Antisense antibacterial compounds and methods

Abstract

Provided are antisense morpholino oligomers targeted against bacterial virulence factors such as genes that contribute to antibiotic resistance or biofilm formation, or essential genes, and related compositions and methods of using the oligomers and compositions, for instance, in the treatment of an infected mammalian subject.

1 . An antisense morpholino oligomer, 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, and having (a) about 10-40 nucleotide bases, and (b) a targeting sequence of sufficient length and complementarity to specifically hybridize to a bacterial mRNA target sequence that encodes a virulence factor; where the oligomer is conjugated to a cell-penetrating peptide (CPP), wherein the target sequence is SEQ ID NO: 22 (GAT ACA GTG AC) or SEQ ID NO: 23 (AAC GAT ATT CC), and wherein thymine (T) bases may be uracil (U) bases. 
     
     
         2 . The antisense morpholino oligomer of  claim 1 , 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; 
         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 - wherein 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 instance of R 1  is —N(R 10 ) 2 R 11  wherein 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  wherein 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, 
         wherein 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: 
       
       
         
           
           
               
               
           
         
         wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that only one instance of G is present, 
       
       wherein the targeting sequence specifically hybridizes to a bacterial mRNA target sequence that encodes a virulence factor. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The antisense morpholino oligomer of claim  4 , wherein the antibiotic resistance protein is selected from one or more of New Delhi metallo-beta-lactamase (NDM-1), serine beta-lactamase (KPC), acridine resistance complex protein AcrA, acridine resistance complex protein AcrB, acridine resistance complex repressor protein AcrR, acridine resistance complex protein TolC, and outer membrane protein A (OmpA). 
     
     
         6 - 16 . (canceled) 
     
     
         17 . The antisense morpholino oligomer of  claim 1 , wherein T is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The antisense morpholino oligomer of  claim 1 , wherein R 2  is selected from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl. 
     
     
         19 . The antisense morpholino oligomer of  claim 1 , wherein T is selected from: 
       
         
           
           
               
               
           
         
       
       and
 R 2  is G. 
 
     
     
         20 . The antisense morpholino oligomer of  claim 1 , wherein T is of the formula: 
       
         
           
           
               
               
           
         
         R 6  is of the formula: 
       
       
         
           
           
               
               
           
         
         and R 2  is G. 
       
     
     
         21 . The antisense morpholino oligomer of  claim 1 , wherein T is of the formula: 
       
         
           
           
               
               
           
         
         and R 2  is G. 
       
     
     
         22 . The antisense morpholino oligomer of  claim 1 , wherein T is of the formula: 
       
         
           
           
               
               
           
         
       
     
     
         23 . The antisense morpholino oligomer according to  claim 1 , wherein R 2  is selected from H, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl. 
     
     
         24 . The antisense morpholino oligomer according to  claim 1 , wherein at least one instance of R 1  is —N(CH 3 ) 2 . 
     
     
         25 . The antisense morpholino oligomer of  claim 24 , wherein each R 1  is —N(CH 3 ) 2 . 
     
     
         26 . The antisense morpholino oligomer according to  claim 1 , wherein the CPP is selected from: 
       
         
           
           
               
               
           
         
         wherein R a  is selected from H, acetyl, benzoyl, and stearoyl. 
       
     
     
         27 . The antisense morpholino oligomer according to  claim 1 , wherein G is selected from: 
       
         
           
           
               
               
           
         
         wherein R a  is selected from H, acetyl, benzoyl, and stearoyl. 
       
     
     
         28 . The antisense morpholino oligomer of  claim 1 , wherein the antisense oligomer is of the formula (VII) selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt of any of the foregoing, 
         wherein R a  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 . 
       
     
     
         29 . The antisense morpholino oligomer of  claim 28 , wherein R a  is acetyl and R b  is H. 
     
     
         30 . The antisense morpholino oligomer according to  claim 17 , wherein the targeting sequence is
 SEQ ID NO: 22 (GAT ACA GTG AC),   
       wherein thymine bases (T) may be uracil bases (U). 
     
     
         31 . The antisense morpholino oligomer according to  claim 1 , wherein the targeting sequence is
 SEQ ID NO: 23 (AAC GAT ATT CC),   
       wherein thymine bases (T) may be uracil bases (U). 
     
     
         32 . (canceled) 
     
     
         33 . A pharmaceutical composition, comprising a pharmaceutically acceptable carrier and an antisense morpholino oligomer according to  claim 1 . 
     
     
         34 . The pharmaceutical composition of  claim 33 , 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; 
         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 - wherein 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 instance of R 1  is —N(R 10  2R 11  wherein 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  wherein 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, 
         wherein 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: 
       
       
         
           
           
               
               
           
         
         wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that only one instance of G is present, 
         wherein the targeting sequence specifically hybridizes to a bacterial mRNA target sequence that encodes a virulence factor. 
       
     
     
         35 - 38 . (canceled) 
     
     
         39 . A method of reducing expression and activity of a virulence factor in a bacterium, comprising contacting the bacterium with an antisense morpholino oligomer according to  claim 1 . 
     
     
         40 . The method of  claim 39 , 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; 
         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 - wherein 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 instance of R 1  is —N(R 10 ) 2 R 11  wherein 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  wherein 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, 
         wherein 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: 
       
       
         
           
           
               
               
           
         
         wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that only one instance of G is present, 
         wherein the targeting sequence specifically hybridizes to a bacterial mRNA target sequence that encodes the virulence factor. 
       
     
     
         41 . The method of  claim 39 , where the bacterium is in a subject, and the method comprises administering the antisense morpholino oligomer to the subject. 
     
     
         42 . The method of  claim 39  or  14 , wherein the bacterium is selected from the genus  Escherichia, Acinetobacter, Klebsiella , and  Burkholderia.    
     
     
         43 . The method of  claim 42 , wherein the bacterium is  Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae , or  Burkholderia cepacia  (complex). 
     
     
         44 - 48 . (canceled)