An Arm-to-Disarm Strategy to Overcome Phenotypic AMR in Mycobacterium tuberculosis.

Most front-line tuberculosis drugs are ineffective against hypoxic non-replicating drug-tolerant Mycobacterium tuberculosis (Mtb) contributing to phenotypic antimicrobial resistance (AMR). This is largely due to the poor permeability in the thick and waxy cell wall of persister cells, leading to diminished drug accumulation and reduced drug-target engagement. Here, using an "arm-to-disarm" prodrug approach, we demonstrate that non-replicating Mtb persisters can be sensitized to Moxifloxacin (MXF), a front-line TB drug. We design and develop a series of nitroheteroaryl MXF prodrugs that are substrates for bacterial nitroreductases (NTR), a class of enzymes that are over-expressed in hypoxic Mtb. Enzymatic activation involves electron-transfer to the nitroheteroaryl compound followed by protonation via water that contributes to the rapid cleavage rate of the protective group by NTR to produce the active drug. Phenotypic and genotypic data are fully consistent with MXF-driven lethality of the prodrug in Mtb with the protective group being a relatively innocuous bystander. The prodrug increased intracellular concentrations of MXF than MXF alone and is more lethal than MXF in non-replicating persisters. Hence, arming drugs to improve permeability, accumulation and drug-target engagement is a new therapeutic paradigm to disarm phenotypic AMR.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Inhibiting respiration as a novel antibiotic strategy.

The approval of the first-in-class antibacterial bedaquiline for tuberculosis marks a breakthrough in antituberculosis drug development. The drug inhibits mycobacterial respiration and represents the validation of a wholly different metabolic process as a druggable target space. In this review, we discuss the advances in the development of mycobacterial respiratory inhibitors, as well as the potential of applying this strategy to other pathogens. The non-fermentative nature of mycobacteria explains their vulnerability to respiration inhibition, and we caution that this strategy may not be equally effective in other organisms. Conversely, we also showcase fundamental studies that reveal ancillary functions of the respiratory pathway, which are crucial to some pathogens' virulence, drug susceptibility and fitness, introducing another perspective of targeting bacterial respiration as an antibiotic strategy.

Rifaximin potentiates clarithromycin against Mycobacterium abscessus in vitro and in zebrafish.

Background: Mycobacterium abscessus is a non-tuberculous mycobacterium (NTM) that causes chronic pulmonary infections. Because of its extensive innate resistance to numerous antibiotics, treatment options are limited, often resulting in poor clinical outcomes. Current treatment regimens usually involve a combination of antibiotics, with clarithromycin being the cornerstone of NTM treatments. Objectives: To identify drug candidates that exhibit synergistic activity with clarithromycin against M. abscessus. Methods: We performed cell-based phenotypic screening of a compound library against M. abscessus induced to become resistant to clarithromycin. Furthermore, we evaluated the toxicity and efficacy of the top compound in a zebrafish embryo infection model. Results: The screen revealed rifaximin as a clarithromycin potentiator. The combination of rifaximin and clarithromycin was synergistic and bactericidal in vitro and potent in the zebrafish model. Conclusions: The data indicate that the rifaximin/clarithromycin combination is promising to effectively treat pulmonary NTM infections.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments.

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas.

Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species.

Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on the Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A noninvasive redox biosensor and a reactive oxygen species (ROS)-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and cotreatment with NAC potentiated the antimycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice, where NAC restricted the emergence of resistance. Thus, NADH-reductive stress contributes to moxifloxacin-mediated killing of M. tuberculosis, and the respiration stimulator (NAC) enhances lethality and suppresses the emergence of drug resistance.

Mycobactin Analogues with Excellent Pharmacokinetic Profile Demonstrate Potent Antitubercular Specific Activity and Exceptional Efflux Pump Inhibition.

In this study, we have designed and synthesized pyrazoline analogues that partially mimic the structure of mycobactin, to address the requirement of novel therapeutics to tackle the emerging global challenge of antimicrobial resistance (AMR). Our investigation resulted in the identification of novel lead compounds 44 and 49 as potential mycobactin biosynthesis inhibitors against mycobacteria. Moreover, candidates efficiently eradicated intracellularly surviving mycobacteria. Thermofluorimetric analysis and molecular dynamics simulations suggested that compounds 44 and 49 bind to salicyl-AMP ligase (MbtA), a key enzyme in the mycobactin biosynthetic pathway. To the best of our knowledge, these are the first rationally designed mycobactin inhibitors to demonstrate an excellent in vivo pharmacokinetic profile. In addition, these compounds also exhibited more potent whole-cell efflux pump inhibition than known efflux pump inhibitors verapamil and chlorpromazine. Results from this study pave the way for the development of 3-(2-hydroxyphenyl)-5-(aryl)-pyrazolines as a new weapon against superbug-associated AMR challenges.

