Antisense antibacterial method and compound
View Patent ↗An antibacterial antisense conjugate and method of using the same for treating a bacterial infection in a mammalian host are disclosed. The conjugate includes an antisense oligonucleotide conjugated to a carrier peptide that significantly enhances the antibacterial activity of the oligonucleotide. The antisense oligonucleotide contains 10-20 nucleotide bases and has a targeting nucleic acid sequence complementary to a target sequence containing or within 10 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes a bacterial protein essential for bacterial replication, where the compound binds to a target mRNA with a T m of between 50° to 60° C. The carrier peptide is an arginine-rich peptide containing between 6 and 12 amino acids.
1. An antibacterial antisense conjugate for use in treating a bacterial infection in a mammalian host, comprising
(a) a substantially uncharged antisense oligonucleotide composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, having between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a target region of the infecting bacteria's mRNA for acyl carrier protein (acpP),
where the target region contains the translational start codon of the bacterial mRNA, or a sequence that is within 20 bases, in a downstream direction, of the translational start codon, and where the oligonucleotide binds to the mRNA to form a heteroduplex having a T m of at least 50° C., thereby to inhibit replication of the bacteria, and
(b) conjugated to the oligonucleotide, a carrier peptide that is (i) represented by the sequence (RFF) n R- or the sequence (RXX) n -, where X is an uncharged amino acid selected from the group consisting of alanine, β-alanine, valine, leucine, isoleucine, serine, threonine, phenyalanine, and tryptophan, and n=2 or 3, and (ii) coupled to the oligonucleotide at the peptide's C terminus.
2. The conjugate of claim 1 , wherein the carrier peptide has the form (RFF) n .
3. The conjugate of claim 1 , wherein the carrier peptide has the form (RFF) n R-.
4. The conjugate of claim 1 , whereinthe carrier peptide is linked at its C-terminus to the 5′ end of the oligonucleotide through a one- or two-amino acid linker.
5. The conjugate of claim 4 , wherein the linker is AhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-alanine.
6. The conjugate of claim 1 , wherein the carrier peptide has the ability, when conjugated to the 5′ end of a phosphorodiamidate-linked morpholino oligomer (PMO) having SEQ ID NO: 66, to enhance the anti-bacterial activity of the PMO by a factor of at least 10, as measured by the reduction in bacterial colony-forming units/ml (CFU/ml) when the peptide-conjugated PMO compound is added at a concentration of 20 μM in a culture to E. coli , strain W3110 at 5×10 7 CFU/ml in Luria broth for a period of 8 hours at 37° C. with aeration, relative to the same activity of the PMO oligonucleotide alone.
7. The conjugate of claim 1 , wherein the carrier peptide has the ability to enhance the anti-bacterial activity of said PMO by a factor of at least 10 2 , under the claimed conditions.
8. The conjugate of claim 1 , wherein the morpholino subunits in the oligonucleotide are joined by phosphorodiamidate linkages, in accordance with the structure:
where Y 1 ═O, Z═O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino.