Efflux Pump Inhibitors Research Articles

Enhancing Antibacterial Efficacy: Combining Novel Bacterial Topoisomerase Inhibitors with Efflux Pump Inhibitors and Other Agents Against Gram-Negative Bacteria

Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs’ minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections.

Biofilm formation and polar lipid biosynthesis in Mycobacterium abscessus are inhibited by naphthylmethylpiperazine

Mycobacterium abscessus is a biofilm-forming, non-tuberculous mycobacterium that is highly resistant to antibiotics. Bacterial efflux pumps contribute to biofilm formation, export of biofilm-associated lipids and antibiotic tolerance. The Resistance Nodulation Cell Division (RND) and ATP-Binding Cassette (ABC) families of efflux pumps export lipids to the mycobacterial cell surface. 1-(1-naphthyl methyl)-piperazine (NMP) is a chemosensitizer that causes membrane destabilization and is an inhibitor of RND efflux pumps. The effects of NMP on biofilm formation and lipid metabolism in M. abscessus biofilms have not been investigated. Plumbagin (PLU) is an inhibitor of ABC efflux pumps that has not been studied for its effects on antibiotic tolerance in M. abscessus biofilms. In this study, we report that the efflux pump inhibitors NMP and PLU inhibit biofilm formation by 50% at sub-MIC levels. We show that NMP inhibits the incorporation of the radiolabeled long-chain fatty acid 14C-palmitate into glycopeptidolipids in cell surface lipids of log-phase M. abscessus. NMP also inhibits the utilization of the radiolabel in the biosynthesis of phosphatidylethanolamine in the cell surface and cellular lipids of M. abscessus cells in log-phase and in biofilms. Incorporation of the radiolabel into cardiolipin in the cellular lipids of M. abscessus biofilms was inhibited by NMP. The incorporation of 14C-acetate into cell surface phosphatidylethanolamine in log-phase and biofilm cells was also inhibited by NMP. Triacylglycerol biosynthesis using 14C-palmitate and 14C-acetate in cellular lipids of log-phase and biofilm cells was increased several folds by NMP. Efflux pump activity in M. abscessus cells was inhibited by 97% and 68% by NMP and PLU respectively. NMP and PLU caused 5-fold decreases in the minimum inhibitory concentrations of ciprofloxacin and clarithromycin against M. abscessus. Our results demonstrate that NMP and PLU affect important physiological processes in M. abscessus associated with its pathogenesis.

Identifying how drug efflux mechanisms impact Acinetobacter baumannii evolutionary paths to ciprofloxacin resistance

Acinetobacter baumannii is a multi-drug resistant pathogen commonly found in clinical settings. This pathogen frequently uses efflux pumps to mitigate antibiotic treatment stress and eliminate the drug. When A. baumannii is exposed to antibiotics, it often develops mutations in the efflux pump regulator genes, causing an increase in efflux pump production. We hypothesize that efflux is a key pathway that leads to treatment failure in A. baumannii infections. The extent to which increasing drug efflux impacts other cellular functions remains unknown. To identify how efflux pump mutations impact growth, resistance, and evolvability, wildtype A. baumannii laboratory strain 17978UN, along with four mutants of this strain, each with a single nucleotide polymorphism (SNP) in an efflux pump regulator (adeL L341R, adeN I49N, adeR D23Y, and adeS R152S), were propagated in the presence of antibiotic and an efflux pump inhibitor to place selective pressure on the isolates. SNPs increase the production of efflux pumps; inhibiting efflux ability will determine if the effect of each SNP is nullified. All evolved populations demonstrated differences in fitness and antibiotic resistance in comparison to their respective ancestors; the extent of adaptation affecting each phenotype was highly dependent on the regulator that was mutated. Counterintuitively, efflux inhibitors also placed stress on wild-type A. baumannii which leads to antibiotic resistance. These results demonstrate that efflux regulator mutations can influence population adaptability and cause treatment failure. Understanding the role that different efflux systems play in treatment failure and drug resistance evolution will be instrumental in developing treatment strategies that hinder the development of antibiotic resistance.