Mevo lectin specificity toward high-mannose structures with terminal αMan(1,2)αMan residues and its implication to inhibition of the entry of Mycobacterium tuberculosis into macrophages.

Mannose-binding lectins can specifically recognize and bind complex glycan structures on pathogens and have potential as antiviral and antibacterial agents. We previously reported the structure of a lectin from an archaeal species, Mevo lectin, which has specificity toward terminal α1,2 linked manno-oligosaccharides. Mycobacterium tuberculosis expresses mannosylated structures including lipoarabinomannan (ManLAM) on its surface and exploits C-type lectins to gain entry into the host cells. ManLAM structure has mannose capping with terminal αMan(1,2)αMan residues and is important for recognition by innate immune cells. Here, we aim to address the specificity of Mevo lectin toward high-mannose type glycans with terminal αMan(1,2)αMan residues and its effect on M. tuberculosis internalization by macrophages. Isothermal titration calorimetry studies demonstrated that Mevo lectin shows preferential binding toward manno-oligosaccharides with terminal αMan(1,2)αMan structures and showed a strong affinity for ManLAM, whereas it binds weakly to Mycobacterium smegmatis lipoarabinomannan, which displays relatively fewer and shorter mannosyl caps. Crystal structure of Mevo lectin complexed with a Man7D1 revealed the multivalent cross-linking interaction, which explains avidity-based high-affinity for these ligands when compared to previously studied manno-oligosaccharides lacking the specific termini. Functional studies suggest that M. tuberculosis internalization by the macrophage was impaired by binding of Mevo lectin to ManLAM present on the surface of M. tuberculosis. Selectivity shown by Mevo lectin toward glycans with terminal αMan(1,2)αMan structures, and its ability to compromise the internalization of M. tuberculosis  in vitro, underscore the potential utility of Mevo lectin as a research tool to study host-pathogen interactions.

14-Residue peptaibol velutibol A from Trichoderma velutinum: its structural and cytotoxic evaluation.

Velutibol A (1), a new 14-residue peptaibol was isolated from the Himalayan cold habitat fungus Trichoderma velutinum. The structural characterization was carried out by 1D and 2D NMR studies, and tandem mass studies, and Marfey's method aided in determining the stereochemistry of the amino acids. The CD analysis revealed folding of the peptide in a 310-helical conformation. The intramolecular H-bonding was determined by an NMR-VT experiment. Cytotoxic evaluation was carried out against a panel of cancer cell lines. The cell cycle assay was carried out on human myeloid leukaemia (HL-60) cells and revealed the formation of apoptotic bodies and DNA damage in a dose-dependent manner. Three other peptaibols namely velutibol B (2), velutibol C (3), and velutibol D (4) were also isolated in trace amounts from the psychotropic fungus and characterized through tandem mass spectroscopy and Marfey's analysis.

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.

Physicochemical, pharmacokinetic, efficacy and toxicity profiling of a potential nitrofuranyl methyl piperazine derivative IIIM-MCD-211 for oral tuberculosis therapy via in-silico-in-vitro-in-vivo approach.

Recent tuberculosis (TB) drug discovery programme involve continuous pursuit for new chemical entity (NCE) which can be not only effective against both susceptible and resistant strains of Mycobacterium tuberculosis (Mtb) but also safe and faster acting with the target, thereby shortening the prolonged TB treatments. We have identified a potential nitrofuranyl methyl piperazine derivative, IIIM-MCD-211 as new antitubercular agent with minimum inhibitory concentration (MIC) value of 0.0072 µM against H37Rv strain. Objective of the present study is to investigate physicochemical, pharmacokinetic, efficacy and toxicity profile using in-silico, in-vitro and in-vivo model in comprehensive manner to assess the likelihood of developing IIIM-MCD-211 as a clinical candidate. Results of computational prediction reveal that compound does not violate Lipinski's, Veber's and Jorgensen's rule linked with drug like properties and oral bioavailability. Experimentally, IIIM-MCD-211 exhibits excellent lipophilicity that is optimal for oral administration. IIIM-MCD-211 displays evidence of P-glycoprotein (P-gp) induction but no inhibition ability in rhodamine cell exclusion assay. IIIM-MCD-211 shows high permeability and plasma protein binding based on parallel artificial membrane permeability assay (PAMPA) and rapid equilibrium dialysis (RED) assay model, respectively. IIIM-MCD-211 has adequate metabolic stability in rat liver microsomes (RLM) and favourable pharmacokinetics with admirable correlation during dose escalation study in Swiss mice. IIIM-MCD-211 has capability to appear into highly perfusable tissues. IIIM-MCD-211 is able to actively prevent progression of TB infection in chronic infection mice model. IIIM-MCD-211 shows no substantial cytotoxicity in HepG2 cell line. In acute toxicity study, significant increase of total white blood cell (WBC) count in treatment group as compared to control group is observed. Overall, amenable preclinical data make IIIM-MCD-211 ideal candidate for further development of oral anti-TB agent.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 µM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

