Combined effect of a peptide–morpholino oligonucleotide conjugate and a cell-penetrating peptide as an antibiotic

Abstract

A cell-penetrating peptide (CPP)-morpholino oligonucleotide (MO) conjugate (PMO) that has an antibiotic effect in culture had some contaminating CPPs in earlier preparations. The mixed conjugate had gene-specific and gene-nonspecific effects. An improved purification procedure separates the PMO from the free CPP and MO. The gene-specific effects are a result of the PMO, and the nonspecific effects are a result of the unlinked, unreacted CPP. The PMO and the CPP can be mixed together, as has been shown previously in earlier experiments, and have a combined effect as an antibiotic. Kinetic analysis of these effects confirm this observation. The effect of the CPP is bacteriostatic. The effect of the PMO appears to be bacteriocidal. An assay for mutations that would alter the ability of these agents to affect bacterial viability is negative.EGS technology | gram-negative bacteria | gram-positive bacteria | RNase P M orpholino oligonucleotides (MOs) covalently linked with a cell-penetrating peptide (CPP) can be used as antibiotics (1). Such compounds are potent in the variety of cells they attack (both Gram-positive and Gram-negative bacteria) but are somewhat less efficient than conventional antibiotics today in terms of the concentrations used to kill cells. These compounds work well in curing internal infections in mice (2-4) and are also potent in preventing the growth and development of the malarial parasite, Plasmodium falciparum (5).A very efficient CPP-MO combination (PMO) has been synthesized from a basic peptide from human T cells (6), which has no biological activity on human cells, and an MO. One significant asset of this conjugate is its relative resistance to mutation that will alter its properties, as more than three noncontiguous mutations are needed to inactivate it. Another feature, as with most nucleic acid-based potential drugs, is its universal application against any target gene through the rational design of the base sequence of its MO (1).Many peptides from a variety of organisms are natural antibiotics (7,8). Several derivatives could be used in humans as antibiotics, but this form of therapy has not become popular. Some peptides that resemble signal peptides in their content of basic amino acid residues, such as arginine and lysine, are also useful as CPPs (2-4).The external guide sequence (EGS) technology relies on an RNA introduced in vivo that is complementary to a pathogenic RNA. The resident and ubiquitous RNase P then cleaves the complex, thereby rendering the pathogenic RNA inactive (9). In an investigation of the PMO that relies on EGS technology for its use, both gene-specific and non-gene-specific effects had been ascribed to this and similar compounds (1). Gel filtration purification yielded the PMO and, separately, unreacted CPP and MO. Using larger bed volume columns in scaling up preparations revealed that earlier preparations did have some contamination by chemically unreacted CPP in the PMO fractions. Reassaying of all these purified components indicated that significant anti...

Regulation of Hfq mRNA and Protein Levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA

“…For instance, a PMO targeting the highly conserved region of the E. coli gyrA was recently shown to effectively inactivate several species of Gram-positive and Gram-negative bacteria, when used in the micro-molar range [36].…”

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

“…Specific EGSs complementary to the selected sites were synthesized. The peptide YARVRRRGPRGYARVRRRGPRR was conjugated to a MO with the same sequence as the EGS to generate the PPMO (13).…”

“…The sequence of the MO thereby decides the fate of the endogenous transcript. For example, MOs with External Guide Sequence (EGS) conjugated to peptides (PPMOs) have been designed to target essential genes of several pathogenic bacteria and have displayed strong antibacterial activity (10)(11)(12)(13). Similarly, PPMOs targeting the P. falciparum PfGyrA and PfPAT RNAs for RNase P-mediated cleavage inhibit parasite growth in the low micromolar range (4,(14)(15)(16).…”

“…For morpholino-based antimicrobial activity, two types of conjugates have been developed, PPMOs and vivo morpholino oligomers (VMOs, octa-guanidinium dendrimer-conjugated MOs; Materials and Methods). PPMOs are produced following conjugation of a specific MO to a cell-penetrating, arginine-rich peptide, whereas VMOs are synthesized as conjugates between a MO molecule and an octa-guanidinium head group (13,17,24). To date, VMOs have been used to downregulate gene expression in human fibroblasts, mice, zebrafish, Xenopus oocytes, Toxoplasma gondii, and other model organisms (20,(25)(26)(27)(28)(29).…”

Synthetic Biology of Antibiotic Production

“…Russell et al [31] recently reported the discovery of a superfamily of bacterial phospholipases that act as antibacterial effectors in inter-and intra-species interactions by degrading the major component of bacterial cell membranes, namely phosphatidylethanolamine. Of special interest from a synthetic biology perspective are developments at the interface of chemistry and biology, such as the establishment of a peptide-morpholino oligonucleotide conjugate which, in combination with a bacteriostatic cell-penetrating peptide, exerts antibiotic activity over a wide range of pathogen species [32].…”