The Role of Orientation of Surface Bound Dihydropyrrol-2-ones (DHP) on Biological Activity.

Quorum sensing (QS) signaling system is important for bacterial growth, adhesion, and biofilm formation resulting in numerous infectious diseases. Dihydropyrrol-2-ones (DHPs) represent a novel class of antimicrobial agents that inhibit QS, and are less prone to develop bacterial resistance due to their non-growth inhibition mechanism of action which does not cause survival pressure on bacteria. DHPs can prevent bacterial colonization and quorum sensing when covalently bound to substrates. In this study, the role of orientation of DHP compounds was investigated after covalent attachment by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling reaction to amine-functionalized glass surfaces via various positions of the DHP scaffold. The functionalized glass surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements and tested for their in vitro biological activity against S. aureus and P. aeruginosa. DHPs attached via the N-1 position resulted in the highest antibacterial activities against S. aureus, while no difference was observed for DHPs attached either via the N-1 position or the C-4 phenyl ring against P. aeruginosa.

The Mosaic of Rottlerin: The Sequel.

Rottlerin (1) is a potent protein kinase C δ inhibitor that possesses a wide range of biological activities. However, the potential of this molecule to be developed as a drug has been restricted by its limited availability. We report herein a gram scale quantity synthesis of rottlerin in a five-step synthetic route that can be completed within 2 days. The methodology was extended by the reaction of the key aminochromene intermediate (15) with various electron-rich arenes, forming novel unsymmetrical methylene-bridged compounds. The X-ray crystal structure revealed the boomerang shape of this kind of molecule for the first time. The direct transformation of rottlerin (1) into the natural product, isorottlerin (35), was observed for the first time, and we named this transformation the "isorottlerin change". In addition, the antibacterial activities of rottlerin (1) and new rottlerin analogues 32-34 were examined against Staphylococcus aureus. The compounds showed MIC values as low as 2.0 µM, which were comparable to the clinically used antibiotic gentamicin.

Dihydropyrrolones as bacterial quorum sensing inhibitors.

Bacteria regulate their pathogenicity and biofilm formation through quorum sensing (QS), which is an intercellular communication system mediated by the binding of signaling molecules to QS receptors such as LasR. In this study, a range of dihydropyrrolone (DHP) analogues were synthesized via the lactone-lactam conversion of lactone intermediates. The synthesized compounds were tested for their ability to inhibit QS, biofilm formation and bacterial growth of Pseudomonas aeruginosa. The compounds were also docked into a LasR crystal structure to rationalize the observed structure-activity relationships. The most active compound identified in this study was compound 9i, which showed 63.1% QS inhibition of at 31.25 µM and 60% biofilm reduction at 250 µM with only moderate toxicity towards bacterial cell growth.

Antibiofilm Nitric Oxide-Releasing Polydopamine Coatings.

The growing number of patient morbidity related to nosocomial infections has placed an importance on the development of new antibacterial coatings for medical devices. Here, we utilize the versatile adhesion property of polydopamine (pDA) to design an antibacterial coating that possesses low-fouling and nitric oxide (NO)-releasing capabilities. To demonstrate this, glass substrates were functionalized with pDA via immersion in alkaline aqueous solution containing dopamine, followed by grafting of low-fouling polymer (poly(ethylene glycol) (PEG)) via Michael addition and subsequent formation of N-diazeniumdiolate functionalities (NO precursors) by purging with NO gas. X-ray photoelectron spectroscopy confirmed the successful grafting of PEG and formation of N-diazeniumdiolate on polydopamine-coated substrates. NO release from the coating was observed over 2 days, and NO loading is tunable by the pDA film thickness. The antibacterial efficiency of the coatings was assessed using Gram-negative Pseudomonas aeruginosa (i.e., wild-type PAO1 and multidrug-resistant PA37) and Gram-positive Staphylococcus aureus (ATCC 29213). The NO-releasing PEGylated pDA film inhibited biofilm attachment by 96 and 70% after exposure to bacterial culture solution for 24 and 36 h, respectively. In contrast, films that do not contain NO failed to prevent biofilm formation on the surfaces at these time points. Furthermore, this coating also showed 99.9, 97, and 99% killing efficiencies against surface-attached PAO1, PA37, and S. aureus bacteria. Overall, the combination of low-fouling PEG and antibacterial activity of NO in pDA films makes this coating a potential therapeutic option to inhibit biofilm formation on medical devices.

Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa.

Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics.

Synthesis and photodynamic activities of integrin-targeting silicon(IV) phthalocyanine-cRGD conjugates.

A series of novel symmetric or unsymmetric silicon (IV) phthalocyanines axially substituted with cyclic Arg-Gly-Asp (cRGD) ligands through different ethylene glycol chains have been synthesized by a facile and mild "click" reaction. All the compounds show efficient photosensitizing activities in N,N-dimethylformamide, and are essentially non-aggregated in RPMI 1640 medium with 0.05% Cremophor EL. Owing to the presence of two cRGD ligands, the conjugate 6b exhibits highest selectivity toward αvß3+ HT-29 cells in photocytotoxicities. It shows higher cellular uptake and ROS generation efficiency toward the αvß3+ HT-29 cells compared with that of αvß3- MCF-7 cells. The competitive cellular uptake and subcellular localization indicate that 6b is internalized mainly through integrin-mediated endocytosis. In addition, the in vivo studies showed that 6b can mainly accumulate in tumor sites and show a significant PDT effect resulting in 75% tumor growth inhibition. The results indicate that 6b is a highly promising photosensitizer for targeted photodynamic therapy.

Design and Synthesis of Lactams Derived from Mucochloric and Mucobromic Acids as Pseudomonas aeruginosa Quorum Sensing Inhibitors.

Bacterial infections, particularly hospital-acquired infections caused by Pseudomonas aeruginosa, have become a global threat with a high mortality rate. Gram-negative bacteria including P. aeruginosa employ N-acyl homoserine lactones (AHLs) as chemical signals to regulate the expression of pathogenic phenotypes through a mechanism called quorum sensing (QS). Recently, strategies targeting bacterial behaviour or QS have received great attention due to their ability to disarm rather than kill pathogenic bacteria, which lowers the evolutionary burden on bacteria and the risk of resistance development. In the present study, we report the design and synthesis of N-alkyl- and N-aryl 3,4 dichloro- and 3,4-dibromopyrrole-2-one derivatives through the reductive amination of mucochloric and mucobromic acid with aliphatic and aromatic amines. The quorum sensing inhibition (QSI) activity of the synthesized compounds was determined against a P. aeruginosa MH602 reporter strain. The phenolic compounds exhibited the best activity with 80% and 75% QSI at 250 µM and were comparable in activity to the positive control compound Fu-30. Computational docking studies performed using the LasR receptor protein of P. aeruginosa suggested the importance of hydrogen bonding and hydrophobic interactions for QSI.

Exploiting the Versatility of Polydopamine-Coated Nanoparticles to Deliver Nitric Oxide and Combat Bacterial Biofilm.

In this study, an antimicrobial platform in the form of nitric oxide (NO) gas-releasing polydopamine (PDA)-coated iron oxide nanoparticles (IONPs) is developed for combating bacterial biofilms. NO is bound to the PDA-coated IONPs via the reaction between NO and the secondary amine moieties on PDA to form N-diazeniumdiolate (NONOate) functionality. To impart colloidal stability to the nanoparticles in aqueous solutions (e.g., phosphate buffered saline (PBS) and bacteria cell culture media M9), a polymer bearing hydrophilic and amine pendant groups, P(OEGMA)-b-P(ABA), is synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization and is subsequently grafted onto the PDA-coated IONPs by employing the Schiff base/Michael addition reaction between o-quinone and a primary amine. These nanoparticles are able to effectively disperse Pseudomonas aeruginosa biofilms (up to 79% dispersal) at submicromolar NO concentrations. In addition, the nanoparticles demonstrate excellent bactericidal activity toward P. aeruginosa planktonic and biofilm cells (up to 5-log10 reduction).

Glyoxylamide-based self-assembly hydrogels for sustained ciprofloxacin delivery.