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.

Identification and characterization of novel small molecule inhibitors of the acetyltransferase activity of Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU).

This study aims at identifying novel chemical scaffolds as inhibitors specific to the acetyltransferase domain of a bifunctional enzyme, Escherichia coli GlmU, involved in the cell wall biosynthesis of Gram-negative organisms. A two-pronged approach was used to screen a 50,000 small-molecule library. Using the first approach, the library was in silico screened by docking the library against acetyltransferase domain of E. coli GlmU studies. In the second approach, complete library was screened against Escherichia coli ATCC 25922 to identify the whole cell active compounds. Active compounds from both the screens were screened in a colorimetric absorbance-based assay to identify inhibitors of acetyltransferase domain of E. coli GlmU which resulted in the identification of 1 inhibitor out of 56 hits identified by in silico screening and 4 inhibitors out of 35 whole cell active compounds on Gram-negative bacteria with the most potent inhibitor showing IC50 of 1.40 ± 0.69 µM. Mode of inhibition studies revealed these inhibitors to be competitive with AcCoA and uncompetitive with GlcN-1-P. These selected inhibitors were also tested for their antibacterial and cytotoxic activities. Compounds 5175178 and 5215319 exhibited antibacterial activity that co-related with GlmU inhibition. These compounds, therefore, represent novel chemical scaffolds targeting acetyltransferase activity of E. coli GlmU.

Nitrofuranyl Methyl Piperazines as New Anti-TB Agents: Identification, Validation, Medicinal Chemistry, and PK Studies.

Whole-cell screening of 20,000 drug-like small molecules led to the identification of nitrofuranyl methylpiperazines as potent anti-TB agents. In the present study, validation followed by medicinal chemistry has been used to explore the structure-activity relationship. Ten compounds demonstrated potent MIC in the range of 0.17-0.0072 µM against H37Rv Mycobacterium tuberculosis (MTB) and were further investigated against nonreplicating and resistant (Rif(R) and MDR) strains of MTB. These compounds were also tested for cytotoxicity. Among the 10 tested compounds, five showed submicromolar to nanomolar potency against nonreplicating and resistant (Rif(R) and MDR) strains of MTB along with a good safety index. Based on their overall in vitro profiles, the solubility and pharmacokinetic properties of five potent compounds were studied, and two analogues, 14f and 16g, were found to have comparatively better solubility than others tested and acceptable pharmacokinetic properties. This study presents the rediscovery of a nitrofuranyl class of compounds with improved aqueous solubility and acceptable oral PK properties, opening a new direction for further development.

Synthesis and Biological Evaluation of Polar Functionalities Containing Nitrodihydroimidazooxazoles as Anti-TB Agents.

Novel polar functionalities containing 6-nitro-2,3-dihydroimidazooxazole (NHIO) analogues were synthesized to produce a compound with enhanced solubility. Polar functionalities including sulfonyl, uridyl, and thiouridyl-bearing NHIO analogues were synthesized and evaluated against Mycobacterium tuberculosis (MTB) H37Rv. The aqueous solubility of compounds with MIC values ≤0.5 µg/mL were tested, and six compounds showed enhanced aqueous solubility. The best six compounds were further tested against resistant (Rif(R) and MDR) and dormant strains of MTB and tested for cytotoxicity in HepG2 cell line. Based on its overall in vitro characteristics and solubility profile, compound 6d was further shown to possess high microsomal stability, solubility under all tested biological conditions (PBS, SGF and SIF), and favorable oral in vivo pharmacokinetics and in vivo efficacy.

