Synchrotron-based X-ray fluorescence microscopy reveals accumulation of polymyxins in single human alveolar epithelial cells.

Intravenous administration of the last-line polymyxins results in poor drug exposure in the lungs and potential nephrotoxicity; while inhalation therapy offers better pharmacokinetics/pharmacodynamics for pulmonary infections by delivering the antibiotic to the infection site directly. However, polymyxin inhalation therapy has not been optimized and adverse effects can occur. This study aimed to quantitatively determine the intracellular accumulation and distribution of polymyxins in single human alveolar epithelial A549 cells. Cells were treated with an iodine-labeled polymyxin probe FADDI-096 (5.0 and 10.0 µM) for 1, 4, and 24 h. Concentrations of FADDI-096 in single A549 cells were determined by synchrotron-based X-ray fluorescence microscopy. Concentration- and time-dependent accumulation of FADDI-096 within A549 cells was observed. The intracellular concentrations (mean ± SEM, n ≥ 189) of FADDI-096 were 1.58 ± 0.11, 2.25 ± 0.10, and 2.46 ± 0.07 mM following 1, 4 and 24 h of treatment at 10 µM, respectively. The corresponding intracellular concentrations following the treatment at 5 µM were 0.05 ± 0.01, 0.24 ± 0.04, and 0.25 ± 0.02 mM (n ≥ 189). FADDI-096 was mainly localized throughout the cytoplasm and nuclear region over 24 h. The intracellular zinc concentration increased in a concentration- and time-dependent manner. This is the first study to quantitatively map the accumulation of polymyxins in human alveolar epithelial cells and provides crucial insights for deciphering the mechanisms of their pulmonary toxicity. Importantly, our results may shed light on the optimization of inhaled polymyxins in patients and the development of new-generation safer polymyxins.

Simulations of octapeptin-outer membrane interactions reveal conformational flexibility is linked to antimicrobial potency.

The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure-activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins interacting with bacterial outer membrane (OM). Specifically, we examined the interaction of octapeptin C4 and FADDI-115, lacking a single hydroxyl group compared with octapeptin C4, with the lipid A-phosphoethanolamine modified OM of Acinetobacter baumannii Octapeptin C4 and FADDI-115 both penetrated into the OM hydrophobic center but experienced different conformational transitions from an unfolded to a folded state that was highly dependent on the structural flexibility of their respective N-terminal fatty acyl groups. The additional hydroxyl group present in the fatty acyl group of octapeptin C4 resulted in the molecule becoming trapped in a semifolded state, leading to a higher free energy barrier for OM penetration. The free energy barrier for the translocation through the OM hydrophobic layer was ∼72 kcal/mol for octapeptin C4 and 62 kcal/mol for FADDI-115. Our results help to explain the lower antimicrobial activity previously observed for octapeptin C4 compared with FADDI-115 and more broadly improve our understanding of the structure-function relationships of octapeptins. These findings may facilitate the discovery of next-generation octapeptins against polymyxin-resistant Gram-negative 'superbugs.'

Molecular dynamics simulations informed by membrane lipidomics reveal the structure-interaction relationship of polymyxins with the lipid A-based outer membrane of Acinetobacter baumannii.

BACKGROUND: MDR bacteria represent an urgent threat to human health globally. Polymyxins are a last-line therapy against life-threatening Gram-negative 'superbugs', including Acinetobacter baumannii. Polymyxins exert antimicrobial activity primarily via permeabilizing the bacterial outer membrane (OM); however, the mechanism of interaction between polymyxins and the OM remains unclear at the atomic level. METHODS: We constructed a lipid A-based OM model of A. baumannii using quantitative membrane lipidomics data and employed all-atom molecular dynamics simulations with umbrella sampling techniques to elucidate the structure-interaction relationship and thermodynamics governing the penetration of polymyxins [B1 and E1 (i.e. colistin A) representing the two clinically used polymyxins] into the OM. RESULTS: Polymyxin B1 and colistin A bound to the A. baumannii OM by the initial electrostatic interactions between the Dab residues of polymyxins and the phosphates of lipid A, competitively displacing the cations from the headgroup region of the OM. Both polymyxin B1 and colistin A formed a unique folded conformation upon approaching the hydrophobic centre of the OM, consistent with previous experimental observations. Polymyxin penetration induced reorientation of the headgroups of the OM lipids near the penetration site and caused local membrane disorganization, thereby significantly increasing membrane permeability and promoting the subsequent penetration of polymyxin molecules into the OM and periplasmic space. CONCLUSIONS: The thermodynamics governing the penetration of polymyxins through the outer leaflet of the A. baumannii OM were examined and novel structure-interaction relationship information was obtained at the atomic and membrane level. Our findings will facilitate the discovery of novel polymyxins against MDR Gram-negative pathogens.