In this study, we report the synthesis of glyoxylamide peptide-mimics as self-assembled gels with well-defined molecular structures for topical delivery of ciprofloxacin (CIP). The glyoxylamide peptide mimics successfully formed hydrogels with critical gel concentrations of 0.02-0.08% (w/v). The mechanical strength, secondary structure, and fiber morphology of these hydrogels can be modulated by varying the N-substituent of the ring-opened isatins. The synthesised hydrogel exhibited a high loading capacity of CIP (40% (w/w)) and a sustained release profile. The CIP-loaded hydrogels were able to release CIP for more than 15 days and the released solution was shown to retain activity against Gram-positive and Gram-negative bacteria. In addition, the hydrogels formed showed low toxicity against Cos7 cells.

Dual-Action Biomaterial Surfaces with Quorum Sensing Inhibitor and Nitric Oxide To Reduce Bacterial Colonization.

Bacterial biofilms on implanted medical devices are a serious problem. At present, no effective strategies are available and the emergence of multidrug resistance has highlighted the need to develop novel antibacterial coatings to combat device-related infections. One approach is to interfere with the bacterial communication pathway or quorum sensing (QS), which is responsible for biofilm formation and virulence factors, by incorporating QS inhibitors (QSIs) such as dihydropyrrolones (DHPs) on biomaterial surfaces. The endogenous biological signaling molecule nitric oxide (NO) is also a potential candidate for prevention of biomedical infections due to its antibiofilm activity. In this study, we have developed dual-action surface coatings based on DHPs and NO. X-ray photoelectron spectroscopy (XPS) and contact angle measurements confirmed successful immobilization of DHPs and NO, and the Griess assay revealed NO release from the coatings at 24 h. Bacterial colonization on the surfaces was assessed by confocal laser scanning microscopy (CLSM), where the DHP+NO surfaces demonstrated significantly higher efficacy in reducing colonization of Staphylococcus aureus and Pseudomonas aeruginosa via a nonbactericidal mechanism than the DHP or NO-releasing coatings alone. The excellent antibacterial activity of the novel coatings suggests the combination of DHP and NO has great potential to combat device-related bacterial infections.

Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents.

Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alternatives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many microbes. AMPs are part of the natural defenses of most living organisms and they also have a unique mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics were synthesized by incorporating the 3'-amino-[1,1'-biphenyl]-3-carboxylic acid backbone to mimic the essential properties of natural AMPs. By altering hydrophobicity and charge, we identified the most potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC = 15.6 µM) and Gram-negative Escherichia coli (MIC = 7.8 µM) bacteria. Cytoplasmic permeability assay results revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using tethered bilayer lipid membranes (tBLMs) and their activity against established biofilms of S. aureus and E. coli.

Biofilm dispersal using nitric oxide loaded nanoparticles fabricated by photo-PISA: influence of morphology.

Polymeric nanoparticles (NPs) of different morphologies (spheres and worms) were synthesized using a visible light mediated polymerization-induced self-assembly (PISA) approach. Spherical and worm-like NPs were subsequently modified to generate diazeniumdiolate functionalized NPs. Interestingly, the NO release rate and the dispersal of biofilms were found to strongly depend on the NP morphology. NPs with a higher aspect ratio (worms) exhibited a slower NO release rate and greater biofilm dispersal after 1 h of incubation.

Facile solvent-free fabrication of nitric oxide (NO)-releasing coatings for prevention of biofilm formation.

We describe a simple and solvent-free method to generate nitric oxide (NO)-releasing coatings by incorporating diazeniumdiolate (NONOate) into allylamine or diallylamine plasma polymer coatings. The resulting coatings demonstrate continuous release of NO for over 48 hours and are effective at reducing the adhesion and biofilm formation of medically-relevant Gram-negative and Gram-positive opportunistic pathogens.

Rational Design of Single-Chain Polymeric Nanoparticles That Kill Planktonic and Biofilm Bacteria.

