Compositions and methods for the delivery of nitric oxide

Abstract

H-NOX proteins are mutated to exhibit improved or optimal kinetic and thermodynamic properties for blood gas NO delivery. The engineered H-NOX proteins comprise mutations that impart altered NO or O 2 ligand-binding relative to the corresponding wild-type H-NOX domain, and are operative as physiologically compatible mammalian blood NO gas carriers. The invention also provides pharmaceutical compositions, kits, and methods that use wild-type or mutant H-NOX proteins for the treatment of any condition for which delivery of NO is beneficial.

1 - 320 . (canceled) 
     
     
         321 . An isolated H-NOX protein comprising at least one mutation that alters the k off , k 1 , or k 2  for NO, the O 2  dissociation constant, the NO dissociation constant, or the NO reactivity compared to that of a corresponding wild type H-NOX protein, wherein the mutant H-NOX protein is not  Thermoanaerobacter tengcongensis  H-NOX Y140L,  T. tengcongensis  H-NOX W9F,  T. tengcongensis  H-NOX F78Y/Y140L,  Legionella pneumophilia  2 H-NOX F142Y,  Rattus norvegicus  sGC β1 H-NOX (1-385) I145Y; and wherein either
 (a) the k off , k 1 , or k 2  for NO of the H-NOX protein is between about 1×10 −4  s −1  and about 10 s −1  at 37° C., and the O 2  dissociation constant of the H-NOX protein is at least about 1 μM at 37° C., or 
 (b) the NO dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and the NO reactivity of the H—NOX protein is at least 10-fold lower than that of human hemoglobin alpha. 
 
     
     
         322 . The isolated H-NOX protein of  claim 321 , wherein the k off , k 1 , or k 2  for NO of the mutant H-NOX protein is between about 1×10 −4  s −1  and about 10 s −1  at 37° C., and wherein the O 2  dissociation constant of the H-NOX protein is at least about 1 μM at 37° C. 
     
     
         323 . The isolated H-NOX protein of  claim 321 , wherein the NO dissociation constant of the mutant H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and wherein the NO reactivity of the mutant H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha. 
     
     
         324 . The isolated H-NOX protein of  claim 321 , wherein the k off , k 1 , or k 2  for NO of the mutant H-NOX protein is between about 1×10 −4  s −1  and about 0.012 s −1  at 37° C. 
     
     
         325 . The isolated H-NOX protein of  claim 321 , wherein the NO reactivity of the mutant H-NOX protein is less than about 700 s −1 . 
     
     
         326 . The isolated H-NOX protein of  claim 321 , wherein the NO reactivity of the mutant H-NOX protein is at least 100-fold lower than that of human hemoglobin alpha. 
     
     
         327 . The isolated H-NOX protein of  claim 321 , wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1  at 37° C. 
     
     
         328 . The isolated H-NOX protein of  claim 321 , wherein the H-NOX protein comprises at least one distal pocket mutation. 
     
     
         329 . The isolated H-NOX protein of  claim 328 , wherein the distal pocket mutation is a mutation of a residue in alpha-helix A, D, E or G. 
     
     
         330 . The isolated H-NOX protein of  claim 328 , wherein the distal pocket mutation corresponds to at least one of Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, and Leu144 of  T. tengcongensis  H-NOX. 
     
     
         331 . The isolated H-NOX protein of  claim 328 , wherein the distal pocket mutation is a mutation in which a residue that corresponds to Tyr140 of  T. tengcongensis  H-NOX is replaced by any other amino acid or a residue that corresponds to Phe142 of  L. pneumophilia  2 H-NOX is replaced by any other amino acid. 
     
     
         332 . The isolated protein of  claim 328 , wherein the distal pocket mutation corresponds to an N74E mutation of  T. tengcongensis , an N74A mutation of  T. tengcongensis , or an N74H mutation of  T. tengcongensis.    
     
     
         333 . The isolated protein of  claim 328 , wherein the distal pocket mutations corresponds to an N74A/Y140H mutation of  T. tengcongensis , or a W9F/N74A mutation of  T. tengcongensis.    
     
     
         334 . The isolated protein of  claim 332 , wherein the distal pocket mutation is an N74E mutation of  T. tengcongensis , an N74A mutation of  T. tengcongensis , or an N74H mutation of  T. tengcongensis.    
     
     
         335 . The isolated protein of  claim 334 , wherein the distal pocket mutations is an N74A/Y140H mutation of  T. tengcongensis , or a W9F/N74A mutation of  T. tengcongensis.    
     
     
         336 . The isolated H-NOX protein of  claim 321 , wherein the H-NOX protein comprises at least one mutation that is not in the distal pocket 
     
     
         337 . The isolated H-NOX protein of  claim 321 , wherein the corresponding wild-type H-NOX protein is a mammalian protein. 
     
     
         338 . The isolated H-NOX protein of  claim 337 , wherein the corresponding wild-type H-NOX protein is a human protein. 
     
     
         339 . The isolated H-NOX protein of  claim 338 , wherein the corresponding wild-type H-NOX protein is β1. 
     
     
         340 . The isolated H-NOX protein of  claim 321 , wherein the corresponding wild-type H-NOX protein is a bacterial protein. 
     
     
         341 . The isolated H-NOX protein of  claim 340 , wherein the corresponding wild-type H-NOX protein is a  T. tengcongensis  protein. 
     
     
         342 . The isolated H-NOX protein of  claim 321 , wherein the H-NOX protein does not have a guanylyl cyclase catalytic domain. 
     
     
         343 . The isolated H-NOX protein of  claim 321 , wherein the H-NOX protein is a fusion protein that includes an H-NOX domain and part or all of another protein. 
     
     
         344 . The isolated H-NOX protein of  claims 321 , wherein the mutant H-NOX protein is covalently bound to another molecule or moiety or is part of a fusion protein. 
     
     
         345 . The isolated H-NOX protein of  claim 344 , wherein the mutant H-NOX protein is covalently bound to polyethylene glycol. 
     
     
         346 . A recombinant nucleic acid encoding an H-NOX protein of  claim 321 . 
     
     
         347 . A vector comprising a nucleic acid of  claim 346 . 
     
     
         348 . A cell comprising a nucleic acid of  claim 346  or the vector of  claim 347 . 
     
     
         349 . A method of producing an H-NOX protein comprising culturing a cell comprising a nucleic acid encoding an H-NOX protein of any one of  claim 321  under conditions suitable for production of the protein. 
     
     
         350 . The method of  claim 349 , further comprising the step of purifying the H-NOX protein.