Compositions and methods for the delivery of oxygen

Abstract

H-NOX proteins are mutated to exhibit improved or optimal kinetic and thermodynamic properties for blood gas O 2 delivery. The engineered H-NOX proteins comprise mutations that impart altered O 2 or NO ligand-binding relative to the corresponding wild-type H-NOX domain, and are operative as physiologically compatible mammalian blood O 2 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 O 2 is beneficial.

1 - 403 . (canceled) 
     
     
         404 . A pharmaceutical blood gas O 2  carrier composition comprising (i) a pharmaceutically effective amount of an H-NOX protein from an aerobic organism, wherein said H-NOX protein binds and delivers O 2  with minimal NO reactivity, wherein the H-NOX protein comprises at least two distal pocket mutations, and wherein the at least two distal pocket mutations comprise a first distal pocket mutation which adds an H-bond donor to the distal pocket and a second distal pocket mutation; and (ii) a pharmaceutically acceptable carrier. 
     
     
         405 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the H-bond donor is a tyrosine residue. 
     
     
         406 . The pharmaceutical blood gas O 2  carrier composition of  claim 405 , wherein the first distal pocket mutation comprises a substitution of an amino acid residue corresponding to a I145Y substitution of a human H-NOX protein. 
     
     
         407 . The pharmaceutical blood gas O 2  carrier composition of  claim 406 , wherein the human H-NOX has the amino acid sequence of SEQ ID NO: 122. 
     
     
         408 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the second distal pocket mutation comprises a mutation of a residue in alpha-helix A, D, E or G of the H-NOX protein. 
     
     
         409 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the second distal pocket mutation comprises a substitution at a residue that corresponds to at least one of Phe4, Ile5, Val8, Leu9, Phe70, Met73, Phe74, Phe75, Phe77, Cys78, or Ile149 of a human H-NOX. 
     
     
         410 . The pharmaceutical blood gas O 2  carrier composition of  claim 406 , wherein the second distal pocket mutation comprises a substitution that corresponds to at least one of Phe4, Ile5, Leu9, Phe74, Phe75, Cys78, Ile142, or Ile149 of a human H-NOX. 
     
     
         411 . The pharmaceutical blood gas O 2  carrier composition of  claim 410 , wherein the human H-NOX has the amino acid sequence of SEQ ID NO: 122. 
     
     
         412 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the H-NOX protein from an aerobic organism is a human H-NOX protein. 
     
     
         413 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the O 2  dissociation constant of the mutant H-NOX protein is between 1 nM and 1 mM at 20° C., and the NO reactivity of the mutant H-NOX protein is less than 700 s −1  at 20° C. 
     
     
         414 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the O 2  dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha. 
     
     
         415 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the O 2  dissociation constant of the H-NOX protein is between 2 nM and 50 μM at 20° C. 
     
     
         416 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the NO reactivity of the H-NOX protein is less than 1 s −1  at 20° C. 
     
     
         417 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the NO reactivity of the H-NOX protein is at least 100-fold lower than that of human hemoglobin alpha. 
     
     
         418 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the k off  for oxygen of the H-NOX protein is between 0.01 s −1  and 200 s −1  at 20° C. 
     
     
         419 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the rate of heme autoxidation of the H-NOX protein is less than 1 h −1  at 37° C. 
     
     
         420 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the H-NOX protein does not contain a guanylyl cyclase catalytic domain. 
     
     
         421 . The pharmaceutical blood gas O 2  carrier composition of  claim 404 , wherein the H-NOX protein is a fusion protein that includes an H-NOX domain and part or all of another protein. 
     
     
         422 . A method of delivering oxygen to an individual comprising administering to the individual in need thereof an H-NOX protein from an aerobic organism in an amount sufficient to deliver an effective amount of oxygen to the individual, wherein the H-NOX protein binds and delivers O 2  with minimal reactivity, wherein the H-NOX protein comprises at least two distal pocket mutations, wherein the at least two distal pocket mutations comprise a first distal pocket mutation which adds an H-bond donor to the distal pocket and a second distal pocket mutation. 
     
     
         423 . The method of  claim 422 , wherein the H-bond donor is a tyrosine residue. 
     
     
         424 . The method of  claim 423 , wherein the first distal pocket mutation comprises a substitution of an amino acid residue corresponding to a I145Y substitution of a human H-NOX protein. 
     
