Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates: Antisense Targeting of Fatty Acid Biosynthesis.

Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge. In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59)-Tat48-60, BF-2A-RXR, and drosocin-RXR are capable of transporting PNA effectively into E. coli (MICs of 1-4 µM). Importantly, presence of the inner-membrane peptide transporter SbmA was not required for antibacterial activity of PNA-AMP conjugates containing Pep-1-K, KLW-9,13-a, or drosocin-RXR (MICs of 2-4 µM).

In vitro inhibition of angiogenesis by heat and low pH stable hydroalcoholic extract of Peganum harmala seeds via inhibition of cell proliferation and suppression of VEGF secretion.

CONTEXT: Progression of cancer cells is completely dependent on its angiogenesis. Inhibition of tumor angiogenesis has shed new light on cancer treatment. As a result, anti-angiogenesis therapy represents one of the most significant advances in clinical oncology. Peganum harmala L. (Zygophyllaceae) is a native plant from the eastern Iranian region, which is used as a traditional folk medicine. Although some biological properties of this plant are determined, its effect on angiogenesis is still unclear. OBJECTIVE: We investigated the anti-angiogenic effects of heat and low pH stable hydroalcoholic extract of P. harmala seeds on endothelial cells (ECs) proliferation and VEGF secretion. MATERIALS AND METHODS: Dried Peganum seeds were purchased from Kermanshah Traditional Bazar in 2011. Hydroalcoholic extract of dried seeds (0, 10, 20, 40, 60, 80, 100, 120, and 150 µg/ml) was used for in vitro evaluation of its cytotoxicity, anti-proliferative, and anti-angiogenic effects on ECs. In vitro effect of the extract on VEGF secretion was assayed using ELISA. RESULTS: Treatment with hydroalcoholic extract at seven different concentrations resulted in significant decrease of ECs proliferation and angiogenesis with an ID50 of ∼ 85 µg/ml. VEGF secretion was (inhibited) decreased by the extracts at concentrations higher than 10 µg/ml. DISCUSSION AND CONCLUSION: Herbal plant extracts still attract attention owing to their fewer side effects comparing to synthetic drug agents. Current study indicated that hydroalcoholic extract of P. harmala seeds contains a potent anti-angiogenic component, which exerts its inhibitory effect mainly through down-regulation of essential mediators such as VEGF.

ß Cell and Autophagy: What Do We Know?

Pancreatic ß cells are central to glycemic regulation through insulin production. Studies show autophagy as an essential process in ß cell function and fate. Autophagy is a catabolic cellular process that regulates cell homeostasis by recycling surplus or damaged cell components. Impaired autophagy results in ß cell loss of function and apoptosis and, as a result, diabetes initiation and progress. It has been shown that in response to endoplasmic reticulum stress, inflammation, and high metabolic demands, autophagy affects ß cell function, insulin synthesis, and secretion. This review highlights recent evidence regarding how autophagy can affect ß cells' fate in the pathogenesis of diabetes. Furthermore, we discuss the role of important intrinsic and extrinsic autophagy modulators, which can lead to ß cell failure.

Extracellular Vesicle Therapy for Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic disorder characterized by immune-mediated destruction of pancreatic insulin-producing ß-cells. The primary treatment for T1D is multiple daily insulin injections to control blood sugar levels. Cell-free delivery packets with therapeutic properties, extracellular vesicles (EVs), mainly from stem cells, have recently gained considerable attention for disease treatments. EVs provide a great potential to treat T1D ascribed to their regenerative, anti-inflammatory, and immunomodulatory effects. Here, we summarize the latest EV applications for T1D treatment and highlight opportunities for further investigation.

Antisense Peptide Nucleic Acid-Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria.

Precision antisense antibacterial agents may be developed into novel antibiotics in the fight against multidrug-resistant Gram-negative bacteria. In this study, a series of diaminobutanoic acid (DAB) dendrons are presented as novel carriers for the delivery of antisense antibacterial peptide nucleic acids (PNAs). The dendron-PNA conjugates targeting the essential acpP gene exhibit specific antisense antimicrobial bactericidal activity against Escherichia coli and Klebsiella pneumoniae at one-digit micromolar concentrations, while showing low toxicity to human cells. One compound selected from a structure-activity relationship series showed high stability in mouse and human serum (t1/2 ≫ 24 h) as well as in vivo activity against a multidrug-resistant, extended spectrum beta-lactamase-producing E. coli in a murine peritonitis model. The compound was also well tolerated in mice upon i.v. administration up to a dose of 20 mg/kg, and in vivo fluorescence imaging indicated clearance via renal excretion with slight accumulation in the kidneys and liver. Thus, DAB-based dendrons constitute a promising new chemistry platform for development of effective delivery agents for antibacterial drugs with possible in vivo use.

Effects of LPS Composition in Escherichia coli on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

Uptake, Stability, and Activity of Antisense Anti-acpP PNA-Peptide Conjugates in Escherichia coli and the Role of SbmA.

PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)3K, targeting essential bacterial genes, such as acpP, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)3K-eg1-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)3K-eg1-PNA by showing that the peptide part of (KFF)3K-eg1-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture (t1/2 < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)3k-eg1-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)3K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)3K-eg1-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).

Antibiotic Potentiation in Multidrug-Resistant Gram-Negative Pathogenic Bacteria by a Synthetic Peptidomimetic.

