Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency against Escherichia coli In Vitro and In Vivo

Abstract

The potency of antisense peptide-phosphorodiamidate morpholino oligomers (PPMOs) was improved by varying the peptide composition. An antisense phosphorodiamidate morpholino oligomer (PMO) complementary to the mRNA of the essential gene acpP (which encodes the acyl carrier protein required for lipid biosynthesis) in Escherichia coli was conjugated to the 5 ends of various cationic membrane-penetrating peptides. Each peptide had one of three repeating sequence motifs: C-N-N (motif 1), C-N (motif 2), or C-N-C (motif 3), where C is a cationic residue and N is a nonpolar residue. Variations in the cationic residues included arginine, lysine, and ornithine (O). Variations in the nonpolar residues included phenylalanine, valine, ␤-alanine (B), and 6-aminohexanoic acid (X). The MICs of the PPMOs varied from 0.625 to >80 M (about 3 to 480 g/ml). Three of the most potent were the (RX) 6 B-, (RXR) 4 XB-, and (RFR) 4 XB-AcpP PMOs, which were further tested in mice infected with E. coli. The (RXR) 4 XB-AcpP PMO was the most potent of the three conjugates tested in mice. The administration of 30 g (1.5 mg/kg of body weight) (RXR) 4 XB-AcpP PMO at 15 min postinfection reduced CFU/ml in blood by 10 2 to 10 3 within 2 to 12 h compared to the numbers in water-treated controls. All mice treated with 30 g/dose of (RXR) 4 XB-AcpP PMO survived infection, whereas all water-treated mice died 12 h postinfection. The reduction in CFU/ml in blood was proportional to the dose of PPMO from 30 to 300 g/ml. In summary, the C-N-C motif was more effective than the other two motifs, arginine was more effective than lysine or ornithine, phenylalanine was more effective than 6-aminohexanoic acid in vitro but not necessarily in vivo, and (RXR) 4 XB-AcpP PMO reduced bacterial infection and promoted survival at clinically relevant doses.

Basic peptide-morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes

“…The effective concentration of the conjugates we used (plural because the PMOs had different base sequences) was ten-to a hundredfold more effective than previous compounds tested. The ability of mice to withstand the administration of similar compounds (4,5) indicates that these conjugates are worthwhile candidates for use in infected animals.…”

“…Among these are the use of basic peptide-morpholino oligonucleotide conjugates. Some practical success has been observed, in particular, in terms of curing mice infected with bacteria and prevention of infection of mammalian tissue by dangerous viruses (4)(5)(6)(7)(8)(9). A previously undescribed basic peptide (cell-penetrating peptide: CPP), derived from a protein found in human T cells, Tyr-AlaArg-Val-Arg-Arg-Arg-Gly-Pro-Arg-Gly-Tyr-Ala-Arg-Val-Arg-ArgArg-Gly-Pro-Arg-Arg, which has no biological activity on its own (10), has been employed to make a previously undescribed conjugate with phosphorodiamidate morpholino oligonucleotides (PMOs) by the chemical method described by Abes et al (11).…”

Peptide Conjugated Phosphorodiamidate Morpholino Oligomers Increase Survival of Mice Challenged with AmesBacillus anthracis

“…However, the sequence-specific effects of the PMO on bacterial gene expression have been significantly improved by attaching a membrane-penetrating peptide to the end of the PMO (Geller et al, 2003;Tilley et al, 2006;Mellbye et al, 2009;Tilley et al, 2007). The peptides themselves are known to traverse through the lipid bilayer, often without causing lysis of the bacterial cell, or without killing the bacteria (Vaara and Porro, 1996).…”

Antisense Antibacterials: From Proof-Of-Concept to Therapeutic Perspectives

“…Validated essential genes among different bacterial species include fbpA/ fbpB/fbpC (Harth & et al, 2002(Harth & et al, , 2007 and glnA1 (Harth & et al, 2000) in Mycobacterium tuberculosis, gyrA/ompA in Klebsiella pneumonia (Kurupati & et al, 2007), inhA in Mycobacterium smegmatis (Kulyte & et al, 2005), oxyR/ahpC in Mycobacterium avium (Shimizu & et al, 2003), NPT/EhErd2 in Entamoeba histolytica (Stock & et al, 2000(Stock & et al, , 2001, gtfB in Streptococcus mutans (Guo & et al, 2006), fmhB/ gyrA/hmrB (Nekhotiaeva & et al, 2004a) and fabI (Ji & et al, 2004) in Staphylococcus aureus, 23S rRNA (Xue-Wen & et al, 2007), 16S rRNA plus lacZ/bla (Good & Nielsen, 1998), and RNAse P (Gruegelsiepe & et al, 2006) in Escherichia coli, and acpP in Burkholderia cepacia (Greenberg & et al, 2010), Escherichia coli (Deere & et al, 2005b;Geller & et al, 2003aGeller & et al, , 2003bGeller & et al, , 2005Mellbye & et al, 2009Mellbye & et al, , 2010Tan & et al, 2005;Tilley & et al, 2007) as well as Salmonella enterica serovar Typhimurium (Mitev & et al, 2009;Tilley & et al, 2006). Mellbye et al, First proof-of-principle evidence was given by White et al in 1997 for successful increasing the bactericidal activity of norfloxacin by antisense inhibiting the marRAB operon in Escherichia coli (White & et al, 1997).…”