Ajoene: a natural compound with enhanced antimycobacterial and antibiofilm properties mediated by efflux pump modulation and ROS generation against M. Smegmatis.

Tuberculosis (TB) continues to be a primary worldwide health concern due to relatively ineffective treatments. The prolonged duration of conventional antibiotic therapy warrants innovative approaches to shorten treatment courses. In response to challenges, the study explores potential of Ajoene, a naturally occurring garlic extract-derived compound, for potential TB treatment. Mycobacterium smegmatis as a model organism for M. tuberculosis (M. tb) to investigate Ajoene's efficiency. In vitro techniques like antimicrobial susceptibility, antibiofilm, EtBr accumulation assay, and ROS assay evaluate the potency of Ajoene and conventional TB drugs against Mycobacterium smegmatis. An in-silico study also investigated the interaction between Ajoene and quorum-sensing proteins, specifically regX3, MSMEG_5244, and MSMEG_3944, which are involved in biofilm formation and sliding activity. In vitro findings revealed that Ajoene exhibited significant antibacterial activity by inhibiting growth and showing bactericidal effects. It also demonstrated additive interactions with common antibiotics such as Isoniazid and Rifampicin. Furthermore, Ajoene demonstrated a comparative interaction with commonly used antibiotics, such as Isoniazid and Rifampicin, and reduced M. smegmatis motility, both alone and in combination with these antibiotics. In silico analysis shows that Ajoene exhibited a higher binding affinity with regX3, a protein orthologous to the regX3 gene in M.tb. Ajoene also demonstrated consistent antibiofilm effects, particularly when combined synergistically with Isoniazid and Rifampicin. Mechanistic investigations demonstrated Ajoene's potential to inhibit efflux pumps and promote ROS generation in bacteria, suggesting a potential direct killing mechanism. Collectively, the findings emphasize Ajoene's effectiveness as a novel antimycobacterial and antibiofilm molecule for TB treatment.

Liposomal formulation with thiazolic compounds against bacterial efflux pumps

This study aimed to evaluate the effects of liposome-encapsulated eugenol-based thiazolic derivatives against efflux pump-carrying bacteria. The Minimum Inhibitory Concentration (MIC) was determined to evaluate the antibacterial activity and antibiotic potentiation against Pseudomonas aeruginosa and Staphylococcus aureus, as well as to analyze the inhibition of efflux pumps in S. aureus strains 1199B and K2068 in the ethidium bromide assay. The direct antibacterial activity analysis showed no clinically relevant results since the compounds presented MICs ≥1024 µg/mL. Regarding the analysis of antibiotic potentiation against multidrug-resistant (MDR) strains of S. aureus, compound LF16 reduced norfloxacin MIC from 128 µg/mL to 64 µg/mL. All associated with gentamicin caused a significant antibiotic MIC reduction. None of the compounds could potentate the activity of norfloxacin against P. aeruginosa. However, all of them potentiated the activity of gentamicin against the same strain. Only the LF 26 caused a significant MIC reduction in the ethidium bromide assay, suggesting efflux inhibition in the S. aureus 1199B strain. Similar results were observed with the K2068 strain. Observing antibiotic MIC reduction S. aureus strains carrying the NorA and MepA proteins brought additional evidence of efflux pump inhibition. Our results indicate that while eugenol-based thiazoles didn’t exhibit direct activity, they can potentiate the antibiotics activity against MDR strains of P. aeruginosa and S. aureus. Among them, compound LF 26 potentiated the inhibitory effects of ethidium bromide and antibiotics against S. aureus strains carrying the NorA and MepA proteins, indicating a potential role of this class of compounds as efflux pump inhibitors.

A Synergistic Interaction Between Citrus Hystrix Peel Essential Oil and Tetracycline and Evaluation of their Antibacterial Mechanism of Action in Vitro and in Silico Against Escherichia Coli.