Evaluation of functional properties of composite flours and sensorial attributes of composite flour biscuits.

The present study was undertaken to develop biscuits from the composite flours. Composite flours were prepared by blending wheat flour with rice flour, green gram flour and potato flour in ratios of 100:0:0:0 (W100), 85:5:5:5 (W85), 70:10:10:10 (W70) and 55:15:15:15 (W55), respectively. The functional properties of composite flours such as swelling capacity, water absorption capacity, oil absorption capacity, emulsion activity, emulsion stability, foam capacity, foam stability, gelatinization temperature, least gelation concentration and bulk density were increased with increase in the incorporation of other flours with wheat flour. Overall acceptability for composite flour biscuits was awarded highest score for W55 followed by W70 and W85 as compared to control biscuits. All biscuits coincided in the range of 'like moderately' to 'like very much' for composite flours biscuits while 'like slightly' to like moderately' for control biscuits.

Synthesis, antimalarial and antitubercular activities of meridianin derivatives.

Meridianins are marine-derived indole alkaloids, known to possess kinase inhibitory and antimalarial activities. A series of N-aryl and heteroaryl sulfonamide derivatives of meridianins were prepared and screened for antimalarial activity against D6 and W2 strains of Plasmodium falciparum. 2-Nitro-4-trifluoromethyl sulfonamide derivative 14v displayed promising antiplasmodial activity against both strains with IC50 values of 2.56 and 3.41 µM, respectively. These compounds were not cytotoxic to mammalian cell lines including VERO (monkey kidney fibroblasts), LLC-PK1 (pig kidney epithelial cells) and four cancer cell lines; SK-MEL (human malignant, melanoma), KB (human epidermal carcinoma), BT-549 (ductal carcinoma), SK-OV-3 (human ovary carcinoma) up to 25 µg/ml. Furthermore, all sulfonamide derivatives along with acyl, alkyl and C-ring modified derivatives of meridianins were screened for antitubercular activity against a sensitive strain (H37Rv) of Mycobacterium tuberculosis (Mtb), wherein several compounds showed MIC values in the range of 5.2-304.8 µM. Meridianin C (3) and meridianin G (7) showed anti-tubercular activity with MIC values of 111.1 and 304.8 µM, respectively. The C-ring modified analog 12 exhibited potent anti-tubercular activity against H37Rv strain of Mtb with MIC of 5.2 µM. Furthermore, the most potent analogs 11b and 12 were screened against two clinical isolates of M. tuberculosis INH(R) and MDR and one laboratory generated mutant strain Rif(R). These two analogs 11b and 12 displayed promising activity against these resistant strains with MIC values in the range of 5.2-187.7 µM. This is the first report on the anti-tubercular activity of this scaffold.

Discovery of 4-acetyl-3-(4-fluorophenyl)-1-(p-tolyl)-5-methylpyrrole as a dual inhibitor of human P-glycoprotein and Staphylococcus aureus Nor A efflux pump.

Polysubstituted pyrrole natural products, lamellarins, are known to overcome multi-drug resistance in cancer via the inhibition of p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) efflux pumps. Herein, a series of simplified polysubstituted pyrroles, prepared via a one-pot domino protocol, were screened for P-gp inhibition in P-gp overexpressing human adenocarcinoma LS-180 cells using a rhodamine 123 efflux assay. Several compounds showed the significant inhibition of P-gp at 50 µM, as indicated by increase in the intracellular accumulation of Rh123 in LS-180 cells. Furthermore, pyrrole 5i decreased the efflux of digoxin, a FDA approved P-gp substrate in MDCK-MDR1 cells with an IC50 of 11.2 µM. In in vivo studies, following the oral administration of a P-gp substrate drug, rifampicin, along with compound , the Cmax and AUC0-∞ of rifampicin was enhanced by 31% and 46%, respectively. All the compounds were then screened for their ability to potentiate ciprofloxacin activity via the inhibition of Staphylococcus aureus Nor A efflux pump. Pyrrole showed the significant inhibition of S. aureus Nor A efflux pump with 8- and 4-fold reductions in the MIC of ciprofloxacin at 50 and 6.25 µM, respectively. The molecular docking studies of compound with the human P-gp and S. aureus Nor A efflux pump identified its plausible binding site and key interactions. Thus, the results presented herein strongly indicate the potential of this scaffold for its use as multi-drug resistance reversal agent or bioavailability enhancer.