Infections caused by multidrug-resistant bacteria are on the rise and, therefore, new antimicrobial agents are required to prevent the onset of a postantibiotic era. In this study, we develop new antimicrobial compounds in the form of single-chain polymeric nanoparticles (SCPNs) that exhibit excellent antimicrobial activity against Gram-negative bacteria (e.g., Pseudomonas aeruginosa) at micromolar concentrations (e.g., 1.4 µM) and remarkably kill ≥99.99% of both planktonic cells and biofilm within an hour. Linear random copolymers, which comprise oligoethylene glycol (OEG), hydrophobic, and amine groups, undergo self-folding in aqueous systems due to intramolecular hydrophobic interactions to yield these SCPNs. By systematically varying the hydrophobicity of the polymer, we can tune the extent of cell membrane wall disruption, which in turn governs the antimicrobial activity and rate of resistance acquisition in bacteria. We also show that the incorporation of OEG groups into the polymer design is essential in preventing complexation with proteins in biological medium, thereby maintaining the antimicrobial efficacy of the compound even in in vivo mimicking conditions. In comparison to the last-resort antibiotic colistin, our lead agents have a higher therapeutic index (by ca. 2-3 times) and hence better biocompatibility. We believe that the SCPNs developed here have potential for clinical applications and the information pertaining to their structure-activity relationship will be valuable toward the general design of synthetic antimicrobial (macro)molecules.

Synthesis and biological evaluation of N-naphthoyl-phenylglyoxamide-based small molecular antimicrobial peptide mimics as novel antimicrobial agents and biofilm inhibitors.

Antimicrobial peptides (AMPs) are a key component of the human immune system. Synthetic AMP mimics represent a novel strategy to counteract the increasing incidence of antimicrobial resistance. Here, we describe the synthesis of novel glyoxamide derivatives via ring-opening reactions of N-hexanoyl, N-benzoyl and N-naphthoylisatins with N,N-dimethylethane-1,2-diamine and N,N-dimethylpropane-1,3-diamine. These were converted to both the hydrochloric acid (HCl) or quaternary ammonium iodide (MeI) salts and their antibacterial activity against Staphylococcus aureus was investigated by their zone-of-inhibition and minimum inhibitory concentration (MIC). The HCl salt 22b exhibited the lowest MIC of 16 µg mL(-1), whereas the corresponding MeI salt 22c had a MIC of 39 µg mL(-1). We also investigated the in vitro toxicity of active compounds against the MRC-5 normal human lung fibroblasts and their activity against established biofilm in S. aureus.

Co-delivery of nitric oxide and antibiotic using polymeric nanoparticles.

The rise of hospital-acquired infections, also known as nosocomial infections, is a growing concern in intensive healthcare, causing the death of hundreds of thousands of patients and costing billions of dollars worldwide every year. In addition, a decrease in the effectiveness of antibiotics caused by the emergence of drug resistance in pathogens living in biofilm communities poses a significant threat to our health system. The development of new therapeutic agents is urgently needed to overcome this challenge. We have developed new dual action polymeric nanoparticles capable of storing nitric oxide, which can provoke dispersal of biofilms into an antibiotic susceptible planktonic form, together with the aminoglycoside gentamicin, capable of killing the bacteria. The novelty of this work lies in the attachment of NO-releasing moiety to an existing clinically used drug, gentamicin. The nanoparticles were found to release both agents simultaneously and demonstrated synergistic effects, reducing the viability of Pseudomonas aeruginosa biofilm and planktonic cultures by more than 90% and 95%, respectively, while treatments with antibiotic or nitric oxide alone resulted in less than 20% decrease in biofilm viability.

Hybrids of acylated homoserine lactone and nitric oxide donors as inhibitors of quorum sensing and virulence factors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen causing a variety of life-threatening diseases such as cystic fibrosis and nosocomial infections in burn victims. The ability of P. aeruginosa to cause infection is attributed to the production of virulence factors such as pyocyanin and elastases. These virulence factors are under the control of quorum sensing (QS) a cell to cell communication process controlled by small diffusible signalling molecules based on N-acyl-homoserine lactones (AHLs) known as autoinducers. The inhibition of QS and thereby virulence factors is seen as a potential new anti-infective strategy. Additionally, the role of nitric oxide (NO) in downstream processes in bacteria such as biofilm dispersal, motility, virulence and antimicrobial defence systems is gaining attention and could be used to control bacterial. Herein we report the design and synthesis of hybrid compounds based on AHL signalling molecules and NO donors as anti-infective agents. A series of AHL-NO hybrids were synthesised and potent inhibitors of QS and virulence factors of P. aeruginosa were identified. This research has led to conversion of agonist AHLs to antagonist AHLs with dual properties of QS inhibition and NO release.

Quorum sensing inhibitory activities of surface immobilized antibacterial dihydropyrrolones via click chemistry.

