A comparative analysis of peptide-delivered antisense antibiotics using diverse nucleotide mimics.

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.

Synthetic Glyconanoparticles Modulate Innate Immunity but Not the Complement System.

Nanoparticles that modulate innate immunity can act as vaccine adjuvants and antigen carriers and are promising alternatives to conventional anticancer therapy. Nanoparticles might, upon contact with serum, activate the complement system that might in turn result in clearance and allergic reactions. Herein, we report that ultrasmall glyconanoparticles decorated with nonimmunogenic α-(1-6)-oligomannans trigger an innate immune response without drastically affecting the complement system. These negatively charged glyconanoparticles (10-15 nm) are stable in water and secrete proinflammatory cytokines from macrophages via the NF-κB signaling pathway. The glyconanoparticles can be used as immunomodulators for monotherapy or in combination with drugs and vaccines.

Targeting and Inhibiting Plasmodium falciparum Using Ultra-small Gold Nanoparticles.

Malaria, a mosquito-borne disease caused by Plasmodium species, claims more than 400,000 lives globally each year. The increasing drug resistance of the parasite renders the development of new anti-malaria drugs necessary. Alternatively, better delivery systems for already marketed drugs could help to solve the resistance problem. Herein, we report glucose-based ultra-small gold nanoparticles (Glc-NCs) that bind to cysteine-rich domains of Plasmodium falciparum surface proteins. Microscopy shows that Glc-NCs bind specifically to extracellular and all intra-erythrocytic stages of P. falciparum. Glc-NCs may be used as drug delivery agents as illustrated for ciprofloxacin, a poorly soluble antibiotic with low antimalarial activity. Ciprofloxacin conjugated to Glc-NCs is more water-soluble than the free drug and is more potent. Glyco-gold nanoparticles that target cysteine-rich domains on parasites may be helpful for the prevention and treatment of malaria.

Vancomycin Derivative Inactivates Carbapenem-Resistant Acinetobacter baumannii and Induces Autophagy.

Vancomycin is a standard drug for the treatment of multidrug-resistant Gram-positive bacterial infections. Albeit, development of resistance (VRE, VRSA) and its inefficacy against persistent infections is a demerit. It is also intrinsically inactive against Gram-negative bacteria. Herein, we report a vancomycin derivative, VanQAmC10, that addresses these challenges. VanQAmC10 was rapidly bactericidal against carbapenem-resistant A. baumannii (6 log10 CFU/mL reduction in 6 h), disrupted A. baumannii biofilms, and eradicated their stationary phase cells. In MRSA infected macrophages, the compound reduced the bacterial burden by 1.3 log10 CFU/mL while vancomycin exhibited a static effect. Further investigation indicated that the compound, unlike vancomycin, promoted the intracellular degradative mechanism, autophagy, in mammalian cells, which may have contributed to its intracellular activity. The findings of the work provide new perspectives on the field of glycopeptide antibiotics.

Aryl-alkyl-lysines: Novel agents for treatment of C. difficile infection.

Clostridium difficile infections (CDIs) are a growing health concern worldwide. The recalcitrance of C. difficile spores to currently available treatments and concomitant virulence of vegetative cells has made it imperative to develop newer modalities of treatment. Aryl-alkyl-lysines have been earlier reported to possess antimicrobial activity against pathogenic bacteria, fungi, and parasites. Their broad spectrum of activity is attributed to their ability to infiltrate microbial membranes. Herein, we report the activity of aryl-alkyl-lysines against C. difficile and associated pathogens. The most active compound NCK-10 displayed activity comparable to the clinically-used antibiotic vancomycin. Indeed, against certain C. difficile strains, NCK-10 was more active than vancomycin in vitro. Additionally, NCK-10 exhibited limited permeation across the intestinal tract as assessed via a Caco-2 bidirectional permeability assay. Overall, the findings suggest aryl-alkyl-lysines warrant further investigation as novel agents to treat CDI.

Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance.

As more antibiotics are rendered ineffective by drug-resistant bacteria, focus must be shifted towards alternative therapies for treating infections. Although several alternatives already exist in nature, the challenge is to implement them in clinical use. Advancements within biotechnology, genetic engineering, and synthetic chemistry have opened up new avenues towards the search for therapies that can substitute for antibiotics. This review provides an introduction to the various promising approaches that have been adopted in this regard. Whilst the use of bacteriophages and antibodies has been partly implemented, other promising strategies, such as probiotics, lysins, and antimicrobial peptides, are in various stages of development. Propitious concepts such as genetically modified phages, antibacterial oligonucleotides, and CRISPR-Cas9 are also discussed.

