efflux pump. METHODS: The broth microdilution test was performed to assess antibacterial activity. Efflux pump inhibition was evaluated in silico by molecular docking and in vitro by fluorometric tests, and the minimum inhibitory concentration (MIC) was determined. The MIC was determined in the association between 1,8-naphthyridine and norfloxacin or ethidium bromide. RESULTS: The 1,8-naphthyridines did not show direct antibacterial activity. However, they effectively reduced the MIC of multidrug-resistant bacteria by associating with norfloxacin and ethidium bromide, in addition to increasing the fluorescence emission. In silico analysis addressing the binding between NorA and 1,8-naphthyridines suggests that hydrogen bonds and hydrophilic interactions represent the interactions with the most favourable binding energy, corroborating the experimental data. CONCLUSION: Our data suggest that 1,8-naphthyridines sulfonamides inhibit bacterial resistance through molecular mechanisms associated with inhibition of the NorA efflux pump in S. aureus strains.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

An Update on Staphylococcus aureus NorA Efflux Pump Inhibitors.

BACKGROUND: Methicillin-resistant and vancomycin-resistant Staphylococcus aureus are pathogens causing severe infectious diseases that pose real public health threats problems worldwide. In S. aureus, the most efficient multidrug-resistant system is the NorA efflux pump. For this reason, it is critical to identify efflux pump inhibitors. OBJECTIVE: In this paper, we present an update of the new natural and synthetic compounds that act as modulators of antibiotic resistance through the inhibition of the S. aureus NorA efflux pump. RESULTS: Several classes of compounds capable of restoring the antibiotic activity have been identified against resistant-S. aureus strains, acting as NorA efflux pump inhibitors. The most promising classes of compounds were quinolines, indoles, pyridines, phenols, and sulfur-containing heterocycles. However, the substantial degree structural diversity of these compounds makes it difficult to establish good structure- activity correlations that allow the design of compounds with more promising activities and properties. CONCLUSION: Despite substantial efforts put forth in the search for new antibiotic adjuvants that act as efflux pump inhibitors, and despite several promising results, there are currently no efflux pump inhibitors authorized for human or veterinary use, or in clinical trials. Unfortunately, it appears that infection control strategies have remained the same since the discovery of penicillin, and that most efforts remain focused on discovering new classes of antibiotics, rather than trying to prolong the life of available antibiotics, and simultaneously fighting mechanisms of bacterial resistance.

A Molecular Modeling Approach to Identify Novel Inhibitors of the Major Facilitator Superfamily of Efflux Pump Transporters.

Multidrug efflux systems play a prominent role in medicine, as they are important contributors to bacterial antibiotic resistance. NorA is an efflux pump transporter from the major facilitator superfamily that expels numerous drug compounds across the inner membrane of Staphylococcus aureus (S. aureus). The design of novel inhibitors to combat drug efflux could offer new opportunities to avoid the problem of antibiotic resistance. In this study, we performed molecular modeling studies in an effort to discover novel NorA efflux pump inhibitors. A group of over 673 compounds from the PubChem database with a high (>80%) level of similarity to the chemical structure of capsaicin was used to study the binding affinity of small molecule compounds for the NorA efflux pump. Ten potential lead compounds displayed a good druggability profile, with one in particular (CID 44330438) providing new insight into the molecular mechanism of the inhibition of major facilitator superfamily (MFS) efflux pump transporters. It is our hope that the overall strategy described in this study, and the structural information of the potential novel inhibitors thus identified, will stimulate others to pursue the development of better drugs to tackle multidrug resistance in S. aureus.

N,N'-disubstituted cinnamamide derivatives potentiate ciprofloxacin activity against overexpressing NorA efflux pump Staphylococcus aureus 1199B strains.

A multi-step procedure has been described which afforded satisfactory yields of N,N'-disubstituted cinnamamides derived from N-Boc-protected amino acids (Boc-Gly, Boc-Val, Boc-Phe). The key step of this synthesis was a regioselective RedAl reduction of an amide function in presence of a carbamate group. Next, these cinnamamides were evaluated in co-admnistration with ciprofloxacin as efflux pump inhibitors against two S. aureus strains, NorA overexpressing SA1199B and wild type SA1199. In parallel, their intrinsic toxicity was appreciated on human lung fibroblast MRC5 cells. Therefore, the cinnamamide combining both carbamate and indol-3-yl groups, was found to be the most active and one of the less toxic EPI and constituted a promising hit.

Studies on 2-phenylquinoline Staphylococcus aureus NorA efflux pump inhibitors: New insights on the C-6 position.

The alarming and rapid spread of antimicrobial resistance among bacteria represents a high risk for global health. Targeting factors involved in resistance to restore the activity of failing antibiotics is a promising strategy to overcome this urgent medical need. Efflux pump inhibitors are able to increase antibiotic concentrations in bacteria, thus they can be considered true antimicrobial resistance breakers. In this work, continuing our studies on inhibitors of the Staphylococcus aureus NorA pump, we designed, synthesized and biologically evaluated novel 2-phenylquinoline derivatives starting from our hits 1 and 2. Two of the synthesized compounds (6 and 7) bearing a C-6 benzyloxy group showed the best NorA inhibition activity, thereby providing an excellent starting point to direct future chemical optimizations.

Searching for Novel Inhibitors of the S.†aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues.

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA).

Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible.

Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus.

Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds.