Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Heterochiral dipeptide d-phenylalanyl- l-phenylalanine (H-D Phe-L Phe-OH) as a potential inducer of metastatic suppressor NM23H1 in p53 wild-type and mutant cells.

In recent years, significant progress has been made to the use-case of small peptides because of their diversified edifice and hence their versatile application scope in cancer therapy. Here we identify the heterochiral dipeptide H-D Phe-L Phe-OH (F1) as a potent inducer of the metastatic suppressor NM23H1. We divulge the effect of F1 on the major EMT/metastasis-associated genes and the implications on the invasion and migration ability of cancer cells. The anti-invasive potential of F1 was directly correlated with NM23H1 expression. Mechanistically, F1 treatment elevated p53 levels as validated by localization and transcriptional studies. In the NM23H1 knockdown condition, F1 failed to induce any p53 expression/nuclear localization, indicating that the upregulation in p53 expression by F1 is NM23H1 dependent. We also demonstrate how the antimetastatic potential of F1 is primarily mediated through NM23H1 irrespective of the p53 status of the cell. However, both NM23H1 and a functional p53 protein in conjunction govern the apoptotic and cytostatic potential of F1. Coimmunoprecipitation studies unraveled the augmentation of the p53 and NM23H1 interaction in p53 wild-type cells. However, in p53 mutated cells, no such enrichment was evidenced. We employed mouse isogenic cell lines (4T-1 and 4T-1 p53) to determine the in vivo efficacy of F1 (spontaneous and experimental models). Decreased tumor volume in the cohort injected with 4T-1 p53 cells demonstrated that while the antimetastatic potential of F1 was reliant on NM23H1, p53 activation was required for ablation of primary tumor burden. Our findings unravel that F1 treatment induces significant abrogation of the migration, invasion and metastatic potential of both p53 wild-type and p53 deficient cancers mediated through NM23H1.

Synergistic antimicrobial and antibiofilm activities of piperic acid and 4-ethylpiperic acid amides in combination with ciprofloxacin.

In the present work, piperic acid and 4-ethylpiperic acid (EPA) amides with amino acids (C1-C8) were bio-evaluated for their antimicrobial activity and biofilm inhibition against Gram-positive and Gram-negative bacterial strains. Among all, EPA-ß3,3-Pip(Bzl)-OMe, C2 displayed the potent antimicrobial activity with MIC of 6.25 µg ml-1 against Gram-negative bacteria Escherichia coli. In combination studies, the FIC indices suggested that C1 and C2 have a synergistic effect with ciprofloxacin against E. coli and Bacillus subtilis, whereas C5 exhibited a synergistic effect with ciprofloxacin against all the tested bacteria. The inhibitory effect of amides C1, C2, and C5 on the biofilm formation of test strains was significantly potentiated by co-administration with ciprofloxacin. Furthermore, the effective concentrations of C2 in combination reduced drastically compared to alone for biofilm inhibition. At these concentrations, C2 showed negligible hemolytic and cytotoxic activities.

Investigation of α/γ hybrid peptide self-assembled structures with antimicrobial and antibiofilm properties.

The present work describes the synthesis and characterization of α/γ hybrid peptides, Boc-Phe-γ4 -Phe-Val-OMe, P1; Boc-Ala-γ4 -Phe-Val-OMe, P2; and Boc-Leu-γ4 -Phe-Val-OMe, P3 together with the formation of self-assembled structures formed by these hybrid peptides in dimethyl sulfoxide (DMSO)/water (1:1). The self-assembled structures were characterized by infrared (IR) spectroscopy, circular dichroism (CD), and scanning electron microscopy (SEM). Further, α/γ hybrid peptide self-assembled structures were evaluated for antibacterial properties. Among all, the self-assembled peptide P1 exhibited the antimicrobial activity against Escherichia coli and Klebsiella pneumoniae, while self-assembled peptide P3 inhibited the biofilms of Salmonella typhimurium and Pseudomonas aeruginosa. In this study, we have shown the significance of self-assembled structures formed from completely hydrophobic α/γ hybrid peptides in exploring the antibacterial properties together with biofilm inhibition.