Antisense antibacterial compounds and methods
View Patent ↗Provided are antisense oligomers targeted against or genes associated with a biochemical pathway and/or cellular process, and related compositions and methods of using the oligomers and compositions to treat an infected mammalian subject, for example, as primary antimicrobials or as adjunctive therapies with classic antimicrobials.
1. An antisense morpholino oligomer of formula (I):
or a pharmaceutically acceptable salt thereof,
where each Nu is a nucleobase which taken together forms a targeting sequence,
where the targeting sequence is set forth in SEQ ID NOS: 2-11, comprises a fragment of at least 10 contiguous nucleotides of SEQ ID NOS: 2-11, or comprises a variant having at least 80% sequence identity to SEQ ID NOS: 2-11, where thymine bases (T) are optionally uracil bases (U);
X is an integer from 9 to 38;
T is selected from OH and a moiety of the formula:
where each R 4 is independently C 1 -C 6 alkyl, and R 5 is selected from an electron pair and H, and R 6 is selected from OH, —N(R 7 )CH 2 C(O)NH 2 , and a moiety of the formula:
where
R 7 is selected from H and C 1 -C 6 alkyl; and
R 8 is selected from G, —C(O)R 9 OH, acyl, trityl, and 4-methoxytrityl, where:
R 9 is of the formula —(O-alkyl) Y - where y is an integer from 3 to 10 and each of the y alkyl groups is independently selected from C 2 -C 6 alkyl;
each of R 1 is =N(R 10 ) 2 R 11 where each R 10 is independently C 1 -C 6 alkyl, and R 11 is selected from an electron pair and H;
R 2 is selected from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, stearoyl, and a moiety of the formula:
where L is selected from —C(O)(CH 2 ) 6 C(O)— and —C(O)(CH 2 ) 2 S 2 (CH 2 ) 2 C(O)—, and each R 12 is of the formula (CH 2 ) 2 OC(O)N(R 14 ) 2 where each R 14 is of the formula —(CH 2 ) 6 NHC(═NH)NH 2 ; and
R 3 is selected from an electron pair, H, and C 1 -C 6 alkyl,
where G is a cell penetrating peptide (“CPP”) and linker moiety selected from —C(O)(CH 2 )hd 5 NH-CPP, —C(O)(CH 2 ) 2 NH-CPP, —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH-CPP, and —C(O)CH 2 NH-CPP, or G is of the formula:
where the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, and
wherein the CPP is selected from:
where Ra is selected from H, acetyl, benzoyl, and stearoyl, with the proviso that R 2 or R 8 is G,
where the targeting sequence specifically hybridizes to a bacterial mRNA target sequence that encodes a protein selected from at least one of a protein associated with a biochemical pathway and/or cellular process and antibiotic resistance.
2. The antisense morpholino oligomer of claim 1 , where T is selected from:
3. The antisense morpholino oligomer of claim 2 , where R 2 is selected from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.
4. The antisense morpholino oligomer of claim 3 , where T is selected from:
and
and R 2 is G.
5. The antisense morpholino oligomer of claim 1 , where T is of the formula:
R 6 is of the formula:
and R 2 is G.
6. The antisense morpholino oligomer of claim 1 , where T is of the formula:
and R 2 is G.
7. The antisense morpholino oligomer of claim 1 , where T is of the formula:
8. The antisense morpholino oligomer of claim 7 , where R 2 is selected from H, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.
9. The antisense morpholino oligomer of claim 8 , where at least one instance of R 1 is —N(CH 3 ) 2 .
10. The antisense morpholino oligomer of claim 9 , where each R 1 is —N(CH 3 ) 2 .
11. The antisense morpholino oligomer of claim 1 , where the CPP is selected from:
where Ra is selected from H, acetyl, benzoyl, and stearoyl.
12. The antisense morpholino oligomer of claim 1 , where G is selected from:
where R a is selected from H, acetyl, benzoyl, and stearoyl.
