Method for the treatment of pulmonary disease and method of producing proteins of use therein

Abstract

Methods of treating a subject with pulmonary disease by administering a therapeutically effective amount of a defensin polypeptide including at least one arginine residue susceptible to ADP-ribosylation and nicotinamide adenine dinucleotide (NAD), are described. The polypeptide and/or NAD can be administered via inhalation. Also disclosed is a pharmaceutical composition including at least one defensin polypeptide and NAD. In vitro methods of producing a polypeptide with altered activity, including contacting the polypeptide with NAD and an arginine-specific mono-ADP-ribosyltransferase to produce a polypeptide including at least one ADP-ribosylated arginine residue, incubating the ADP-ribosylated polypeptide under conditions sufficient for conversion of at least one ADP-ribosylated arginine residue to ornithine, and isolating the ornithine-containing polypeptide, are also described. Methods of treating a subject with pulmonary disease by administering a therapeutically effective amount of a modified defensin polypeptide including at least one ornithine residue in place of an arginine residue are also disclosed.

1 . A method of treating a subject with pulmonary disease, comprising administering to the subject a therapeutically effective amount of a defensin polypeptide and nicotinamide adenine dinucleotide (NAD), wherein the defensin polypeptide comprises at least one arginine residue that is susceptible to ADP-ribosylation, and wherein the NAD is administered by inhalation. 
     
     
         2 . The method of  claim 1 , wherein the defensin polypeptide comprises a human neutrophil peptide-1 (HNP-1) polypeptide. 
     
     
         3 . The method of  claim 2 , wherein the HNP-1 polypeptide comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         4 . The method of  claim 3 , wherein the HNP-1 polypeptide comprises an arginine residue that is susceptible to ADP-ribosylation at amino acid position 14, 24, or a combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the defensin polypeptide is administered by inhalation. 
     
     
         6 . The method of  claim 1 , further comprising administering to the subject a therapeutically effective amount of an arginine-specific mono-ADP-ribosyltransferase (ART) polypeptide. 
     
     
         7 . The method of  claim 6 , wherein the ART polypeptide is ART1 or ART5. 
     
     
         8 . The method of  claim 1 , wherein the pulmonary disease is cystic fibrosis, emphysema, asthma, sarcoidosis, chronic bronchitis, bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia, or adult respiratory distress syndrome. 
     
     
         9 . An in vitro method of producing a polypeptide with altered activity, comprising:
 contacting a polypeptide comprising at least one arginine residue susceptible to ADP-ribosylation with NAD and an arginine-specific mono-ADP-ribosyltransferase to produce a polypeptide comprising at least one arginine residue that is ADP-ribosylated;   incubating the ADP-ribosylated polypeptide under conditions sufficient for conversion of the at least one ADP-ribosylated arginine residue to ornithine; and   isolating the ornithine-containing polypeptide, thereby producing the polypeptide with altered activity.   
     
     
         10 . The method of  claim 9 , wherein the polypeptide comprises a defensin polypeptide. 
     
     
         11 . The method of  claim 10 , wherein the defensin polypeptide comprises a human neutrophil peptide-1 (HNP-1) polypeptide. 
     
     
         12 . The method of  claim 11 , wherein the HNP-1 polypeptide comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         13 . The method of  claim 12 , wherein the ornithine-containing HNP-1 polypeptide comprises ornithine at amino acid position 14, 24, or a combination thereof. 
     
     
         14 . The method of  claim 9 , wherein the conditions sufficient for conversion of the at least one ADP-ribosylated arginine residue to ornithine comprise:
 (i) incubating the ADP-ribosylated defensin polypeptide at approximately pH 7 to pH 9;   (ii) incubating the ADP-ribosylated defensin polypeptide at about 30° C. to 37° C.;   (iii) incubating the ADP-ribosylated defensin polypeptide for about 4 to 24 hours;   (iv) incubating the ADP-ribosylated defensin polypeptide at about pH 9 for about 24 hours at about 30° C.; or   (v) any combination of (i) to (iv).   
     
     
         15 . The method of  claim 9 , further comprising administering the polypeptide with modified activity to a subject with pulmonary disease. 
     
     
         16 . A pharmaceutical composition comprising a therapeutically effective amount of a defensin polypeptide and NAD, wherein the defensin polypeptide comprises at least one arginine residue that is susceptible to ADP-ribosylation. 
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein the defensin polypeptide comprises human neutrophil peptide-1 (HNP-1). 
     
     
         18 . The pharmaceutical composition of  claim 17 , wherein the HNP-1 comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         19 . The pharmaceutical composition of  claim 16 , further comprising a therapeutically effective amount of an arginine-specific mono-ADP-ribosyltransferase (ART) polypeptide. 
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the ART polypeptide is ART1 or ART5.