De Novo Designed Cortisol Biosensor

Patent metadata

Field	Value
Publication number	US-2025154213-A1
Application number	US-202418945961-A
Country	US
Kind code	A1
Filing date	Nov 13, 2024
Priority date	Nov 15, 2023
Publication date	May 15, 2025
Grant date	—

Abstract

Polypeptides are disclosed having an amino acid sequence at least 50% identical to the amino acid sequence of SEQ ID NO:1, wherein, relative to SEQ ID NO:1, residue 43 is I, residue 95 is Q, and residue 128 is L, fusion proteins thereof, kits thereof, and methods for using the polypeptides for treating a disorder associated with cortisol, or for detecting cortisol in a biological sample.

First claim

We claim:

1 . A polypeptide comprising an amino acid sequence at least 50% identical to the amino acid sequence of SEQ ID NO:1, wherein, relative to SEQ ID NO:1, residue 43 is I, residue 95 is Q, and residue 128 is L.

2 . The polypeptide of claim 1 , wherein substitutions relative to SEQ ID NO:1 are selected from the residues shown in the substitution column on Table 1.

3 . The polypeptide of claim 1 , wherein, relative to SEQ ID NO:1, the polypeptide is identical at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 of the identified residues that interact with cortisol.

4 . The polypeptide of claim 1 , wherein substitutions relative to the SEQ ID NO:1 are conservative amino acid substitutions.

5 . A fusion protein comprising the polypeptide of claim 1 fused to one or more functional domains.

6 . A polypeptide comprising an amino acid sequence at least 50% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:2-21.

7 . The polypeptide of claim 6 , wherein, relative to the reference amino acid sequence, the polypeptide is identical at 1, 2, 3, 4, 5, 6, 7, 8, or all of the identified residues that form an interface with (a) cortisol and (b) a polypeptide comprising an amino acid sequence at least 50% identical to the amino acid sequence of SEQ ID NO:1, wherein, relative to SEQ ID NO:1, residue 43 is I, residue 95 is Q, and residue 128 is L.

8 . The polypeptide of claim 6 , wherein substitutions relative to the reference sequence are conservative amino acid substitutions.

9 . A fusion protein comprising the polypeptide of claim 6 fused to one or more functional domains.

10 . A nucleic acid encoding the polypeptide of claim 1 .

11 . An expression vector comprising the nucleic acid of claim 10 operatively linked to a suitable control sequence.

12 . A host cell comprising the expression vector of claim 11 .

13 . A pharmaceutical composition, comprising:
(a) the polypeptide of claim 1 ; and (b) a pharmaceutically acceptable carrier.

14 . A kit, comprising:
(a) one or more first polypeptides of claim 1 , or a nucleic acid encoding the one or more first polypeptides; and (b) one or more second polypeptides comprising an amino acid sequence at least 50%, identical to the amino acid sequence selected from SEQ ID NO:2-21, or a nucleic acid encoding the one or more second polypeptides.

15 . A method for treating a disorder associated with cortisol, comprising administering to a subject in need thereof an amount effective to treat the disorder of the polypeptide of claim 1 .

16 . A method for detecting cortisol in a biological sample, comprising
(a) contacting the biological sample with an amount of the polypeptide of claim 1 effective bind to cortisol in the biological sample to produce a binding complex, and (b) detecting the binding complex in the biological sample, thereby detecting cortisol in the biological sample.

17 . A method for detecting cortisol in a biological sample, comprising
(a) contacting the biological sample with:
(i) one or more first polypeptides of claim 1 ; and
(ii) one or more second polypeptides comprising an amino acid sequence at least 50%, identical to the amino acid sequence selected from SEQ ID NO:2-21; and
(b) detecting a ternary binding complex in the biological sample between (i) the first polypeptide or fusion protein, (ii) the second polypeptide or fusion protein, and (iii) cortisol present in the biological sample, thereby detecting cortisol in the biological sample.

18 . The method of claim 17 , wherein the detectable protein is selected from the group consisting of, bioluminescence resonance energy transfer (BRET) reporters, bimolecular fluorescence complementation (BiFC) reporters, fluorescence resonance energy transfer (FRET) reporters, colorimetry reporters, cell survival reporters, electrochemical reporters, radioactive reporters, and molecular barcode reporters.

Assignees

• Univ Washington

Inventors

• Yeh Hsien-Wei
• Baker David

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• G01N2333/575

  Hormones · CPC title

• G01N33/743

  Steroid hormones · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title