Aryl-Alkyl-Lysines Interact with Anionic Lipid Components of Bacterial Cell Envelope Eliciting Anti-Inflammatory and Antibiofilm Properties.

The emergence of bacterial resistance and hesitance in approving new drugs has bolstered research on membrane-active agents such as antimicrobial peptides and their synthetic derivatives as therapeutic alternatives against bacterial infections. Herein, we document the action of aryl-alkyl-lysines on liposomes mimicking bacterial membranes using solid-state nuclear magnetic resonance spectroscopy. A significant perturbation of the lipid thickness and order parameter of the lipid membrane was observed upon treatment with this class of compounds. Encouraged by these results, the ability of the most active compound (NCK-10) to interact with aggregates of lipopolysaccharides (LPSs) was studied. In vitro experiments showed that NCK-10 was able to prevent the LPS-induced stimulation of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The compound could also disrupt the biofilms of Pseudomonas aeruginosa in vitro and bring down the bacterial burden by more than 99% in a mice model of burn infections caused by the biofilms of P. aeruginosa.

l-Lysine based lipidated biphenyls as agents with anti-biofilm and anti-inflammatory properties that also inhibit intracellular bacteria.

l-Lysines were conjugated to lipidated biphenyls using simple synthetic chemistry to obtain selective membrane-active antibacterial agents that inhibit cell-wall biosynthesis. The most selective compound bore promising activity against biofilm-related infections and intracellular bacteria, and also suppressed the stimulation of TNF-α induced by lipoteichoic acid. Belligerent to resistance development, it was active in a murine model of MRSA infection.

Aryl-alkyl-lysines: Membrane-Active Fungicides That Act against Biofilms of Candida albicans.

Mortality due to pathogenic fungi has been exacerbated by the rapid development of resistance to frontline antifungal drugs. Fungicidal compounds with novel mechanisms of action are urgently needed. Aryl-alkyl-lysines, which are membrane-active small molecules, were earlier shown to be broad-spectrum antibacterial agents with potency in vitro and in vivo. Herein, we report the antifungal properties of aryl-alkyl-lysines. After identifying the most active compound (NCK-10), we tested its activity against a panel of clinically relevant pathogenic fungi and examined NCK-10's effect against immature and mature biofilms of Candida albicans. NCK-10 was capable of inhibiting the growth of various species of fungi (including Candida spp., Cryptococcus spp., and Aspergillus fumigatus) at concentrations similar to those of antifungal drugs used clinically. It was observed that polarization and permeability of the fungal cell membrane were compromised upon addition of NCK-10, indicating its mechanism is disruption of the fungal cell membrane. In addition to interfering with the growth of planktonic fungi, NCK-10 demonstrated the ability to both inhibit biofilm formation and reduce the metabolic activity of cells in C. albicans biofilm. Additionally, our compound was capable of crossing the blood-brain barrier in an in vitro model, expanding the potential antifungal applications for NCK-10. Overall, aryl-alkyl-lysines were found to be excellent compounds that warrant further investigation as novel antifungal agents.

Aryl-alkyl-lysines: small molecular membrane-active antiplasmodial agents.

Due to emerging resistance there is a steady need for new antimalarial drugs. Here, we report a new class of water soluble, non-toxic compounds, aryl-alkyl-lysines, with promising activity against the ring stage of Plasmodium falciparum. The optimal compound perturbed the plasma membrane potential and the digestive vacuole of parasites. In the murine model of malaria (Plasmodium berghei ANKA) the compound was able to increase the survival of mice by at least 5 days by an intra-peritoneal route. Further, the compounds showed no apparent toxicity to mice at the concentration tested.

A review on cell wall synthesis inhibitors with an emphasis on glycopeptide antibiotics.

Cell wall biosynthesis inhibitors (CBIs) have historically been one of the most effective classes of antibiotics. They are the most extensively used class of antibiotics and their importance is exemplified by the ß-lactams and glycopeptide antibiotics. However, this class of antibiotics has not received impunity from resistance development. In the wake of this predicament, this review presents the progress of CBIs, especially glycopeptide derivatives as antibiotics to confront antibacterial resistance. The various strategies used for the development of CBIs, their clinical status and possible directions in which this field can evolve have also been discussed.

