Compositions and methods for the delivery of nitric oxide
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. A pharmaceutical blood gas NO carrier composition comprising (i) a pharmaceutically effective amount of an H-NOX protein, wherein said H-NOX protein binds and delivers NO 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 NO carrier composition of claim 1 , wherein 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 0.012 s −1 at 37° C.
3. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein 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 wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 .
4. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein 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 wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C.
5. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C., and wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C.
6. The pharmaceutical blood gas NO 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., and wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 .
7. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein the distal pocket mutation alters the NO dissociation constant, the k off for NO, the k 1 for NO, the k 2 for NO, the O 2 dissociation constant, the NO stability, the rate of heme autoxidation, or any combination of two or more of the foregoing compared to that of a corresponding wild-type protein.
8. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein the H-NOX protein is a mammalian protein or is derived from a mammalian protein.
9. The pharmaceutical blood gas NO carrier composition of claim 1 , 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.
10. The pharmaceutical blood gas NO carrier composition of claim 1 , comprising one or more liposomes or nanoparticles that comprise the H-NOX protein.
11. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein the H-NOX protein is covalently bound to another molecule or moiety.
12. The pharmaceutical blood gas NO carrier composition of claim 11 , wherein the H-NOX protein is covalently bound to polyethylene glycol.
13. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein the NO 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.
14. The pharmaceutical blood gas NO carrier composition of claim 1 , wherein 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 wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C.
15. A kit comprising (i) a pharmaceutical composition of an H-NOX protein, wherein said H-NOX protein binds and delivers NO 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 NO to an individual.
16. The kit of claim 15 , wherein 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 wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C.
17. The kit of claim 15 , wherein the H-NOX protein binds and delivers NO with minimal NO reactivity, and wherein the NO 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.
18. A method of delivering NO 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 NO to the individual, wherein said H-NOX protein binds and delivers NO 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.
19. The method of claim 18 , wherein 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 0.012 s −1 at 37° C.
20. The method of claim 18 , wherein 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 −4 at 37° C., and wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C.
21. The method of claim 18 , wherein 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 wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 .
22. The method of claim 18 , wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C., and wherein the NO reactivity of the H-NOX protein is less than about 700 s −1 .
23. The method of claim 18 , wherein 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 wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C.
24. The method of claim 18 , wherein the H-NOX protein comprises at least one mutation that alters the NO dissociation constant, the k off for NO, the k 1 for NO, the k 2 for NO, the O 2 dissociation constant, the NO stability, the rate of heme autoxidation, or any combination of two or more of the foregoing compared to that of a corresponding wild-type protein.
25. The method of claim 18 , wherein the H-NOX protein is a mammalian protein or is derived from a mammalian protein.
26. The method of claim 18 , 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.
27. The method of claim 18 , wherein one or more liposomes or nanoparticles comprise the H-NOX protein.
28. The method of claim 18 , wherein the H-NOX protein is covalently bound to another molecule or moiety.
29. The method of claim 28 , wherein the H-NOX protein is covalently bound to polyethylene glycol.
30. The method of claim 18 , wherein the individual is a human.
31. The method of claim 18 , wherein the individual is suffering from or at risk for a cardiovascular condition, hypertension, a condition exacerbated by hypertension, a vasoconstrictive condition, stroke, a functional NO deficiency, heart failure, or renal failure.
32. The method of claim 18 , wherein the NO 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.
33. The method of claim 18 , wherein 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 wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C.