COMPOSITIONS AND METHODS FOR THE DELIVERY OF OXYGEN
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.