Antiviral Screening of Multiple Compounds against Ebola Virus.

In light of the recent outbreak of Ebola virus (EBOV) disease in West Africa, there have been renewed efforts to search for effective antiviral countermeasures. A range of compounds currently available with broad antimicrobial activity have been tested for activity against EBOV. Using live EBOV, eighteen candidate compounds were screened for antiviral activity in vitro. The compounds were selected on a rational basis because their mechanisms of action suggested that they had the potential to disrupt EBOV entry, replication or exit from cells or because they had displayed some antiviral activity against EBOV in previous tests. Nine compounds caused no reduction in viral replication despite cells remaining healthy, so they were excluded from further analysis (zidovudine; didanosine; stavudine; abacavir sulphate; entecavir; JB1a; Aimspro; celgosivir; and castanospermine). A second screen of the remaining compounds and the feasibility of appropriateness for in vivo testing removed six further compounds (ouabain; omeprazole; esomeprazole; Gleevec; D-LANA-14; and Tasigna). The three most promising compounds (17-DMAG; BGB324; and NCK-8) were further screened for in vivo activity in the guinea pig model of EBOV disease. Two of the compounds, BGB324 and NCK-8, showed some effect against lethal infection in vivo at the concentrations tested, which warrants further investigation. Further, these data add to the body of knowledge on the antiviral activities of multiple compounds against EBOV and indicate that the scientific community should invest more effort into the development of novel and specific antiviral compounds to treat Ebola virus disease.

A Biodegradable Polycationic Paint that Kills Bacteria in Vitro and in Vivo.

Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and ß-lactam-resistant Klebsiella pneumoniae. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive S. aureus and Gram-negative E. coli, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15-20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under in vivo conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections.

Designing Simple Lipidated Lysines: Bifurcation Imparts Selective Antibacterial Activity.

In the global effort to thwart antimicrobial resistance, lipopeptides are an important class of antimicrobial agents, especially against Gram-negative infections. In an attempt to circumvent their synthetic complexities, we designed simple membrane-active agents involving only one amino acid and two lipid tails. Herein we show that the use of two short lipid tails instead of a single long one significantly increases selective antibacterial activity. This study yielded several selective antibacterial compounds, and investigations into the properties of this compound class were conducted with the most active compound. Fluorescence spectroscopic studies revealed the capacity of the representative compound to cause depolarization and permeabilization of bacterial cell membranes. This membrane-active nature of the compound imparts superior activity against persister cells, biofilms, and planktonic cells. Topical application of the compound decreased bacterial burden in mice inflicted with burn-infections caused by Acinetobacter baumannii. We anticipate that the design principles described herein will direct the development of several antimicrobial agents of clinical importance.

Aryl-alkyl-lysines: Membrane-Active Small Molecules Active against Murine Model of Burn Infection.

Infections caused by drug-resistant Gram-negative pathogens continue to be significant contributors to human morbidity. The recent advent of New Delhi metallo-ß-lactamase-1 (blaNDM-1) producing pathogens, against which few drugs remain active, has aggravated the problem even further. This paper shows that aryl-alkyl-lysines, membrane-active small molecules, are effective in treating infections caused by Gram-negative pathogens. One of the compounds of the study was effective in killing planktonic cells as well as dispersing biofilms of Gram-negative pathogens. The compound was extremely effective in disrupting preformed biofilms and did not select resistant bacteria in multiple passages. The compound retained activity in different physiological conditions and did not induce any toxic effect in female Balb/c mice until concentrations of 17.5 mg/kg. In a murine model of Acinetobacter baumannii burn infection, the compound was able to bring the bacterial burden down significantly upon topical application for 7 days.

Correction: Selective and broad spectrum amphiphilic small molecules to combat bacterial resistance and eradicate biofilms.

Correction for 'Selective and broad spectrum amphiphilic small molecules to combat bacterial resistance and eradicate biofilms' by Jiaul Hoque et al., Chem. Commun., 2015, 51, 13670-13673.

Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.

