Design, Synthesis and Biological Evaluation of Novel Anthraniloyl-AMP Mimics as PQS Biosynthesis Inhibitors Against Pseudomonas aeruginosa Resistance.

The Pseudomonas quinolone system (PQS) is one of the three major interconnected quorum sensing signaling systems in Pseudomonas aeruginosa. The virulence factors PQS and HHQ activate the transcription regulator PqsR (MvfR), which controls several activities in bacteria, including biofilm formation and upregulation of PQS biosynthesis. The enzyme anthraniloyl-CoA synthetase (PqsA) catalyzes the first and critical step in the biosynthesis of quinolones; therefore, it is an attractive target for the development of anti-virulence therapeutics against Pseudomonas resistance. Herein, we report the design and synthesis of novel triazole nucleoside-based anthraniloyl- adenosine monophosphate (AMP) mimics. These analogues had a major impact on the morphology of bacterial biofilms and caused significant reduction in bacterial aggregation and population density. However, the compounds showed only limited inhibition of PQS and did not exhibit any effect on pyocyanin production.

Gemini Cationic Amphiphiles Control Biofilm Formation by Bacterial Vaginosis Pathogens.

Antibiotic resistance and recurrence of bacterial vaginosis (BV), a polymicrobial infection, justify the need for novel antimicrobials to counteract microbial resistance to conventional antibiotics. Previously, two series of cationic amphiphiles (CAms) which self-assemble into supramolecular nanostructures with membrane-lytic properties were designed with hydrophilic head groups and nonpolar domains. The combination of CAms and commonly prescribed antibiotics is suggested as a promising strategy for targeting microorganisms that are resistant to conventional antibiotics. Activities of the CAms against Gardnerella vaginalis ATCC 14018, a representative BV pathogen, ranged from 1.1 to 24.4 µM. Interestingly, the tested healthy Lactobacillus species, especially Lactobacillus plantarum ATCC 39268, were significantly more tolerant of CAms than the selected pathogens. In addition, CAms prevented biofilm formation at concentrations which did not influence the normal growth ability of G. vaginalis ATCC 14018. Furthermore, the biofilm minimum bactericidal concentration (MBC-Bs) of CAms against G. vaginalis ATCC 14018 ranged from 58.8 to 425.6 µM, while much higher concentrations (≥850 µM) were required to produce ≥3-log reductions in the number of biofilm-associated lactobacilli. The conventional antibiotic metronidazole synergized strongly with all tested CAms against planktonic cells and biofilms of G. vaginalis ATCC 14018. The synergism between CAms and the tested conventional antibiotic may be considered a new, effective, and beneficial method of controlling biofilm-associated bacterial vaginosis.

Synthesis of {[5-(adenin-9-yl)-2-furyl]methoxy}methyl phosphonic acid and evaluations against human adenylate kinases.

AMP mimics constitute an important class of therapeutic derivatives to treat diseases where the pool of ATP is involved. A new phosphonate derivative of 9-(5-hydroxymethylfuran-2-yl)adenine was synthesized in a multi-step sequence from commercially available adenosine. Its ability to behave as a substrate of human adenylate kinases 1 and 2 was assessed. The phosphonate was shown to be a moderate but selective substrate of the mitochondrial human AK2, better than well-known antiviral acyclic phosphonates 9-(2-phosphonomethoxyethyl)adenine (PMEA, Adefovir) and (R)-9-(2-phosphonomethoxypropyl)adenine (PMPA, Tenofovir). Putative binding mode within adenylate kinase NMP site revealed by molecular docking in comparison to AMP native substrate allowed to illustrate this selective behavior.

Evaluation of the Antimicrobial Activity in Host-Mimicking Media and In Vivo Toxicity of Antimicrobial Polymers as Functional Mimics of AMPs.

Activity tests for synthetic antimicrobial compounds are often limited to the minimal inhibitory concentration assay using standard media and bacterial strains. In this study, a family of acrylamide copolymers that act as synthetic mimics of antimicrobial peptides were synthesized and shown to have a disruptive effect on bacterial membranes and structural integrity through microscopy techniques and membrane polarization experiments. The polymers were tested for their antimicrobial properties using media that mimic clinically relevant conditions. Additionally, their activity was compared in two different strains of the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeruginosa. We showed that the medium composition can have an important influence on the polymer activity as there was a considerable reduction in minimal inhibitory concentrations against S. aureus grown in synthetic wound fluid (SWF), and against P. aeruginosa grown in synthetic cystic fibrosis sputum media (SCFM), compared to the concentrations in standard testing media. In contrast, we observed a complete loss of activity against P. aeruginosa in the serum-containing SWF. Finally, we made use of an emerging invertebrate in vivo model, using Galleria mellonella larvae, to assess toxicity of the polymeric antimicrobials, showing a good correlation with cell line toxicity measurements and demonstrating its potential in the evaluation of novel antimicrobial materials.

Amphiphilic Cationic Macromolecules Highly Effective Against Multi-Drug Resistant Gram-Positive Bacteria and Fungi With No Detectable Resistance.