Citrus hystrix essential oil (CHEO) have shown various pharmacological properties including antibacterial activity. This EO also possessed antibacterial effect against foodborne pathogens. There is less information available about the synergy interaction between CHEO and tetracycline, as well as their mechanism of action. Therefore, this study was conducted to evaluate the synergistic effect of CHEO and tetracycline against clinical isolate of Escherichia coli. Antibiofilm, bacteriolytic, and efflux pump inhibitor activities were also performed. The chemical composition of CHEO was analysed using GC-MS. Three major compounds, D-limonene (25.02 %), β-pinene (23.37 %), and β-sabinene (22.20 %) were identified. CHEO exhibited moderate antibacterial activity with MIC value of 250 μg/mL. The combination of CHEO (7.8 μg/mL) and tetracycline (62.5 μg/mL) produced a synergistic effect on E. coli with fractional inhibitory concentration index of 0.5. This mixture inhibited biofilm formation in E. coli. The combination of 7.8 μg/mL CHEO and 62.5 μg/mL tetracycline demonstrated bacteriolytic activity. In addition, the CHEO at 250 μg/mL showed a significant effect in inhibiting efflux pump. D-limonene has a binding free energy value of -20.13 kcal/mol with ompA transmembrane domain of E. coli. This finding indicates that CHEO has a potency to be developed as natural antibacterial against E. coli.

Efflux Pump Inhibitors as a Broad-Spectrum Strategy to Combat Multidrug Resistance Acenitobacter Baumanii

The World Health Organization (WHO) identifies A. baumannii as one of the most resistant pathogens. Over the past few years, there has been a significant rise in its resistance to a wide array of antibiotics. Multidrug resistance is attributed to several mechanisms, with efflux pumps being the most notable. Recently, the development of new treatment strategies to tackle A. baumannii infections has shown great promise. This review focuses on the various families of efflux pumps in A. baumannii and explores innovative therapeutic options that have emerged over the past four years. Specifically, we emphasize synthetic and natural efflux pump inhibitors (EPIs), nanoparticles, bacteriophage therapy, and antimicrobial peptides (AMPs) as part of these novel strategies.

In vitro and in silico effect of meldrum's acid-derived compounds on Staphylococcus aureus strains as NorA efflux pump inhibitors

The misuse of antibiotics has led to an alarming increase in bacterial strains resistant to these drugs. Efflux pumps, which expel antibiotics from bacterial cells, have emerged as one of the key mechanisms of bacterial resistance. In the quest to combat and mitigate bacterial resistance, researchers have turned their attention to efflux pump inhibitors as a potential solution. Meldrum's acid, a synthetic molecule widely utilized in the synthesis of bioactive compounds, has garnered significant interest in this regard. Hence, this study aims to investigate the antibacterial activity and evaluate the efficacy of three derivatives of meldrum's acid in inhibiting efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed through rigorous broth microdilution testing. While the derivatives themselves did not exhibit direct antibacterial activity, they demonstrated remarkable potential in potentiating the effects of antibiotics. Additionally, fluorescence emission assays using ethidium bromide (EtBr) revealed fluorescence levels comparable to the positive control, indicating a possible blockade of efflux pumps. Molecular docking studies conducted in silico further supported these findings by revealing binding interactions similar to norfloxacin and CCCP, known efflux pump inhibitors. These results underscore the potential of meldrum's acid derivatives as effective inhibitors of efflux pumps. By inhibiting these mechanisms, the derivatives hold promise in enhancing the effectiveness of antibiotics and combatting bacterial resistance. This study contributes valuable insights into the development of novel strategies to address the pressing issue of bacterial resistance and paves the way for further research and exploration in this field.

Guanethidine Restores Tetracycline Sensitivity in Multidrug-Resistant Escherichia coli Carrying tetA Gene.