Discovery of Novel Polymyxin-Like Antibiotics.

The antimicrobial lipopeptides polymyxin B and colistin (polymyxin E) are used as a 'last-line' therapy for infections caused by multidrug-resistant (MDR) Gram-negative pathogens. However, their effective use as antibiotic drugs in the clinical setting is still plagued by significant toxicity issues, in particular their potential for nephrotoxicity. Furthermore, resistance to the polymyxins has begun to emerge in the clinic, which implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative 'superbugs'. This chapter details our current understanding of polymyxin structure-activity relationships as well as recent pre-clinical and clinical drug development efforts aimed at generating new polymyxin antibiotics with improved safety and efficacy.

History, Chemistry and Antibacterial Spectrum.

Polymyxins are naturally occurring cyclic lipopeptides that were discovered more than 60 years ago. They have a narrow antibacterial spectrum, which is mainly against Gram-negative pathogens. The dry antibiotic pipeline, together with the increasing incidence of bacterial resistance in the clinic, has been dubbed 'the perfect storm'. This has forced a re-evaluation of 'old' antibiotics, in particular the polymyxins, which retain activity against many multidrug-resistant (MDR) Gram-negative organisms. As a consequence, polymyxin B and colistin (polymyxin E) are now used as the last therapeutic option for infections caused by 'superbugs' such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. This chapter covers the history, chemistry and antibacterial spectrum of these very important last-line lipopeptide antibiotics.

Methionine Ameliorates Polymyxin-Induced Nephrotoxicity by Attenuating Cellular Oxidative Stress.

Polymyxins are a last line of defense against multidrug-resistant Gram-negative pathogens. Recent pharmacological data show that intravenous polymyxins can cause nephrotoxicity in up to 60% of patients, and the plasma concentrations of polymyxins achieved with the currently recommended dosage regimens are suboptimal in a large proportion of patients. Simply increasing the daily dose of polymyxins is not possible due to nephrotoxicity. This study aimed to examine the protective effect of methionine against polymyxin-induced nephrotoxicity. Methionine (400 mg/kg of body weight), polymyxin B (35 mg/kg), a combination of methionine (100 or 400 mg/kg) and polymyxin B, and saline were administered to mice twice daily over 3.5 days. Kidneys were collected immediately at the end of the experiment for histological examination. The effect of methionine on the pharmacokinetics of polymyxin B was investigated in rats. The attenuation of polymyxin B (0.75 mM)-induced mitochondrial superoxide production by methionine (10.0 mM) was examined in rat kidney (NRK-52E) cells. Histological results revealed that the polymyxin-induced nephrotoxicity in mice was ameliorated by methionine in a dose-dependent manner. The methionine doses were well tolerated in the mice and rats, and the pharmacokinetics of polymyxin B in rats were not affected by methionine. In the group receiving polymyxin B-methionine, the total body clearance of polymyxin B was very similar to that in the group receiving polymyxin B alone (3.71 ± 0.57 versus 3.12 ± 1.66 ml/min/kg, P > 0.05). A substantial attenuation of polymyxin-induced mitochondrial superoxide production in NRK-52E cells was observed following pretreatment with methionine. Our results demonstrate that coadministration of methionine significantly ameliorated polymyxin-induced nephrotoxicity and decreased mitochondrial superoxide production in renal tubular cells.

Functional Characterization of the Unique Terminal Thioesterase Domain from Polymyxin Synthetase.

