Emerging Non-Traditional Approaches to Combat Antibiotic Resistance.

An increasing number of bacterial pathogens are acquiring resistance to the commonly used antibiotics. This has spurred a global threat leading to a resistance era and has penetrated the consciousness of the common people and the clinicians alike. The delay in discovering new antibiotics has exacerbated the resistance problem, forcing researchers to focus on unconventional antimicrobial therapeutics that differ from conventional antibiotics. Alternative therapies have emerged in recent years, including antimicrobial peptides, phage therapy, efflux pump inhibitors, antibodies, and immunomodulatory agents, which have produced impressive results in both laboratory and in clinical trials. Additionally, ultra-narrow-spectrum therapeutics such as CRISPR-Cas system and peptide nucleic acids aided in the development of sequence-specific antimicrobials. Moreover, combinatorial therapies that combine these new approaches have been efficient enough to get approval for clinical use and have accelerated the discovery of novel combination approaches that enhance the performance of already in-use antibiotics. In this review, we provide an overview of these approaches along with studies that focus on the uncharted microbial territories that have been able to deliver some of the important new antibiotics of recent times. It is hoped that the information gathered in this article will provide an update on the current antibiotic resistance threat and encourage profound research.

Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

Validation of Suitable Carrier Molecules and Target Genes for Antisense Therapy Using Peptide-Coupled Peptide Nucleic Acids (PNAs) in Streptococci.

Antisense peptide nucleic acids (PNAs) targeting genes involved in metabolism or virulence are a possible means to treat infections or to investigate pathogenic bacteria. Potential targets include essential genes, virulence factor genes, or antibiotic resistance genes. For efficient cellular uptake, PNAs can be coupled to cell-penetrating peptides (CPPs). CPPs are peptides that serve as molecular transporters and are characterized by a comparably low cytotoxicity. So far, there is only limited information about CPPs that mediate PNA uptake by Gram-positive bacteria. Here, we describe two methods to identify suitable CPP-antisense PNA conjugates, novel carrier molecules, and efficient target genes for streptococcal species and to evaluate their antimicrobial efficiency.

Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens.

There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.

Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin-resistant Staphylococcus aureus by antisense peptide nucleic acid.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to "druggable" target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ7° (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. METHODOLOGY/PRINCIPAL FINDINGS: By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)3K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ7° in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ7° were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. CONCLUSIONS: The present result suggested that RNAP primary σ7° is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.

Inhibiting gene expression with peptide nucleic acid (PNA)--peptide conjugates that target chromosomal DNA.

Peptide nucleic acids (PNAs) are nonionic DNA/RNA mimics that can recognize complementary sequences by Watson-Crick base pairing. The neutral PNA backbone facilitates the recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use, it will be necessary to develop straightforward strategies for introducing them into cells. Here, we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both 13 and 19 base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moieties yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.

A peptide nucleic acid-neamine conjugate that targets and cleaves HIV-1 TAR RNA inhibits viral replication.

The neamine part of the aminoglycoside antibiotic neomycin B was conjugated to a 16 mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. Attachment of the neamine core allows cellular uptake of the PNA and results in potent inhibition of HIV-1 replication. The polycationic neamine moiety imparts greater solubility to the PNA and also confers a unique RNA cleavage property to the conjugate which is specific to its target site and functional at physiological concentrations of Mg(2+). These properties suggest a potential therapeutic application for this class of compounds.

Peptide nucleic acid-DNA decoy chimeras targeting NF-kappaB transcription factors: Induction of apoptosis in human primary osteoclasts.

Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary osteoclasts. Our data suggest that PDP-based induction of osteoclast apoptosis could be a therapeutic approach for disorders in which bone resorption is inappropriately excessive.

Synthesis and DNA binding properties of dioxime-peptide nucleic acids.

Peptide nucleic acids (PNAs) C- or N-modified with dioxime ligands were prepared by solid-phase synthesis using iron(II)-clathrochelates as protected dioxime building blocks. These PNA bind complementary DNA sequence specifically, though with much reduced affinity in comparison with nonmodified PNA. The dioxime-PNA conjugates bind Cu2+ and Ni2+ at microM concentration.

Intrathecal administration of PNA targeting galanin receptor reduces galanin-mediated inhibitory effect in the rat spinal cord.

Peptide nucleic acids (PNA) are nucleic acid analogues containing neutral amide backbone, forming stable and tight complexes with complementary DNA/RNA. However, it is unclear whether unmodified PNA can efficiently penetrate neuronal tissue in order to act as antisense reagent. Here we show that intrathecal (i.t.) injection of an unmodified antisense PNA complementary to the rat galanin receptor type 1 (GalR1) mRNA is able to block the inhibitory effect of i.t. administered galanin on spinal nociceptive transmission. Autoradiographic ligand binding studies using [125I]galanin show that the unmodified PNA is able to reduce the density of galanin binding sites in the dorsal horn. Thus, unmodified PNA applied i.t. appears to function as an effective antisense reagent in rat spinal cord in vivo.

Antisense oligonucleotides for target validation in the CNS.

Although antisense oligonucleotides have been used in cell-based antisense experiments for nearly two decades, studies to investigate the function of CNS proteins in living animals were not successfully conducted until recently. Oligonucleotides are not transported across the blood-brain barrier to any appreciable extent. Consequently, these molecules need to be administered directly into the brain. Antisense approaches may be especially suited to investigation of CNS proteins. Due to their specificity, antisense sequences can more easily and selectively distinguish between closely related proteins, such as receptor subtypes, in contrast to the more traditional pharmacological agents such as small molecule ligands. This review discusses some unique technical aspects surrounding oligonucleotide delivery to the brain, and summarizes some of the more noteworthy applications of antisense tools to the study of CNS protein function during the past two years.