The worrying issue of antibiotic resistance in pathogenic bacteria is aggravated by the scarcity of novel therapeutic agents. Antibiotic adjuvants offer a promising solution due to their cost-effectiveness and high efficacy in addressing this issue, such as the β-lactamase inhibitor sulbactam (a β-lactam adjuvant) and the dihydrofolate reductase inhibitor trimethoprim (a sulfonamide adjuvant). This study aimed to discover potential adjuvants for tetracyclines from a list of previously approved drugs to restore susceptibility to Escherichia coli carrying the tetA gene. We have screened guanethidine, a compound from the Chinese pharmacopoeia, which effectively potentiates the activity of tetracyclines by reversing resistance in tetA-positive Escherichia coli, enhancing its antibacterial potency, and retarding the development of resistance. Guanethidine functions via the inhibition of the TetA efflux pump, thereby increasing the intracellular concentration of tetracyclines. Our findings suggest that guanethidine holds promise as an antibiotic adjuvant.

Virtual screening, docking, molecular dynamics study of efflux pump inhibitors against Helicobacter pylori

Virtual screening, docking, molecular dynamics study of efflux pump inhibitors against Helicobacter pylori

Discovery of clinical isolation of drug-resistant Klebsiella pneumoniae with overexpression of OqxB efflux pump as the decisive drug resistance factor.

Background emergence of multidrug-resistant (MDR) bacterial strains is a public health concern that threatens global and regional security. Efflux pump-overexpressing MDR strains from clinical isolates are the best subjects for studying the mechanisms of MDR caused by bacterial efflux pumps. A Klebsiella pneumoniae strain overexpressing the OqxB-only efflux pump was screened from a clinical strain library to explore reverse OqxB-mediated bacterial resistance strategies. We identified non-repetitive clinical isolated K. pneumoniae strains using a matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry clinical TOF-II (Clin-TOF-II) and susceptibility test screening against levofloxacin and ciprofloxacin. And the polymorphism analysis was conducted using pulsed-field gel electrophoresis. Efflux pump function of resistant strains is obtained by combined drug sensitivity test of phenylalanine-arginine beta-naphthylamide (PaβN, an efflux pump inhibitor) and detection with ethidium bromide as an indicator. The quantitative reverse transcription PCR was performed to assess whether the oqxB gene was overexpressed in K. pneumoniae isolates. Additional analyses assessed whether the oqxB gene was overexpressed in K. pneumoniae isolates and gene knockout and complementation strains were constructed. The binding mode of PaβN with OqxB was determined using molecular docking modeling. Among the clinical quinolone-resistant K. pneumoniae strains, one mediates resistance almost exclusively through the overexpression of the resistance-nodulation-division efflux pump, OqxB. Crystal structure of OqxB has been reported recently by N. Bharatham, P. Bhowmik, M. Aoki, U. Okada et al. (Nat Commun 12:5400, 2021, https://doi.org/10.1038/s41467-021-25679-0). The discovery of this strain will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and builds on the foundation for addressing the threat posed by quinolone resistance.IMPORTANCEThe emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of K. pneumoniae infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the K. pneumoniae strain GN 172867 exhibited a higher oqxB gene expression compared to the reference strain ATCC 43816. Deletion of oqxB led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with oqxB rescued antibiotic resistance in the oqxB mutant strain. We demonstrated that the overexpression of the OqxB efflux pump plays an important role in quinolone resistance. The discovery of strain GN 172867 will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and promotes further study of antimicrobial resistance.

Deciphering the possible role of MmpL7 efflux pump in SQ109 resistance in Mycobacterium tuberculosis