Polymyxins remain one of the few antibiotics available for treating antibiotic resistant bacteria. Here we describe polymyxin B thioesterase which performs the final step in polymyxin B biosynthesis. Isolated thioesterase catalyzed cyclization of an N-acetylcystamine polymyxin B analogue to form polymyxin B. The thioesterase contained a catalytic cysteine unlike most thioesterases which possess a serine. Supporting this, incubation of polymyxin B thioesterase with reducing agents abolished enzymatic activity, while mutation of the catalytic cysteine to serine significantly decreased activity. NMR spectroscopy demonstrated that uncyclized polymyxin B was disordered in solution, unlike other thioesterase substrates which adopt a transient structure similar to their product. Modeling showed the thioesterase substrate-binding cleft was highly negatively charged, suggesting a mechanism for the cyclization of the substrate. These studies provide new insights into the role of polymyxin thioesterase in polymyxin biosynthesis and highlight its potential use for the chemoenzymatic synthesis of polymyxin lipopeptides.

Rediscovering the octapeptins.

Covering: 1975 up to the end of 2016The decline in the discovery and development of novel antibiotics has resulted in the emergence of bacteria that are resistant to almost all available antibiotics. Currently, polymyxin B and E (colistin) are being used as the last-line therapy against life-threatening infections, unfortunately resistance to polymyxins in both the community and hospital setting is becoming more common. Octapeptins are structurally related non-ribosomal lipopeptide antibiotics that do not exhibit cross-resistance with polymyxins and have a broader spectrum of activity that includes Gram-positive bacteria. This makes them a precious and finite resource for the development of new antibiotics against these problematic polymyxin-resistant Gram-negative pathogens, in particular Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. This review surveys the progress in understanding octapeptin chemistry, mechanisms of antibacterial activity and biosynthesis. With the lack of cross-resistance and their broad antibacterial activity, the octapeptins represent ideal candidates for the development of a new generation of polymyxin-like lipopeptide antibiotics targeting polymyxin-resistant 'superbugs'.

The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity.

The first total synthesis of a polypeptin, PE2, as well as its solution structure is reported. Synthesis in optically pure form confirms the proposed stereochemistry of the polypeptins at the 3-position on the 3-hydroxy depsipeptide moiety. We have also determined the NMR structure of PE2 in aqueous solution, showing it to form a stable ring conformation. The synthetic peptide shows anti-bacterial activity consistent with reports for naturally derived counterparts.

Design and Evaluation of Novel Polymyxin Fluorescent Probes.

Polymyxins (polymyxin B and colistin) are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-negative "superbugs". In the present study, two novel fluorescent polymyxin probes were designed through regio-selective modifications of the polymyxin B core structure at the N-terminus and the hydrophobic motif at positions 6 and 7. The resulting probes, FADDI-285 and FADDI-286 demonstrated comparable antibacterial activity (MICs 2-8 mg/L) to polymyxin B and colistin (MICs 0.5-8 mg/L) against a panel of gram-negative clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. These probes should prove to be of considerable utility for imaging cellular uptake and mechanistic investigations of these important last-line antibiotics.

Human oligopeptide transporter 2 (PEPT2) mediates cellular uptake of polymyxins.

OBJECTIVES: Polymyxins are a last-line therapy to treat MDR Gram-negative bacterial infections. Nephrotoxicity is the dose-limiting factor for polymyxins and recent studies demonstrated significant accumulation of polymyxins in renal tubular cells. However, little is known about the mechanism of polymyxin uptake into these cells. Oligopeptide transporter 2 (PEPT2) is a solute carrier transporter (SLC) expressed at the apical membrane of renal proximal tubular cells and facilitates drug reabsorption in the kidney. In this study, we examined the role of PEPT2 in polymyxin uptake into renal tubular cells. METHODS: We investigated the inhibitory effects of colistin and polymyxin B on the substrate uptake mediated through 15 essential SLCs in overexpressing HEK293 cells. The inhibitory potency of both polymyxins on PEPT2-mediated substrate uptake was measured. Fluorescence imaging was employed to investigate PEPT2-mediated uptake of the polymyxin fluorescent probe MIPS-9541 and a transport assay was conducted with MIPS-9541 and [(3)H]polymyxin B1. RESULTS: Colistin and polymyxin B potently inhibited PEPT2-mediated [(3)H]glycyl-sarcosine uptake (IC50 11.4 ± 3.1 and 18.3 ± 4.2 µM, respectively). In contrast, they had no or only mild inhibitory effects on the transport activity of the other 14 SLCs evaluated. MIPS-9541 potently inhibited PEPT2-mediated [(3)H]glycyl-sarcosine uptake (IC50 15.9 µM) and is also a substrate of PEPT2 (Km 74.9 µM). [(3)H]polymyxin B1 was also significantly taken up by PEPT2-expressing cells (Km 87.3 µM). CONCLUSIONS: Our study provides the first evidence of PEPT2-mediated uptake of polymyxins and contributes to a better understanding of the accumulation of polymyxins in renal tubular cells.

