Novel polypeptides with improved proteolytic stability, and methods of preparing and using same
AssigneesTufts University, Tufts Medical Center, Inc.
InventorsKRISHNA KUMAR, VITTORIO MONTANARI, MARTIN BEINBORN, VENKATA RAMAN
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Abstract
The present invention includes methods of improving proteolytic stability of a polypeptide, comprising alkylating at least one selected from the group consisting of a N-terminus amino group, the NH group of the N-terminus first internal amide bond, another primary amino group, a thiol group and a thioether group within the polypeptide. The present invention further includes polypeptides incorporating such chemical modifications.
Claims32
A chemically modified polypeptide, or a salt or solvate thereof, wherein the polypeptide has at least one of the following chemical modifications:
- (i) at least one selected from the group consisting of an N-terminus amino group, the NH of the N-terminus first internal amide bond, other free primary amino group, thiol group and thioether group of the polypeptide is independently derivatized with optionally substituted C1-C16 alkyl, optionally substituted C3-C16 cycloalkyl, optionally substituted C3-C16 aryl, optionally substituted C2-C16 alkenyl or optionally substituted C2-C16 alkynyl;
- and
- (ii) the NH group of at least one selected from the group consisting of the N-terminus amino group and the N-terminus first internal amide bond is derivatized with X, wherein each X is independently selected from the group consisting of optionally substituted phenyl, optionally substituted benzyl, optionally substituted —(CR2)1-6-phenyl, →O, —OH, —OR, alkoxy, NH2, optionally substituted NH(C1-C16 alkyl) and optionally substituted N(C1-C16 alkyl)(C1-C16 alkyl), where R is independently selected at each occurrence from hydrogen or optionally substituted C1-C16 alkyl;
- wherein the chemically modified polypeptide has essentially the same biological activity and/or is more resistant to proteolytic degradation, as compared to the corresponding non-chemically modified polypeptide.
3-6. (canceled)
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15-19. (canceled)
23-24. (canceled)
A chemically modified polypeptide selected from the group consisting parathyroid hormone (PTH), PYY3-36, cholecystokinin, PYY1-36, corticotropin releasing hormone receptor 1 or 2, growth hormone releasing factor, glucagon, Exendin, GLP-1, gastric inhibitory peptide, Liraglutide, prealbumin, peptide HI-27, PACAP, secretin, and vasoactive intestinal peptide (VIP),
- wherein the N-terminus amino group is derivatized with (i) one selected from the group consisting of optionally substituted C1-C16 alkyl, optionally substituted C3-C16 cycloalkyl, optionally substituted C2-C16 alkenyl and optionally substituted C2-C16 alkynyl, or (ii) one selected from the group consisting of optionally substituted phenyl, optionally substituted benzyl, →O, —OH, alkoxy, NH2, NH(C1-C16 alkyl) and N(C1-C16 alkyl)(C1-C16 alkyl),
- wherein the derivatization stabilizes the polypeptide against degradation by at least one selected from the group consisting of Acylamino-acid-releasing enzyme, DPP4, DPP2, DPP8, DPP9, all S9B family Oligopropyl peptidases and a protease with ≥50% homology to DPP4 and/or DPP2, without reducing the polypeptide's biological activity relative to the corresponding non-chemically modified protein.
A method of increasing the in vivo half-life of a polypeptide improving the blood-brain barrier permeability or oral bioavailability as compared to the corresponding non-chemically modified polypeptide, wherein the method comprises at least one the following chemical modifications:
- (i) chemically modifying at least one substituent selected from the group consisting of an N-terminus amino group, the NH group of the N-terminus first internal amide bond, other free primary amino group, thiol group and thioether group of the polypeptide, wherein each chemical modification independently comprises derivatizing the substituent with an optionally substituted C1-C16 alkyl, optionally substituted C3-C16 cycloalkyl, optionally substituted C3-C16 aryl, optionally substituted C2-C16 alkenyl or optionally substituted C2-C16 alkynyl, and
- (ii) chemically modifying at least one substituent selected from the N-terminus amino group and the NH of the independently comprises derivatizing the substituent with the group X, wherein each occurrence of X is independently selected from the group consisting of optionally substituted phenyl, optionally substituted benzyl, optionally substituted —(CR2)1-6-phenyl, →O, —OH, alkoxy, NH2, NH(C1-C16 alkyl) and N(C1-C6 alkyl)(C1-C16 alkyl), where R is independently selected at each occurrence from hydrogen or optionally substituted C1-C16 alkyl.
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A method for characterizing a gastrinoma in a subject, the method comprising administering to the subject a chemically modified secretin polypeptide of claim 24, and detecting an increase in gastrin in the subject, thereby characterizing the presence or absence of a gastrinoma in the subject.
48-50. (canceled)
Dependencies are inferred from the claim text.