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Details for Patent: 5,580,723

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Details for Patent: 5,580,723

Title: Method for identifying active domains and amino acid residues in polypeptides and hormone variants
Abstract:The invention provides methods for the systematic analysis of the structure and function of polypeptides by identifying active domains which influence the activity of the polypeptide with a target substance. Such active domains are determined by substituting selected amino acid segments of the polypeptide with an analogous polypeptide segment from an analog to the polypeptide. The analog has a different activity with the target substance as compared to the parent polypeptide. The activities of the segment-substituted polypeptides are compared to the same activity for the parent polypeptide for the target. A comparison of such activities provides an indication of the location of the active domain in the parent polypeptide. The invention also provides methods for identifying the active amino acid residues within the active domain of the parent polypeptide. The method comprises substituting a scanning amino acid for one of the amino acid residues within the active domain of the parent polypeptide and assaying the residue-substituted polypeptide so formed with a target substance. The invention further provides polypeptide variants comprising segment-substituted and residue-substituted growth hormones, prolactins and placental lactogens.
Inventor(s): Wells; James A. (Burlingame, CA), Cunningham; Brian C. (Piedmont, CA)
Assignee: Genetech, Inc. (South San Francisco, CA)
Filing Date:Feb 02, 1994
Application Number:08/190,723
Claims:1. A method for identifying at least a first unknown active domain in a region of known amino acid sequence of a naturally occurring parent polypeptide, which parent polypeptide has been cloned and has a preidentified biological activity, said active domain being capable of interacting with a first target when said parent polypeptide is in its native folded form, which interaction is responsible for said biological activity, said method comprising:

(a) comparing amino acid sequence or polypeptide structure in the region of known amino acid sequence of the parent polypeptide with amino acid sequence or polypeptide structure in a region of known amino acid sequence of an analog polypeptide to the parent polypeptide, said parent polypeptide and said analog both interacting with said first target, resulting in said biological activity, but having different interactions with said first target, or said analog interacting with a different target with which said parent polypeptide also interacts;

(b) substituting DNA encoding a first analogous polypeptide segment from the analog to said parent polypeptide into DNA encoding substantially the full length parent polypeptide and expressing a first segment-substituted polypeptide;

(c) contacting said first segment-substituted polypeptide with said first target to determine the interaction, if any, between said first target and said segment-substituted polypeptide;

(d) repeating steps b) and c) using a second analogous polypeptide segment from an analog to said parent polypeptide to form at least a second segment-substituted polypeptide containing said second analogous polypeptide segment, which is different from said first analogous polypeptide segment; and

(e) comparing the difference, if any, between the activity relative to said first target of said parent polypeptide and said first and second segment-substituted polypeptides as an indication of the location of said first active domain in said parent polypeptide.

2. The method of claim 1 wherein said unknown active domain comprises at least two discontinuous amino acid segments in the primary amino acid sequence of said parent polypeptide.

3. The method of claim 1 wherein at least a first selected polypeptide segment of said parent polypeptide replaced by said first analogous polypeptide segment of said analog contains at least one amino acid residue located on the surface of the native-folded form of said parent polypeptide.

4. The method of claim 3 further comprising repeating steps b) and c) until substantially all of the amino acid residues on said surface of said parent polypeptide have been substituted by said analogous polypeptide segments.

5. The method of claim 1 further comprising repeating steps b) and c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of analogous polypeptide segments covering about 15-100% of the amino acid sequence of said parent polypeptide.

6. The method of claim 1 further comprising repeating steps b) and c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of analogous-polypeptide segments covering about 60-100% of the amino acid sequence of said parent polypeptide.

7. The method of claim 1 further comprising identifying a second unknown active domain of said parent polypeptide, said second active domain interacting with a second target, said method comprising repeating steps b) through e) with said second target.

8. The method of claim 1 further comprising identifying at least a first active amino acid residue within said first active domain, said method comprising:

f) substituting a scanning amino acid for a different first amino acid residue within said first active domain to form a first residue-substituted polypeptide;

g) contacting said first residue-substituted polypeptide with said first target to determine the interaction, if any, between said target and said residue-substituted polypeptide;

h) repeating steps f) and g) to substitute a scanning amino acid for at least a second amino acid residue within said first active domain to form at least a second residue-substituted polypeptide; and

i) comparing the difference, if any, between the activity relative to said first target of the parent polypeptide and each of said first and second residue-substituted polypeptides as an indication of the location of said first active amino acid residue in said first active domain.

9. The method of claim 8 further comprising repeating steps (b) through (i) with a second target to identify a second active domain and at least one active amino acid residue within said second active domain.