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes.

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity.

Significant accumulation of polymyxin in single renal tubular cells: a medicinal chemistry and triple correlative microscopy approach.

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our approach represents a significant methodological advancement in determination of drug uptake for single-cell pharmacology.

Imaging the distribution of polymyxins in the kidney.

OBJECTIVES: Dose-limiting nephrotoxicity remains the Achilles' heel of polymyxin B and polymyxin E (also known as colistin), which are important last-line antibiotics used against infections caused by MDR Gram-negative 'superbugs'. An understanding of the mechanisms of nephrotoxicity, including renal tissue distribution, is crucial for the development of safer polymyxin lipopeptide antibiotics. This is the first study to visualize the kidney distribution of polymyxin B using a mouse nephrotoxicity model and in situ immunostaining of kidney sections. METHODS: Polymyxin B nephrotoxicity in mice was induced over the course of 3 days (accumulated intravenous dose 175 mg/kg) and kidneys were harvested and frozen sectioned. The sections were fixed in cold acetone, dried and treated with 1% hydrogen peroxide. Endogenous mouse immunoglobulins were blocked and the tissue sections were treated with anti-polymyxin B mouse IgM antibody. The sections were incubated with a biotinylated anti-mouse secondary antibody conjugate followed by an Alexa Fluor 647-streptavidin conjugate. Polymyxin B distribution in the kidney sections was then visualized using a fluorescence scanning microscope. Kidney sections were also subjected to haematoxylin and eosin staining to assess pathological damage from the polymyxin-induced nephrotoxicity. RESULTS: Immunostaining of kidney sections from a mouse with polymyxin B-induced nephrotoxicity revealed that polymyxin B distributed predominantly within the renal cortex. More specifically, polymyxin B accumulated within the proximal tubular cells. CONCLUSIONS: The observed accumulation of polymyxin B within proximal tubular cells is consistent with the extensive renal reabsorption of polymyxins and the likely cause of the associated nephrotoxicity.

Probing the penetration of antimicrobial polymyxin lipopeptides into gram-negative bacteria.

The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative 'superbugs' has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains.

Polymyxins and analogues bind to ribosomal RNA and interfere with eukaryotic translation in vitro.

Looking for targets: while the bactericidal activity of polymyxins is attributed to changes in membrane permeation, we show that these antibiotics can bind prokaryotic and eukaryotic A-sites, domains responsible for translational decoding. Polymyxin B, colistin and analogues also hinder eukaryotic translation in vitro. These new targets and effects might be partially responsible for the plethora of adverse effects by these potent bactericidal agents.

Transcriptomic Responses to Polymyxin B and Analogues in Human Kidney Tubular Cells.