BackgroundSQ109 is a promising candidate drug for the treatment of patients with drug-resistant tuberculosis (DR-TB). The purpose of this study was to investigate the activity of SQ109 against clinical isolates of Mycobacterium tuberculosis (MTB) from patients with multidrug-resistant TB (MDR-TB) and pre-extensively drug-resistant TB (pre-XDR-TB), and to explore new drug-resistant mechanisms of SQ109.MethodsWe evaluated the in vitro activity of SQ109 against clinical isolates from patients with MDR-TB and pre-XDR-TB using minimal inhibitory concentration (MIC) assay. The drug-resistant gene, mmpL3 of SQ109-resistant strains was sequenced, and a quantitative real-time PCR assay was used to analyze 28 efflux pump genes in SQ109-resistant strains without mmpL3 mutations. The role of candidate efflux pumps mmpL5 and mmpL7 on the MIC of SQ109 was evaluated using recombinantly cloned MmpL5 and MmpL7 expressed in Mycobacterium smegmatis.ResultsThe MIC90, MIC95 and MIC99 values of SQ109 for 225 clinical isolates of MTB were 0.25 mg/L, 0.5 mg/L and 1.0 mg/L, respectively. Among the pre-XDR strains, six showed resistance to SQ109 despite the absence of gene mutations in mmpL3. In six resistant pre-XDR strains, the MIC of SQ109 decreased with the use of an efflux pump inhibitor, and there was significant upregulation of mmpL5 and mmpL7 in two strains after exposure to SQ109. The presence of MmpL7 in Mycobacterium smegmatis resulted in decreased susceptibility to SQ109, with the MIC increasing from 16 mg/L to 32 mg/L.ConclusionsOur data demonstrated that SQ109 exhibited excellent levels of in vitro activity against MTB. MmpL7 may be a potential gene for MTB resistance to SQ109, providing a useful target for detecting SQ109 resistance in MTB.

Survival of infection with TEM β-lactamase-producing Escherichia coli with Pan-β-lactam resistance

Antimicrobial resistance is a critical global health issue, significantly contributing to patient mortality. Recent antibiotic developments have aimed to counteract carbapenemase-producing Enterobacterales; however, the impact of their use on the emergence of antibiotic resistance is unknown. This study investigates the first case of a non-carbapenemase-producing, pan-β-lactam-resistant Escherichia coli strain from a patient previously treated with ceftolozane-tazobactam and cefiderocol. This study describes the clinical progression of a 39-year-old ICU patient who developed multiple infections, culminating in the isolation of a pan-β-lactam-resistant E. coli strain (EC554). The resistance profile was characterised through MIC determination, whole-genome sequencing, the use of the β-lactam inactivation method, RT-qPCR, efflux pump inhibition assays, outer membrane protein analysis, and blaTEM transformation. The EC554 isolate displayed resistance to all tested β-lactams and β-lactam-β-lactamase inhibitor combinations. Whole-genome sequencing revealed four plasmids in EC554, with the only β-lactamase gene being blaTEM-252 on the pEC554-PBR-X1-X1 plasmid. We found that the extremely resistant phenotype was attributable to a combination of different mechanisms: a high expression of TEM-252, efflux pump activity, porin loss, and PBP3 mutations. The findings illustrate the complex interplay of multiple resistance mechanisms in E. coli, highlighting the potential for high-level resistance even without carbapenemase production. This study underscores the importance of comprehensively characterising resistance mechanisms in order to inform effective treatment strategies and mitigate the spread of resistant strains.

Stimuli-responsive gelatin-coated alginate nanocarriers: Targeted delivery of efflux pump inhibitor and antibacterial agents to control multidrug resistant P. aeruginosa

Multidrug resistance is an inescapable obstacle in healthcare settings. As the over-expression of efflux pumps is an important mediator of bacterial drug resistance, stimuli-responsive co-delivery nanosystems were developed for the sequential release of efflux pump inhibitors (EPI) and antibacterial agents. Gelatin-coated alginate nanocarriers (AlNCs@Gel) were developed containing ciprofloxacin (Cip) and quercetin (Que) as antibacterial/antibiofilm agents and EPI, respectively. Heat plot maps revealed a synergistic interaction of Cip with Que as the minimum inhibitory concentration value of Cip was reduced in the presence of EPI (quercetin) against P. aeruginosa. The encapsulation efficiency of 82 ± 1 % was observed for Cip while it was 70 ± 1 % for Que in Cip/AlNCs@Que/Gel nanocarriers. FTIR analyses confirmed an interaction between alginate and gelatin by the occurrence of a blue shift that is associated with electrostatic interactions between alginate and gelatin. These nanocarriers loaded with quercetin as EPI would release it preferentially in the presence of gelatinase enzyme and subsequently prevent the efflux of ciprofloxacin from bacterial cells. Membrane-permeability assay confirmed no permeabilization while viability studies exhibited superior bacterial inhibition, as Cip/EPI combination encapsulated in AlNCs@Gel effectively controlled the growth of P. aeruginosa as compared to antibiotic alone in AlNCs@Gel and free drug combinations. In vitro, cytotoxicity-analysis confirmed the biocompatibility of the developed core-shell nanocarriers. Furthermore, gelatin coating improved the physico-chemical properties of nanocarriers and the on-demand-release of EPI and antibiotic molecules. Thus, smartly designed AlNCs@Gel nanocarriers for the co-delivery of EPI and antibiotics present an innovative solution for preventing drug efflux from P. aeruginosa to control resistant infections.