The ever increasing threats of Gram-positive superbugs such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococccus faecium (VRE) are serious matter of concern worldwide toward public health. Such pathogens cause repeated recurrence of infections through the formation of biofilms which consist of metabolically inactive or slow growing dormant bacterial population in vast majority. Concurrently, dispersal of biofilms originates even more virulent dispersed cells responsible for pathogenesis. Along with this, fungal infections most commonly associated with Candida albicans also created a major complicacy in human healthcare. Moreover, concomitant survival of C. albicans and MRSA in a multispecies biofilms created extremely complicated polymicrobial infections. Surprisingly, infections associated with single species biofilm as well as multiple species biofilm (co-existence of MRSA and C. albicans) are almost untreatable with conventional antibiotics. Therefore, the situation demands an urgent development of antimicrobial agent which would tackle persistent infections associated with bacteria, fungi and their biofilms. Toward this goal, herein we developed a new class of branched polyethyleneimine based amphiphilic cationic macromolecules (ACMs) bearing normal alkyl, alkyl ester and alkyl amide moieties. An optimized compound with dual activity against drug-resistant bacteria (MIC = 2-4 µg/mL) and fungi (MIC = 4-8 µg/mL) was identified with minimal toxicity toward human erythrocytes (HC50 = 270 µg/mL). The lead compound, ACM-AHex (12) displayed rapid bactericidal and fungicidal kinetics (>5 log CFU/mL reduction within 1-4 h). It also killed metabolically dormant stationary (MRSA and VRE) and persister (S. aureus) cells. Moreover, this compound was able to disrupt the preformed biofilm of MRSA and reduced the bacterial burden related to the dispersed cells. It showed significant proficiencies to eliminate polymicrobial biofilms of MRSA and C. albicans. Bacteria also could not develop any resistant against this class of membrane active molecules even after 15 days of successive passages. Taken together this class of macromolecule can be developed further as a dual therapeutic agent to combat infections associated with bacterial and fungal co-existence.

Amino Acid Conjugated Polymers: Antibacterial Agents Effective against Drug-Resistant Acinetobacter baumannii with No Detectable Resistance.

An optimum hydrophilic/hydrophobic balance has been recognized as a crucial parameter in designing cationic polymers that mimic antimicrobial peptides (AMPs). To date, this balance was achieved either by hydrophilicity variation through altering the nature and the number of cationic charges or by hydrophobicity modulation through incorporation of alkyl groups of different chain lengths. However, how the hydrophobicity variation through AMPs' building blocks-amino acids-influences the antibacterial efficacy of AMP-mimicking cationic polymers has rarely been explored. Toward this goal, herein we report a class of amino acid conjugated polymers (ACPs) with tunable antibacterial activity through a simple post-polymer-functionalization strategy. Our polymeric design comprised a permanent cationic charge in every repeating unit, whereby the hydrophobicity was tuned through incorporation of different amino acids. Our results revealed that the amino acid alteration has a strong influence on antibacterial efficacy. Upon increasing the amino acid side-chain hydrophobicity, both the antibacterial activity (against broad spectrum of bacteria) and toxicity increased. However, the distinct feature of this class of polymers was their good activity against Acinetobacter baumannii-the top most critical pathogen according to WHO, which has created an alarming situation worldwide, causing the majority of infections in humans. A nontoxic (no hemolysis even at 1000 µg/mL) ACP including a glycine residue (ACP-1 (Gly)) showed very good activity (MIC = 8-16 µg/mL) against both drug-sensitive and drug-resistant strains of A. baumannii, including clinical isolates. This polymer not only was rapidly bactericidal against growing planktonic A. baumannii but also killed nondividing stationary-phase cells instantaneously (<2 min). Moreover, it eradicated the established biofilm formed by drug-resistant A. baumannii clinical isolates. No propensity for bacterial resistance development against this polymer was seen even after 14 continuous passages. Taken together, the results highlight that hydrophobicity modulation through incorporation of amino acids in cationic polymers will provide a significant opportunity in designing new ACPs with potent antibacterial activity and minimum toxicity toward mammalian cells. More importantly, the excellent anti-A. baumannii efficacy of the optimized lead polymer indicates its immense potential for being developed as therapeutic agent.

Cationic amphiphiles against Gardnerella vaginalis resistant strains and bacterial vaginosis-associated pathogens.

Antibiotic resistance and infection recurrence are critical issues in treating bacterial vaginosis, the most common vaginal disorder in women of reproductive age. Novel alternatives to traditional antibiotics, such as peptidomimetics, have the potential to address this challenge. Previously, two series of cationic amphiphiles (CAms) were developed with both hydrophilic head groups and non-polar domains, giving them the ability to self-assemble into supramolecular nanostructures with membrane-lytic properties. Those CAms were shown to be effective against biofilms of Gardnerella vaginalis while preserving the commensal microbiota. Two new series of CAms were designed with varying levels of flexibility between the hydrophilic head groups and the hydrophobic domains. Activities against the vaginal pathogen G. vaginalis ranged from 1.3 to 18.5 µM, while the tested vaginal lactobacilli were significantly more tolerant of CAms, with minimal inhibitory concentration values as high as 208 µM. Minimal biofilm bactericidal concentrations of the tested CAms ranged from 21.47 to <388.3 µM, and were lowest against resistant forms of G. vaginalis, while Lactobacillus biofilms were tolerant of concentrations ≥687 µM. Safety aspects of the CAms were also investigated, and they were found to be safe for use against vaginal ectocervical tissue.