US-20260126438-A1 - SYSTEMS AND METHOD FOR TARGET ENGAGEMENT

Abstract

Provided herein are systems and methods for the detection and quantification of a target protein via bioluminescence and/or bioluminescent resonance energy transfer (BRET). In particular, provided herein are assay methods using a multi-component biochemical system that assembles into a detectable in vitro chemical complex at a protein target of interest.

Inventors

• Cesear Corona
• Ming Chen
• Wenhui Zhou

Assignees

• PROMEGA CORPORATION

Dates

Publication Date

20260507

Application Date

20250808

Claims (20)

1. 1 . A system comprising: (a) a bioluminescent protein or a bioluminescent complex tethered to a target protein; (b) fluorophore tethered to a reference ligand for the target protein; (c) a test ligand for the target protein; and (d) a substrate for the bioluminescent protein or complex; wherein the bioluminescent protein or complex emits a bioluminescent signal at a first wavelength in the presence of the substrate; wherein the fluorophore absorbs light at the first wavelength and emits a fluorescent signal at the second wavelength; wherein when the reference ligand is bound to the target protein the fluorophore is in proximity to the bioluminescent protein or complex to allow bioluminescence resonance energy transfer (BRET) from the bioluminescent protein or complex; and wherein if the test ligand is capable of binding to the target protein the test ligand competes with the reference ligand for binding to the target protein, thereby decreasing the fluorescent signal resulting from the BRET.
2. 2 - 3 . (canceled)
3. 4 . The system of claim 1 , wherein the bioluminescent protein is an Oplophorous -derived polypeptide that comprises at least 70% sequence identity with SEQ ID NO: 1.
4. 5 . (canceled)
5. 6 . The system of claim 1 , wherein the bioluminescent protein is a circularly permuted variant of an Oplophorous -derived polypeptide.
6. 7 . The system of claim 1 , wherein the bioluminescent protein or a bioluminescent complex tethered to a target protein is a bioluminescent complex; wherein a first component of the bioluminescent complex is tethered to the target protein; wherein a second component of the bioluminescent complex is not tethered to the target protein.
7. 8 . The system of claim 7 , wherein the first and second components of the bioluminescent complex form an active bioluminescent complex upon binding to each other; and wherein the first and second components of the bioluminescent complex require facilitation to form an active bioluminescent complex.
8. 9 . The system of claim 8 , wherein the first and second components of the bioluminescent complex collectively comprise at least 70% sequence identity with SEQ ID NO: 1 or 2.
9. 10 . The system of claim 9 , wherein the first component of the bioluminescent complex comprises at least 70% sequence identity with SEQ ID NO: 3.
10. 11 . (canceled)
11. 12 . The system of claim 10 , wherein the second component of the bioluminescent complex comprises at least 70% sequence identity with SEQ ID NO: 5.
12. 13 . The system of claim 1 , wherein the bioluminescent protein or complex is fused to the target protein directly or by a linker peptide/polypeptide.
13. 14 . The system of claim 1 , wherein the bioluminescent protein or complex is tethered to an affinity molecule capable of binding to the target protein or an affinity tag thereon.
14. 15 . The system of claim 14 , wherein the affinity molecule is an antibody or antibody fragment, and the target protein is tethered to an affinity tag to which the antibody or antibody fragment is capable of binding.
15. 16 . The system of claim 1 , wherein the bioluminescent protein or complex is fused a capture agent capable of covalently binding to a capture ligand tethered to the target protein.
16. 17 . The system of claim 14 , wherein the capture agent is a modified dehalogenase capable of forming a covalent bond to a haloalkyl moiety and the capture ligand is a haloalkyl moiety and wherein the modified dehalogenase comprises at least 70% sequence identity to SEQ ID NO: 8.
17. 18 . (canceled)
18. 19 . The system of claim 1 , wherein the bioluminescent protein or complex is tethered to a secondary affinity molecule capable of binding to a primary affinity molecule capable of binding to the target protein or an affinity tag thereon.
19. 20 . The system of claim 1 wherein the substrate for the bioluminescent protein or complex is a coelenterazine molecule.
20. 21 . (canceled)

