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. A pharmaceutical blood gas O 2 carrier composition comprising (i) a pharmaceutically effective amount of an H-NOX protein, wherein said H-NOX protein binds and delivers O 2 with minimal NO reactivity, wherein the H-NOX protein comprises a distal pocket mutation, and wherein the H-NOX protein does not comprise a guanylyl cyclase catalytic domain; and (ii) a pharmaceutically acceptable carrier.
2. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the O 2 dissociation constant of the H-NOX protein is between about 2 nM and about 50 μM at 20° C.
3. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the NO reactivity of the H-NOX protein is less than about 1 s −1 at 20° C.
4. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the NO reactivity of the H-NOX protein is at least 100-fold lower than that of hemoglobin.
5. The pharmaceutical blood gas O 2 carrier composition of claim 4 , wherein the hemoglobin is Homo sapiens hemoglobin alpha.
6. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the k off for oxygen of the H-NOX protein is between about 0.01 s −1 and about 200 s −1 at 20° C.
7. The pharmaceutical blood gas O 2 carrier composition of claim 6 , wherein the k off for oxygen of the H-NOX protein is between about 1.0 s −1 and about 16.0 s −1 at 20° C.
8. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the k off for oxygen of the protein is less than or equal to about 0.65 s −1 at 20° C.
9. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C.
10. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the H-NOX protein comprises at least one mutation that is not in the distal pocket.
11. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the H-NOX protein is a mammalian protein or is derived from a mammalian protein, wherein the H-NOX protein is a bacterial protein or is derived from a bacterial protein, wherein the H-NOX protein is an insect protein or is derived from an insect protein, or wherein the H-NOX protein is a worm protein or is derived from a worm protein.
12. The pharmaceutical blood gas O 2 carrier composition of claim 1 , comprising one or more liposomes or nanoparticles that comprise the H-NOX protein.
13. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the H-NOX protein is covalently bound to another molecule or moiety.
14. The pharmaceutical blood gas O 2 carrier composition of claim 13 , wherein the H-NOX protein is covalently bound to polyethylene glycol.
15. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 at 20° C., wherein the O 2 dissociation constant of the H-NOX protein is between about 2 nM and about 50 μM at 20° C., wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C., and wherein the k off for oxygen of the H-NOX protein is between about 0.01 and about 200 s −1 at 20° C.
16. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the O 2 dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of hemoglobin.
17. The pharmaceutical blood gas O 1 carrier composition of claim 1 , wherein the O 2 dissociation constant of the H-NOX protein is between about 1 nM and about 1 mM at 20° C., and wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 at 20° C.
18. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the distal pocket mutation is at an amino acid position corresponding to position 144 of T. tengcongensis H-NOX of SEQ ID NO:54.
19. The pharmaceutical blood gas O 18 carrier composition of claim 18 , wherein the distal pocket mutation is a L144F mutation.
20. The pharmaceutical blood gas O 2 carrier composition of claim 1 , wherein the H-NOX protein is a T. tengcongensis H-NOX protein comprising a L144F mutation in SEQ ID NO: 54.
21. A kit comprising (i) a pharmaceutical composition comprising a pharmaceutically effective amount of an H-NOX protein and a pharmaceutically acceptable carrier, wherein the H-NOX protein binds and delivers O 2 with minimal NO reactivity, wherein the H-NOX protein comprises a distal pocket mutation, and wherein the H-NOX protein does not comprise a guanylyl cyclase catalytic domain, and (ii) instructions for using the kit to deliver oxygen to an individual.
22. The kit of claim 21 , wherein the k off for oxygen of the H-NOX protein is between about 1.0 s −1 and about 16.0 s −1 at 20° C.
23. The kit of claim 21 , wherein said H-NOX protein binds and delivers O 2 with minimal NO reactivity, and wherein the O 2 dissociation constant of the H-NOX protein is between about 1 nM and about 1 mM at 20° C., and wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 at 20° C.
24. The kit of claim 21 , wherein said H-NOX protein binds and delivers O 2 with minimal NO reactivity, and wherein the O 2 dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of hemoglobin.
25. The kit of claim 21 , wherein the distal pocket mutation is at an amino acid position corresponding to position 144 of T. tengcongensis H-NOX of SEQ ID NO:54.
26. The kit of claim 25 , wherein the distal pocket mutation is a L144F mutation.
27. The kit of claim 25 , wherein the H-NOX protein is a T. tengcongensis H-NOX protein comprising a L144F mutation.
28. A method of delivering oxygen to an individual comprising administering to an individual in need thereof an H-NOX protein 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 NO reactivity, wherein the H-NOX protein comprises a distal pocket mutation, and wherein the H-NOX protein does not comprise a guanylyl cyclase catalytic domain.
29. The method of claim 28 , wherein the individual is suffering from or at risk for hemorrhagic shock or hemodilution.
30. The method of claim 28 , wherein the O 2 dissociation constant of the H-NOX protein is between about 2 nM and about 50 μM at 20° C.
31. The method of claim 28 , wherein the NO reactivity of the H-NOX protein is less than about 1 s −1 at 20° C.
32. The method of claim 28 , wherein the NO reactivity of the H-NOX protein is at least 100-fold lower than that of hemoglobin.
33. The method of claim 28 , wherein the k off for oxygen of the H-NOX protein is between about 0.01 s −1 and about 200 s −1 at 20° C.
34. The method of claim 33 , wherein the k off for oxygen of the H-NOX protein is between about 1.0 s −1 and about 16.0 s −1 at 20° C.
35. The method of claim 28 , wherein the k off for oxygen of the protein is less than or equal to about 0.65 s −1 at 20° C.
36. The method of claim 28 , wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C.
37. The method of claim 28 , wherein the H-NOX protein comprises at least one mutation that is not in the distal pocket.
38. The method of claim 28 , wherein the H-NOX protein is a mammalian protein or is derived from a mammalian protein, wherein the H-NOX protein is a bacterial protein or is derived from a bacterial protein, wherein the H-NOX protein is an insect protein or is derived from an insect protein, or wherein the H-NOX protein is a worm protein or is derived from a worm protein.
39. The method of claim 28 , wherein the hemoglobin is Homo sapiens hemoglobin alpha.
40. The method of claim 28 , wherein the O 2 dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of hemoglobin.
41. The method of claim 28 , wherein the O 2 dissociation constant of the H-NOX protein is between about 1 nM and about 1 mM at 20° C., and wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 at 20° C.
42. The method of claim 28 , wherein the distal pocket mutation is at an amino acid position corresponding to position 144 of T. tengcongensis H-NOX of SEQ ID NO:54.
43. The method of claim 42 , wherein the distal pocket mutation is a L144F mutation.
44. The method of claim 42 , wherein the H-NOX protein is a T. tengcongensis H-NOX protein comprising a L144F mutation.