     
         425 . The method of  claim 422 , wherein the O 2  dissociation constant of the mutant H-NOX protein is between 1 nM and 1 mM at 20° C., and the NO reactivity of the mutant H-NOX protein is less than 700 s −1  at 20° C. 
     
     
         426 . The method of  claim 422 , wherein the O 2  dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha. 
     
     
         427 . The method of  claim 422 , wherein the H-NOX protein from an aerobic organism is a human H-NOX protein. 
     
     
         428 . An isolated H-NOX protein from an aerobic organism, wherein the H-NOX protein comprises at least two distal pocket mutations, wherein the at least two distal pocket mutations comprise a first distal pocket mutation which adds an H-bond donor to the distal pocket and a second distal pocket mutation. 
     
     
         429 . The isolated H-NOX protein of  claim 428 , wherein the H-bond donor is a tyrosine residue. 
     
     
         430 . The isolated H-NOX protein of  claim 429 , wherein the first distal pocket mutation comprises a substitution of an amino acid residue corresponding to a I145Y substitution of a human H-NOX protein. 
     
     
         431 . The isolated H-NOX protein of  claim 430 , wherein the human H-NOX has the amino acid sequence of SEQ ID NO: 122. 
     
     
         432 . The isolated H-NOX protein of  claim 428 , wherein the second distal pocket mutation comprises a mutation of a residue in alpha-helix A, D, E or G. 
     
     
         433 . The isolated H-NOX protein of  claim 429 , wherein the second distal pocket mutation comprises a substitution that corresponds to at least one of Phe4, Ile5, Val8, Leu9, Phe70, Met73, Phe74, Phe75, Phe77, Cys78, or Ile149 of a human H-NOX. 
     
     
         434 . The isolated H-NOX protein of  claim 430 , wherein the second distal pocket mutation is comprises a substitution that corresponds to at least one of Phe4, Ile5, Leu9, Phe74, Phe75, Cys78, Ile142, or Ile149 of a human H-NOX. 
     
     
         435 . The isolated H-NOX protein of  claim 434 , wherein the human H-NOX has the amino acid sequence of SEQ ID NO: 122. 
     
     
         436 . The isolated H-NOX protein of  claim 428 , wherein the H-NOX protein from an aerobic organism is a human H-NOX protein. 
     
     
         437 . The isolated H-NOX protein of  claim 428 , wherein the O 2  dissociation constant of the mutant H-NOX protein is between 1 nM and 1 mM at 20° C., and the NO reactivity of the mutant H-NOX protein is less than 700 s −1  at 20° C. 
     
     
         438 . The isolated H-NOX protein of  claim 428 , wherein the O 2  dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha. 
     
     
         439 . The isolated H-NOX protein of  claim 428 , wherein the O 2  dissociation constant of the H-NOX protein is between 2 nM and 50 μM at 20° C. 
     
     
         440 . The isolated H-NOX protein of  claim 428 , wherein the NO reactivity of the H-NOX protein is less than 1 s −1  at 20° C. 
     
     
         441 . The isolated H-NOX protein of  claim 428 , wherein the NO reactivity of the H-NOX protein is at least 100-fold lower than that of human hemoglobin alpha. 
     
     
         442 . The isolated H-NOX protein of  claim 428 , wherein the k off  for oxygen of the H-NOX protein is between 0.01 s −1  and 200 s −1  at 20° C. 
     
     
         443 . The isolated H-NOX protein of  claim 428 , wherein the rate of heme autoxidation of the H-NOX protein is less than 1 h −1  at 37° C. 
     
     
         444 . The isolated H-NOX protein of  claim 428 , wherein the H-NOX protein does not contain a guanylyl cyclase catalytic domain. 
     
     
         445 . The isolated H-NOX protein of  claim 428 , wherein the H-NOX protein is a fusion protein that includes an H-NOX domain and part or all of another protein. 
     
     
         446 . A recombinant nucleic acid encoding an H-NOX protein of  claim 428 . 
     
     
         447 . A vector comprising a nucleic acid of  claim 433 . 
     
     
         448 . A cell comprising a nucleic acid of  claim 433 . 
     
     
         449 . A method of producing an H-NOX protein comprising culturing a cell comprising a nucleic acid encoding an H-NOX protein  claim 428  under conditions suitable for production of the protein. 
     
     
         450 . The method of  claim 436 , further comprising the step of purifying the H-NOX protein.