Description

CROSS-REFERENCE This application claims the benefit of U.S. Provisional Patent Application No. 63/681,677, filed Aug. 9, 2024, and Provisional Patent Application No. 63/688,120, filed Aug. 28, 2024; each of which is incorporated by reference herein in its entirety. SEQUENCE LISTING PARAGRAPH The text of the computer readable sequence listing filed herewith, titled “PRMG_43257.203_SequenceListing.xml”, created Sep. 12, 2025, having a file size of 8,886 bytes, is hereby incorporated by reference in its entirety. FIELD Provided herein are systems and methods for the detection and quantification of a target protein via bioluminescence and/or bioluminescent resonance energy transfer (BRET). In particular, provided herein are assay methods using a multi-component biochemical system that assembles into a detectable in vitro chemical complex at a protein target of interest. BACKGROUND Identification of protein binding compounds is a fundamental need in drug discovery. These binding compounds are often identified through established screening methods that require a degree of specificity, sensitivity, and robustness to support high throughput. Current assay methods are reliant on the binding of a manageable subset members of a compound library binding with sufficient affinity to be detected, isolated, and identified. Often, especially in the case of challenging or “intractable” targets, identified hit compounds function sufficiently to serve as tool compounds but fail to mature into viable clinical candidates, leading to a shortage of chemical scaffolds for future medicinal chemistry efforts. SUMMARY Provided herein are systems and methods for the detection and quantification of a target protein via bioluminescence and/or bioluminescent resonance energy transfer (BRET). In particular, provided herein are assay methods using a multi-component biochemical system that assembles into a detectable in vitro chemical complex at a protein target of interest. In some embodiments, provided herein are systems comprising: (a) a bioluminescent protein or a bioluminescent complex tethered to a target protein; (b) fluorophore tethered to a reference ligand for the target protein; (c) a test ligand for the target protein; and (d) a substrate for the bioluminescent protein or complex; wherein the bioluminescent protein or complex emits a bioluminescent signal at a first wavelength in the presence of the substrate; wherein the fluorophore absorbs light at the first wavelength and emits a fluorescent signal at the second wavelength; wherein when the reference ligand is bound to the target protein the fluorophore is in proximity to the bioluminescent protein or complex to allow bioluminescence resonance energy transfer (BRET) from the bioluminescent protein or complex; and wherein if the test ligand is capable of binding to the target protein the test ligand competes with the reference ligand for binding to the target protein, thereby decreasing the fluorescent signal resulting from the BRET. In some embodiments, the bioluminescent protein or a bioluminescent complex tethered to a target protein is a bioluminescent protein. In some embodiments, the bioluminescent protein is a luciferase. In some embodiments, the luciferase is an Oplophorous-derived polypeptide that comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 1. In some embodiments, the luciferase comprises 100% sequence identity with SEQ ID NO: 1. In some embodiments, the luciferase is a circularly permuted variant of an Oplophorous-derived polypeptide. In some embodiments, the bioluminescent protein or a bioluminescent complex tethered to a target protein is a bioluminescent complex; wherein a first component of the bioluminescent complex is tethered to the target protein; wherein a second component of the bioluminescent complex is not tethered to the target protein. In some embodiments, the first and second components of the bioluminescent complex form an active bioluminescent complex upon binding to each other; and wherein the first and second components of the bioluminescent complex require facilitation to form an active bioluminescent complex. In some embodiments, the first and second components of the bioluminescent complex collectively comprise at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 1 or 2. In some embodiments, the first component of the bioluminescent complex comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 3. In some embodiments, the first component of the bioluminescent complex has 100% sequence identity with SEQ ID NO: 3. In some embodiments, the second component of the bioluminescent complex comprises at least 70% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 5. In