Efflux pumps mediate changes to fundamental bacterial physiology via membrane potential.

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.

Short Communication: Novel Di- and Triselenoesters as Effective Therapeutic Agents Inhibiting Multidrug Resistance Proteins in Breast Cancer Cells.

Breast cancer has the highest incidence rate among all malignancies worldwide. Its high mortality is mainly related to the occurrence of multidrug resistance, which significantly limits therapeutic options. In this regard, there is an urgent need to develop compounds that would overcome this phenomenon. There are few reports in the literature that selenium compounds can modulate the activity of P-glycoprotein (MDR1). Therefore, we performed in silico studies and evaluated the effects of the novel selenoesters EDAG-1 and EDAG-8 on BCRP, MDR1, and MRP1 resistance proteins in MCF-7 and MDA-MB-231 breast cancer cells. The cytometric analysis showed that the tested compounds (especially EDAG-8) are inhibitors of BCRP, MDR1, and MRP1 efflux pumps (more potent than the reference compounds-novobiocin, verapamil, and MK-571). An in silico study correlates with these results, suggesting that the compound with the lowest binding energy to these transporters (EDAG-8) has a more favorable spatial structure affecting its anticancer activity, making it a promising candidate in the development of a novel anticancer agent for future breast cancer therapy.

Assessment In vitro and In silico of the Activity of Thiadiazines as NorA Efflux Pump Inhibitors.

Antimicrobials fight microorganisms, preventing and treating infectious diseases. However, antimicrobial resistance (AMR) is a growing concern due to the inappropriate and excessive use of these drugs. Several mechanisms can lead to resistance, including efflux pumps such as the NorA pump in Staphylococcus aureus, which reduces the effectiveness of fluoroquinolones. Thiadiazines are heterocyclic compounds whose chemical structure resembles that of cephalosporins. Therefore, these compounds and their derivatives have been studied for their potential in combating increased bacterial resistance. To analyze this hypothesis, direct activity assays, antibiotic action-modifying activity, fluorescence assays to evaluate the retention of ethidium bromide inside bacteria, and molecular docking were carried out. These experiments involved serial dilutions in microplates against Staphylococcus aureus strain 1199B under the influence of six thiadiazine derivatives (IJ10, IJ11, IJ21, IJ22, IJ23, and IJ25). The tests revealed that, despite not showing effective direct activity, some thiadiazine derivatives (IJ11, IJ21, and IJ22) inhibited the function of the bromide pump both in microdilution tests and in fluorescence and docking assays. Particularly, the IJ11 compound stood out for its activity similar to efflux inhibitors, as well as its inhibition of the norfloxacin pump of this bacterium. Among the results of this study, it deserves to be highlighted for anchoring future experiments, as it represents the first investigation of this group of thiadiazine derivatives against the NorA pump.

PLGA Nanoplatform for the Hypoxic Tumor Delivery: Folate Targeting, Therapy, and Ultrasound/Photoacoustic Imaging.

