Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo

Abstract

Objectives: Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogues that can inhibit bacterial growth by a gene-specific, antisense mechanism. Attaching cationic peptides to PMOs enables efficient penetration through the Gram-negative outer membrane. We hypothesized that cationic groups attached directly to the PMO would obviate the need to attach peptides. Methods: PMOs with identical 11-base sequence (AcpP) targeted to acpP (an essential gene) of Escherichia coli were synthesized with various numbers of either piperazine (Pip) or N-(6-guanidinohexanoyl)piperazine (Gux) coupled to the phosphorodiamidate linker. Peptide-PMO conjugates were made using the membrane-penetrating peptide (RXR)4XB (X is 6-aminohexanoic acid; B is β-alanine). Results: MICs (μM/mg/L) were measured using E. coli:3+Pip-AcpP, 160/653; 6+Pip-AcpP, 160/673; 2+Gux-AcpP, 20/88; 5+Gux-AcpP, 10/49; 8+Gux-AcpP, 10/56; 3+Pip-AcpP-(RXR)4XB, 0.3/2; and 5+Gux-AcpP-(RXR)4XB, 0.6/4. In cell-free protein synthesis reactions, all PMOs inhibited gene expression approximately the same. These results suggested that Pip-PMOs inefficiently penetrated the outer membrane. Indeed, the MICs of 3+Pip-AcpP and 6+Pip-AcpP were reduced to 0.6 and 2.5 μM (1.2 and 10.5 mg/L), respectively, using as indicator a strain with a 'leaky' outer membrane. In vivo, mice were infected intraperitoneally with E. coli. Intraperitoneal treatment with 50 mg/kg 3+Pip-AcpP, 15 mg/kg 5+Gux-AcpP or 0.5 mg/kg 3+Pip-AcpP-(RXR)4XB, or subcutaneous treatment with 15 mg/kg 5+Gux-AcpP or (RXR)4XB-AcpP reduced bacteria in blood and increased survival. Conclusions: Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux-PMOs were more effective than Pip-PMOs. However, neither was as effective as the equivalent PMO-peptide conjugates. Subcutaneous treatment showed that 5+Gux-AcpP or (RXR)4XB-AcpP entered the circulatory system, reduced infection and increased survival. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.