The peptidomimetic H-[NLys-tBuAla]6-NH2 (CEP-136), which exhibits low inherent antimicrobial activity against Gram-negative bacteria (MIC = 16-64 µM), was shown to significantly potentiate the antibacterial activity of several clinically important antibiotics against the human pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Thus, the antibacterial spectrum of rifampicin, clarithromycin, and azithromycin could be extended to include also these Gram-negative bacteria. Additionally, the potentiation effect was demonstrated in a panel of clinically relevant multidrug-resistant isolates including extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing as well as colistin-resistant strains. For some peptidomimetic-antibiotic combinations, the strong synergy corresponded to a more than 50-fold reduction of the minimal inhibitory concentration of the antibiotic. Mechanistic studies indicate that the potentiation arises from a permeabilization effect exerted on the outer membrane lipopolysaccharide layer of the Gram-negative bacteria without significant disruption of the inner membrane. Furthermore, the peptidomimetic enhancer exhibited only a marginal effect on the viability of mammalian HepG2 cells even at concentrations 100-fold higher than that enabling the antibiotic enhancement. Also, a low hemolytic activity combined with limited in vivo acute toxicity of CEP-136 in healthy mice allowed in vivo validation of the potentiation effect on both rifampicin and azithromycin treatment in a murine peritonitis model. Thus, CEP-136 is an interesting hit compound for further development of effective adjuvants for repurposing antibiotics for use against infections by multidrug-resistant Gram-negative bacteria.

Approaches for Systemic Delivery of Dystrophin Antisense Peptide Nucleic Acid in the mdx Mouse Model.

Antisense-mediated exon skipping constitutes a promising new modality for treatment of Duchenne Muscular Dystrophy (DMD), which is caused by gene mutations that typically introduce a translation stop codon in the dystrophin gene, thereby abolishing production of functional dystrophin protein. The exon removal can restore translation to produce a shortened, but still partially functional dystrophin protein. Peptide nucleic acid (PNA) as a potential antisense drug has previously been shown to restore the expression of functional dystrophin by splice modulation in the mdx mouse model of DMD. In this study, we compare systemic administration of a 20-mer splice switching antisense PNA oligomer through intravenous (i.v.) and subcutaneous (s.c.) routes in the mdx mice. Furthermore, the effect of in situ forming depot technology (BEPO®) and PNA-oligonucleotide formulation was studied. In vivo fluorescence imaging analysis showed fast renal/bladder excretion of the PNA (t½ ∼ 20 min) for i.v. administration, while s.c. administration showed a two to three times slower excretion. The release from the BEPO depot exhibited biphasic kinetics with a slow release (t½ ∼ 10 days) of 50% of the dose. In all cases, some accumulation in kidneys and liver could be detected. Formulation of PNA as a duplex hybridization complex with a complementary phosphorothioate oligonucleotide increased the solubility of the PNA. However, none of these alternative administration methods resulted in significantly improved antisense activity. Therefore, either more sophisticated formulations such as designed nanoparticles or conjugation to delivery ligands must be utilized to improve both pharmacokinetics as well as tissue targeting and availability. On the other hand, the results show that s.c. and BEPO depot administration of PNA are feasible and allow easier, higher, and less frequent dosing, as well as more controlled release, which can be exploited both for animal model studies as well as eventually in the clinic in terms of dosing optimization.

PNA Length Restriction of Antibacterial Activity of Peptide-PNA Conjugates in Escherichia coli Through Effects of the Inner Membrane.

Peptide Nucleic Acid (PNA)-peptide conjugates targeting essential bacterial genes are showing promise as antisense antimicrobials in drug discovery. Optimization has focused on selection of target genes and exact localization around the ribosome binding site, but surprisingly a length optimum around 10-12 nucleobases has been found. Addressing this observation, we have investigated the relationship between PNA-length, PNA-RNA duplex stability and antimicrobial activity in E. coli in more detail. For PNAs of identical length of ten nucleobases the expected reverse correlation between the thermal stability (Tm) of the PNA-RNA duplex and the MIC for single mismatched PNAs was found. Also the expected direct correlation between the length of the PNA and the PNA-RNA duplex stability was found. Nonetheless, 10-mer PNAs [in a 6-18 mer extension series of (KFF)3K- and (RXR)4 conjugates] were the most active as antisense antimicrobials in both wild type E. coli MG1655 and AS19, suggesting that the size constraint is related to the bacterial uptake of PNA-peptide conjugates. This conclusion was supported by flow cytometry data showing higher bacterial uptake of shorter PNA fluorophore labeled conjugates. Interestingly, the size-limited uptake seems independent on outer membrane integrity (AS19), and thus the results suggest that the inner membrane limits the molecular size for peptide-PNA passage.

eNOS 4a/b polymorphism and its interaction with eNOS G894T variants in type 2 diabetes mellitus: modifying the risk of diabetic nephropathy.

To investigate the possible association between eNOS 4a/b polymorphism and the risk of developing type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) 173 T2DM patients with and without DN and 101 healthy subjects with ethnic background of Kurds were examined for the frequency of eNOS variants using PCR-RFLP method. The frequency of eNOS 4a/b genotypes between T2DM and controls was not significantly difference. Studying eNOS 4a/b variants alone indicated that the presence of eNOS 4a allele was not associated with the risk of developing DN. However, considering both polymorphisms of eNOS 4a/b and G894T indicated that the risk of macroalbuminuria significantly increased in the presence of either eNOS 4a or 894T allele by 2.45 times (p=0.014) and 3.7-fold (p=0.016), respectively. However, the concomitant presence of both alleles was not associated with the risk of macroalbuminuria. In microalbuminuric patients, in the presence of each allele, the risk of microalbuminuria increased 2.2 times (p=0.028) and 2.72-fold (p=0.057) for eNOS 4a and 894T alleles, respectively. However, the combined presence of both eNOS 894T and 4a alleles was not associated with the risk of microalbuminuria. The present study indicates the absence of association between eNOS 4a/b variants and the risk of developing T2DM and DN. Also, we demonstrated that eNOS 4a or 894T allele alone increased the risk of developing DN but this effect was modified by the concomitant presence of both alleles.