Device-related infection remains a major barrier to the use of biomaterial implants as life-saving devices. This study aims to examine the effectiveness and mechanism of action of surface attached dihydropyrrolones (DHPs), a quorum sensing (QS) inhibitor, against bacterial colonization. DHPs were covalently attached on glass surfaces via copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) click reaction. The covalent attachment of DHP surfaces was confirmed by X-ray photoelectron spectroscopy (XPS) and contact angle measurements, and the antimicrobial efficacy of the DHP coatings was assessed by confocal laser scanning microscopy (CLSM) and image analysis. The results demonstrated that covalently bound DHP compounds are effective in reducing the adhesion by up to 97% (p < 0.05) for both Pseudomonas aeruginosa and Staphylococcus aureus. Furthermore, using the green fluorescent protein (Gfp)-based reporter technology, it is demonstrated that surface attached DHPs were able to repress the expression of a lasB-gfp reporter fusion of P. aeruginosa by 72% (p < 0.001) without affecting cell viability. This demonstrates the ability of the covalently bound QS inhibitor to inhibit QS and suggests the existence of a membrane-based pathway(s) for QS inhibition. Hence, strategies based on incorporation of QS inhibitors such as DHPs represent a potential approach for prevention of device-related infections.

Characterization of chemoselective surface attachment of the cationic peptide melimine and its effects on antimicrobial activity.

Antimicrobial peptides (AMPs) are promising alternatives to current treatments for bacterial infections. However, our understanding of the structural-functional relationship of tethered AMPs still requires further investigation to establish a general approach for obtaining consistent antimicrobial surfaces. In this study, we have systematically examined the effects of surface orientation of a broad-spectrum synthetic cationic peptide, melimine, on its antibacterial activity against Gram-positive and Gram-negative bacteria. The attachment of melimine to maleimide-functionalized glass was facilitated by addition of a single cysteine amino acid into the peptide sequence at the N-terminus (CysN) or C-terminus (CysC), or at position 13 (Cys13, approximately central). The successful attachment of the modified melimine was monitored using X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principle component analysis. The ToF-SIMS analysis clearly demonstrated structural difference between the three orientations. The peptide density for the modified surfaces was found to be between 3.5-4.0×10(-9)molcm(-2) using a modified Bradford assay. The ability of the surfaces to resist Pseudomonas aeruginosa and Staphylococcus aureus colonization was compared using fluorescence confocal microscopy. Reductions in total P. aeruginosa and S. aureus adhesion of 70% (p<0.001) and 83% (p<0.001), respectively, after 48h were observed for the melimine samples when compared to the blank control. We found that melimine attached via the N-terminus was the most effective in reducing total bacterial adhesion and bacterial viability with two- and four times (p<0.001) more activity than melimine attached via the C-terminus for P. aeruginosa and S. aureus, respectively. Furthermore, for Cys13, despite having the highest measured peptide density of the three surfaces, the higher concentration did not confer the greatest antibacterial effect. This highlights the importance of orientation of the peptides on the surface to efficacy. Our results suggest that the optimal orientation of the cationic residues is essential for maximum surface activity, whereby the optimal activity is obtained when the cationic portion is more available to interact with colonizing bacteria.

Characterisation and in vitro activities of surface attached dihydropyrrol-2-ones against Gram-negative and Gram-positive bacteria.

Bacterial infection of biomedical devices is still a major barrier to their use. This is compounded by increasing antibiotic resistance. Here, the specific covalent attachment of a series of dihydropyrrol-2-one (DHP), analogues of bacterial quorum sensing inhibitors, to surfaces via a Michael-type addition reaction is described. Differences in efficiency of attachment related to the substituent groups were found by X-ray photoelectron spectroscopy. The physical characteristics of the surfaces were further explored by atomic force microscopy and contact angle measurements. The ability of these coatings to prevent the formation of a biofilm by Pseudomonas aeruginosa and Staphylococcus aureus was examined using confocal laser scanning microscopy and image analysis. The DHP-treated surfaces showed significant reductions in bacterial adhesion without increased killing for both strains of bacteria (p < 0.001). 5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one was identified as having broad spectrum activity and consequently represents an excellent candidate for the development of novel surfaces for the prevention of biomedical device infections.