Polymyxins are last-line antibiotics for the treatment of Gram-negative 'superbugs'. However, nephrotoxicity can occur in up to 60% of patients administered intravenous polymyxins. The mechanisms underpinning nephrotoxicity remain unclear. To understand polymyxin-induced nephrotoxicity, human renal proximal tubule cells were treated for 24 h with 0.1 mM polymyxin B or two new analogues, FADDI-251 or FADDI-287. Transcriptomic analysis was performed, and differentially expressed genes (DEGs) were identified using ANOVA (FDR < 0.2). Cell viability following treatment with polymyxin B, FADDI-251 or FADDI-287 was 66.0 ± 5.33%, 89.3 ± 3.96% and 90.4 ± 1.18%, respectively. Transcriptomics identified 430, 193 and 150 DEGs with polymyxin B, FADDI-251 and FADDI-287, respectively. Genes involved with metallothioneins and Toll-like receptor pathways were significantly perturbed by all polymyxins. Only polymyxin B induced perturbations in signal transduction, including FGFR2 and MAPK signaling. SIGNOR network analysis showed all treatments affected essential regulators in the immune system, autophagy, cell cycle, oxidative stress and apoptosis. All polymyxins caused significant perturbations of metal homeostasis and TLR signaling, while polymyxin B caused the most dramatic perturbations of the transcriptome. This study reveals the impact of polymyxin structure modifications on transcriptomic responses in human renal tubular cells and provides important information for designing safer new-generation polymyxins.

Critical Role of Position 10 Residue in the Polymyxin Antimicrobial Activity.

Polymyxins (polymyxin B and colistin) are lipopeptide antibiotics used as a last-line treatment for life-threatening multidrug-resistant (MDR) Gram-negative bacterial infections. Unfortunately, their clinical use has been affected by dose-limiting toxicity and increasing resistance. Structure-activity (SAR) and structure-toxicity (STR) relationships are paramount for the development of safer polymyxins, albeit very little is known about the role of the conserved position 10 threonine (Thr) residue in the polymyxin core scaffold. Here, we synthesized 30 novel analogues of polymyxin B1 modified explicitly at position 10 and examined the antimicrobial activity against Gram-negative bacteria and in vivo toxicity and performed molecular dynamics simulations with bacterial outer membranes. For the first time, this study revealed the stereochemical requirements and role of the ß-hydroxy side chain in promoting the correctly folded conformation of the polymyxin that drives outer membrane penetration and antibacterial activity. These findings provide essential information for developing safer and more efficacious new-generation polymyxin antibiotics.

Structure-Interaction Relationship of Polymyxins with Lung Surfactant.

Multidrug-resistant Gram-negative bacteria present an urgent and formidable threat to the global public health. Polymyxins have emerged as a last-resort therapy against these 'superbugs'; however, their efficacy against pulmonary infection is poor. In this study, we integrated chemical biology and molecular dynamics simulations to examine how the alveolar lung surfactant significantly reduces polymyxin antibacterial activity. We discovered that lung surfactant is a phospholipid-based permeability barrier against polymyxins, compromising their efficacy against target bacteria. Next, we unraveled the structure-interaction relationship between polymyxins and lung surfactant, elucidating the thermodynamics that govern the penetration of polymyxins through this critical surfactant layer. Moreover, we developed a novel analog, FADDI-235, which exhibited potent activity against Gram-negative bacteria, both in the presence and absence of lung surfactant. These findings shed new light on the sequestration mechanism of lung surfactant on polymyxins and importantly pave the way for the rational design of new-generation lipopeptide antibiotics to effectively treat Gram-negative bacterial pneumonia.

An Intelligent Strategy with All-Atom Molecular Dynamics Simulations for the Design of Lipopeptides against Multidrug-Resistant Pseudomonas aeruginosa.

Multidrug-resistant Gram-negative bacteria seriously threaten modern medicine due to the lack of efficacious therapeutic options. Their outer membrane (OM) is an essential protective fortress to exclude many antibiotics. Unfortunately, current structural biology methods are not able to resolve the membrane structure and it is difficult to examine the specific interaction between the OM and small molecules. These limitations hinder mechanistic understanding of antibiotic penetration through the OM and antibiotic discovery. Here, we developed biologically relevant OM models by quantitatively determining membrane lipidomics of Pseudomonas aeruginosa and elucidated how lipopolysaccharide modifications and OM vesicles mediated resistance to polymyxins. Supported by chemical biology and pharmacological assays, our multiscale molecular dynamics simulations provide an intelligent platform to quantify the membrane-penetrating thermodynamics of peptides and predict their antimicrobial activity. Through experimental validations with our in-house polymyxin analogue library, our computational strategy may have significant potential in accelerating the discovery of lipopeptides against bacterial "superbugs".