13. The antisense morpholino oligomer of claim 1 , where the antisense oligomer is of the formula (VII) selected from:
or a pharmaceutically acceptable salt of any of the foregoing,
where Ra is selected from H, acetyl, benzoyl, and stearoyl, R b is selected from H, acetyl, benzoyl, stearoyl, trityl, and 4-methoxytrityl, and X and Nu are as defined in claim 1 .
14. The antisense morpholino oligomer of claim 13 , where R a is acetyl and R b is H.
15. The antisense morpholino oligomer of claim 1 , where the targeting sequence is selected from:
b) SEQ ID NO: 2 (ATATCGCTCAC) where X is 9;
c) SEQ ID NO: 3 (ATTCTCCTCAT) where X is 9;
d) SEQ ID NO: 4 (CACAGGAATTC) where X is 9;
e) SEQ ID NO: 5 (TTGCCATTAGC) where X is 9;
f) SEQ ID NO: 6 (CTGTAGTGATTTCACCA) where X is 15;
g) SEQ ID NO: 7 (TTATCTACCAT) where X is 9;
h) SEQ ID NO: 8 (GCACGTTTCAT) where X is 9;
i) SEQ ID NO: 9 (AGAAAACCCAT) where X is 9;
j) SEQ ID NO: 10 (TTGATAGTCAT) where X is 9; and
k) SEQ ID NO: 11 (GCTTTTTTCAT) where X is 9,
where thymine bases (T) may be uracil bases (U).
16. A pharmaceutical composition, wherein the antisense morpholino oligomer is of formula (I):
or a pharmaceutically acceptable salt thereof,
where each Nu is a nucleobase which taken together forms a targeting sequence,
where the targeting sequence is set forth in SEQ ID NOS: 2-11, comprises a fragment of at least 10 contiguous nucleotides of SEQ ID NOS: 2-11, or comprises a variant having at least 80% sequence identity to SEQ ID NOS: 2-11, where thymine bases (T) are optionally uracil bases (U);
X is an integer from 9 to 38;
T is selected from OH and a moiety of the formula:
where each R 4 is independently C 1 -C 6 alkyl, and R 5 is selected from an electron pair and H, and R 6 is selected from OH, —N(R 7 )CH 2 C(O)NH 2 , and a moiety of the formula:
where
R 7 is selected from H and C 1 -C 6 alkyl; and
R 8 is selected from G, —C(O)—R 9 OH, acyl, trityl, and 4-methoxytrityl, where:
R 9 is of the formula —(O-alkyl) Y - where y is an integer from 3 to 20 and each of the y alkyl groups is independently selected from C 2 -C 6 alkyl,
each of R 1 is —N(R 10 ) 2 R 11 where each R 10 is independently C 1 -C 6 alkyl, and
R 11 is selected from an electron pair and H;
R 2 is selected from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, stearoyl, and a moiety of the formula:
where L is selected from —C(O)(CH 2 ) 6 C(O)— and —C(O)(CH 2 ) 2 S 2 (CH 2 ) 2 C(O)—, and each R 12 is of the formula —(CH 2 ) 2 OC(O)N(R 14 ) 2 where each R 14 is of the formula —(CH 2 ) 6 NHC(═NH)NH 2 ; and
R 3 is selected from an electron pair, H, and C 1 -C 6 alkyl,
where G is a cell penetrating peptide (“CPP”) and linker moiety selected from —C(O)(CH 2 ) 5 NH-CPP, —C(O)(CH 2 ) 2 NH-CPP, —C(O)(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH-CPP, and —C(O)CH 2 NH-CPP, or G is of the formula:
where the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, and
wherein the CPP is selected from:
where Ra is selected from H, acetyl, benzoyl, and stearoyl, with the proviso that R 2 or R 8 is G,
where the targeting sequence specifically hybridizes to a bacterial mRNA target sequence that encodes a protein selected from at least one of a protein associated with a biochemical pathway and/or cellular process and antibiotic resistance.