Bacterial biofilms represent the root-cause of chronic or persistent infections in humans. Gram-negative bacterial infections due to nosocomial and opportunistic pathogens such as Acinetobacter baumannii are more difficult to treat because of their inherent and rapidly acquiring resistance to antibiotics. Due to biofilm formation, A. baumannii has been noted for its apparent ability to survive on artificial surfaces for an extended period of time, therefore allowing it to persist in the hospital environment. Here we report, maleic anhydride based novel cationic polymers appended with amide side chains that disrupt surface established multi-drug resistant A. baumannii biofilms. More importantly, these polymers significantly (p < 0.0001) decrease the bacterial burden in mice with chronic A. baumannii burn wound infection. The polymers also show potent antibacterial efficacy against methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci (VRE) and multi-drug resistant clinical isolates of A. baumannii with minimal toxicity to mammalian cells. We observe that optimal hydrophobicity dependent on the side chain chemical structure of these polymers dictate the selective toxicity to bacteria. Polymers interact with the bacterial cell membranes by causing membrane depolarization, permeabilization and energy depletion. Bacteria develop rapid resistance to erythromycin and colistin whereas no detectable development of resistance occurs against these polymers even after several passages. These results suggest the potential use of these polymeric biomaterials in disinfecting biomedical device surfaces after the infection has become established and also for the topical treatment of chronic bacterial infections.

Aryl-Alkyl-Lysines: Agents That Kill Planktonic Cells, Persister Cells, Biofilms of MRSA and Protect Mice from Skin-Infection.

Development of synthetic strategies to combat Staphylococcal infections, especially those caused by methicillin resistant Staphyloccus aureus (MRSA), needs immediate attention. In this manuscript we report the ability of aryl-alkyl-lysines, simple membrane active small molecules, to treat infections caused by planktonic cells, persister cells and biofilms of MRSA. A representative compound, NCK-10, did not induce development of resistance in planktonic cells in multiple passages and retained activity in varying environments of pH and salinity. At low concentrations the compound was able to depolarize and permeabilize the membranes of S. aureus persister cells rapidly. Treatment with the compound not only eradicated pre-formed MRSA biofilms, but also brought down viable counts in bacterial biofilms. In a murine model of MRSA skin infection, the compound was more effective than fusidic acid in bringing down the bacterial burden. Overall, this class of molecules bears potential as antibacterial agents against skin-infections.

Structure-Activity Relationship of Amino Acid Tunable Lipidated Norspermidine Conjugates: Disrupting Biofilms with Potent Activity against Bacterial Persisters.

The emergence of bacterial resistance and biofilm associated infections has created a challenging situation in global health. In this present state of affairs where conventional antibiotics are falling short of being able to provide a solution to these problems, development of novel antibacterial compounds possessing the twin prowess of antibacterial and antibiofilm efficacy is imperative. Herein, we report a library of amino acid tunable lipidated norspermidine conjugates that were prepared by conjugating both amino acids and fatty acids with the amine functionalities of norspermidine through amide bond formation. These lipidated conjugates displayed potent antibacterial activity against various planktonic Gram-positive and Gram-negative bacteria including drug-resistant superbugs such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and ß-lactam-resistant Klebsiella pneumoniae. This class of nontoxic and fast-acting antibacterial molecules (capable of killing bacteria within 15 min) did not allow bacteria to develop resistance against them after several passages. Most importantly, an optimized compound in the series was also capable of killing metabolically inactive persisters and stationary phase bacteria. Additionally, this compound was capable of disrupting the preformed biofilms of S. aureus and E. coli. Therefore, this class of antibacterial conjugates have potential in tackling the challenging situation posed by both bacterial resistance as well as drug tolerance due to biofilm formation.

Membrane-Active Small Molecules: Designs Inspired by Antimicrobial Peptides.

Infectious diseases continue to be one of the major contributors to human morbidity. The rapid rate at which pathogenic microorganisms have developed resistance against frontline antimicrobials has compelled scientists to look for new alternatives. Given their vast antimicrobial repertoire, substantial research effort has been dedicated toward the development of antimicrobial peptides (AMPs) as alternative drugs. However, inherent limitations of AMPs have driven substantial efforts worldwide to develop synthetic mimics of AMPs. This review focuses on the progress that has been made toward the development of small molecules that emulate the properties of AMPs, both in terms of design and biological activity. Herein we provide an extensive discussion of the structural features of various designs and we examine biological properties that have been exploited. Furthermore, we raise a number of questions for which the field has yet to provide solutions and discuss possible future research directions that remain either unexploited or underexploited.