10. The method of claim 9 further comprising the step of substituting at least one of said active amino acid residues in said first active domain with a different amino acid to produce a polypeptide variant having a modified interaction with said first target but which retains substantially all of the interaction of said parent polypeptide with said second target.

11. The method of claim 10 further comprising the step of substituting at least one of said active amino acid residues in said second active domain with a different amino acid to produce a polypeptide variant having a modified interaction with said first and said second target.

12. The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further comprising substituting at least said one common active amino acid residue with a different amino acid to produce a polypeptide variant having modified interactions with each of said first and said second targets.

13. The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further comprising substituting at least one amino acid residue in said first active domain, other than said at least one common active amino acid residue, with a different amino acid to produce a polypeptide variant having a modified interaction with said first target.

14. The method of claim 1 further comprising:

(f) substituting DNA encoding a scanning amino acid for DNA encoding a first amino acid residue at residue number N within DNA encoding substantially the full length parent polypeptide and expressing an N-substituted polypeptide;

(g) substituting a scanning amino acid for each of the amino acid residues at residue numbers N+1 and N-1 to said first residue to form respectively N+1- and N-1-substituted polypeptides;

(h) contacting each of said substituted polypeptides with said first target to determine the interaction, if any, between said first target and said substituted polypeptides;

(i) comparing the difference, if any, between the activity relative to said first target of the parent polypeptide and said substituted polypeptides; and

(j) repeating steps (g) through (i) for increasing residue numbers if said activity difference between said first target and said N+1-substituted polypeptide is greater than two-fold and for decreasing residue numbers if said activity difference between said first target and said N-1-substituted polypeptide is greater than two-fold.

15. The method of claim 14 wherein steps (g) through (i) are repeated until at least four substituted polypeptides containing ,the substitution of a scanning amino acid at four consecutive residues are identified having less than a two-fold activity difference as compared to said parent polypeptide.

16. The method of claim 1, 8 or 14 wherein said parent polypeptide is selected from the group consisting of human growth hormone, human prolactin, .alpha.-interferon, .gamma.-interferon, tissue plasminogen activator, IGF-1, TGH-.beta..sub.1, EGF, CD-4, TNF, GMCSF, TGF, follicle stimulating hormone, luteinizing hormone, atrial naturietic peptide and placental lactogen.

17. The method of claim 16 wherein said parent polypeptide is selected from the group consisting of human growth hormone, human placental lactogen and human prolactin.

18. The method of claim 8 or 14 wherein said scanning amino acid is an isosteric amino acid.

19. The method of claim 8 or 14 wherein said scanning amino acid is a neutral amino acid.

20. The method of claim 19 wherein said neutral amino acid is selected from the group consisting of alanine, serine, glycine and cysteine.

21. The method of claim 20 wherein said scanning amino acid is alanine.

22. The method of claim 1, 8 or 14 wherein said activity is measured in an in vitro or in vivo assay.

23. The method of claim 22 wherein said parent polypeptide is a hormone and said activity is measured in an in vitro assay using a soluble hormone receptor.

24. The method of claim 23 wherein said hormone is human growth hormone and said soluble hormone receptor is shGHr.

25. The method of claim 23 wherein said hormone is human growth hormone and said soluble hormone receptor is shPRLr.

26. The method of claims 1, 8 or 14 wherein said interaction between said first target and said parent polypeptide involves either binding of said target to said parent polypeptide or catalysis of said target by said parent polypeptide.

27. The method of claim 26 wherein the activity between said first target and any of said substituted polypeptides is increased greater than two-fold as compared to said parent polypeptide.

28. The method of claim 26 wherein the activity between said first target and any of said substituted polypeptides is decreased greater than two-fold as compared to said parent polypeptide.

29. The method of claim 1, wherein the analog has at least 15% amino acid sequence homology with the parent polypeptide.

30. The method of claim 1, wherein the analog is naturally occurring.

31. The method of claim 1, wherein the analog is a tertiary analog.

32. The method of claim 14 wherein said active domain comprises at least two discontinuous polypeptide segments in the primary amino acid sequence of said parent polypeptide.

33. The method of claim 1 wherein said parent polypeptide is human growth hormone and said analog is selected from the group consisting of human placental lactogen, porcine growth hormone, and human prolactin.

34. The method of claim 1 wherein the biological activity of the parent polypeptide is of clinical utility.

35. The method of claim 1 wherein the parent polypeptide is selected from the group consisting of a hormone, enzyme, antigen, receptor, enzyme substrate, binding protein, and enzyme inhibitor.

36. The method of claim 1 wherein said first target is selected from the group consisting of a hormone, enzyme, antibody, antigen, receptor, enzyme substrate, binding protein, and enzyme inhibitor.
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