Effective targeting of breast tumors is critical for improving therapeutic outcomes in breast cancer treatment. Additionally, hypoxic breast cancers are difficult to treat due to resistance toward chemotherapeutics, poor vascularity, and enhanced angiogenesis, which complicate effective drug delivery and therapeutic response. Addressing this formidable challenge requires designing a drug delivery system capable of targeted delivery of the anticancer agent, inhibition of efflux pump, and suppression of the tumor angiogenesis. Here, we have introduced Palbociclib (PCB)-loaded PLGA nanoparticles (NPs) consisting of chitosan-folate (CS-FOL) for folate receptor-targeted breast cancer therapy. The developed NPs were below 219 nm with a smooth, spherical surface shape. The entrapment efficiencies of NPs were achieved up to 85.78 ± 1.8%. Targeted NPs demonstrated faster drug release at pH 5.5, which potentiated the therapeutic efficacy of NPs due to the acidic microenvironment of breast cancer. In vitro cellular uptake study in MCF-7 cells confirmed the receptor-mediated endocytosis of targeted NPs. In vivo ultrasound and photoacoustic imaging studies on rats with hypoxic breast cancer showed that targeted NPs significantly reduced tumor growth and hypoxic tumor volume, and suppressed angiogenesis.

Thiazine-derived compounds in inhibiting efflux pump in Staphylococcus aureus K2068, mepA gene expression, and membrane permeability alteration

Staphylococcus aureus is a bacterium responsible for resistance to multiple drugs and the efflux system is widely studied among the resistance mechanisms developed by this species. The present study evaluates the inhibition of the MepA efflux pump by thiadiazine-derived compounds. For this purpose, thiadiazine-derived compounds (IJ-14 to IJ-20) were tested against S. aureus K2068 strains. Microdilution tests were initially conducted to assess the Minimum Inhibitory Concentration (MIC) of the compounds and their efflux pump inhibition activity. In addition, fluorimetry tests were performed using BrEt emission and tests were conducted to inhibit the expression of the mepA gene. This involved comparing the bacterial gene expression with the antibiotic alone to the gene expression after combining compounds (IJ-17 and IJ-20) with the antibiotic. Furthermore, membrane permeability assessment tests and in silico molecular docking tests were performed. It was observed that the IJ17 and IJ20 compounds exhibited direct activity against the tested strain. The IJ17 compound produced significant results in the gene inhibition tests, which was also evidenced through the membrane permeability alteration test. These findings suggest that thiadiazine-derived compounds have promising effects against one of the main resistance mechanisms, with the IJ17 compound presenting observable mechanisms of action.

Bioflavonoid Baicalein Modulates Tetracycline Resistance by Inhibiting Efflux Pump in Staphylococcus aureus.

The rise in antibiotic resistance among bacterial pathogens, particularly Staphylococcus aureus, has become a critical global health issue, necessitating the search for novel antimicrobial agents. S. aureus uses various mechanisms to resist antibiotics, including the activation of efflux pumps, biofilm formation, and enzymatic modification of drugs. This study explores the potential of baicalein, a bioflavonoid from Scutellaria baicalensis, in modulating tetracycline resistance in S. aureus by inhibiting efflux pumps. The synergistic action of baicalein and tetracycline was evaluated through various assays. The minimum inhibitory concentration (MIC) of baicalein and tetracycline against S. aureus was 256 and 1.0 μg/mL, respectively. Baicalein at 64 μg/mL reduced the MIC of tetracycline by eightfold, indicating a synergistic effect (fractional inhibitory concentration index: 0.375). Time-kill kinetics demonstrated a 1.0 log CFU/mL reduction in bacterial count after 24 hours with the combination treatment. The ethidium bromide accumulation assay showed that baicalein mediated significant inhibition of efflux pumps, with a dose-dependent increase in fluorescence. In addition, baicalein inhibited DNA synthesis by 73% alone and 92% in combination with tetracycline. It also markedly reduced biofilm formation and the invasiveness of S. aureus into HeLa cells by 52% at 64 μg/mL. These findings suggest that baicalein enhances tetracycline efficacy and could be a promising adjunct therapy to combat multidrug-resistant S. aureus infections.