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WO-2026090735-A1 - SPLIT LUCIFERASE-COUPLED SYSTEM FOR DETECTING PROTEIN-PROTEIN INTERACTIONS

WO2026090735A1WO 2026090735 A1WO2026090735 A1WO 2026090735A1WO-2026090735-A1

Abstract

System for detecting interactions between a first a second protein comprising: (a) a first construct comprising the first protein fused to a first handle comprising a β10 peptide or a β9 peptide of a nanoluciferase fused to the N-terminal of a modified N-terminus intein (IN) of GP41-1; and (b) a second construct comprising the second protein fused to a second handle comprising a modified C-terminus intein (IC) of GP41-1 fused at its C-terminal to a β9 peptide when the β10 is fused to the modified IN, or to the β10 peptide when the β9 peptide is fused to the modified IN. Interaction between the first and second proteins reconstitutes the IN and IC into the GP41-1 that induces splicing of the reconstituted GP41-1 from the first handle and the second handle, ligating the first and the second proteins and arranging the β10 peptide and β9 peptide in a fused tandem.

Inventors

  • YAO, Zhong
  • STAGLJAR, IGOR

Assignees

  • THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO

Dates

Publication Date
20260507
Application Date
20251028
Priority Date
20241029

Claims (20)

  1. CLAIMS
  2. Therefore, what is claimed is:
  3. 1. A system for detecting interactions between a first protein or fragment thereof (bait protein) and a second protein or fragment thereof (prey protein), the system comprising two separate constructs:
  4. (a) a first construct comprising the bait protein and a first handle connected to the bait protein, the first handle comprising (i) a β10 peptide of a nanoluciferase (NanoLuc) fused to the N-terminal side of a modified N-terminus intein of GP41-1 (IN), or (ii) a β9 peptide of the NanoLuc fused to the N-terminal side of the modified IN; and
  5. (b) a second construct comprising the prey protein and a second handle fused to the prey protein, the second handle comprising a modified C-terminus intein of GP41-1 (IC) fused at the C-terminal side (i) to the β9 peptide of the NanoLuc when the β10 peptide of the NanoLuc is fused to the N-terminal side of the modified IN, or (ii) to the β10 peptide of the NanoLuc when the β9 peptide of the NanoLuc is fused to the N-terminal side of the modified IN.
  6. 2. The system of claim 1, wherein the first handle comprises the β10 peptide of the NanoLuc fused to the N-terminal side of the modified IN; and the second handle comprises the modified IC fused at the C-terminal side to the β9 peptide of the NanoLuc.
  7. 3. The system of claim 1 or claim 2, wherein
  8. (i) the modified IC includes amino acid residues at positions 13 to 37 of wild type IC of GP41-1, and the modified IN includes amino acid residues at positions 1 to 88 of the wild type IN of GP41-1 fused to amino acid residues 1 to 12 of wild type IC of GP41 -1 (C25 GP41 -1 split intein), or (ii) the modified IC includes amino acids at positions 14 to 37 of wild type IC of GP41-1 and the modified IN includes amino acid residues at positions 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 13 of wild type IC of GP41-1 (C24 GP41-1 split intein), or (iii) the modified IC includes amino acids at positions 15 to 37 of wild type IC of GP41-1 and the modified IN includes amino acid residues at positions 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 14 of wild type IC of GP41-1 (C23 GP41-1 split intein).
  9. 4. The system of claim 1 or claim 2, wherein the modified IN comprises SEQ ID NO: 3 and the modified IC comprises SEQ ID NO: 4, or the modified IN comprises SEQ ID NO: 5 and the modified IC comprises SEQ ID NO: 6, or the modified IN comprises SEQ ID NO: 7 and the modified IC comprises SEQ ID NO: 8.
  10. 5. The system according to any one of claims 1 to 4, wherein the β9 peptide comprises SEQ ID NO: 12 and the β10 peptide comprises SEQ ID NO: 14.
  11. 6. The system according to any one of claims 2 to 4, wherein the first handle comprises SEQ ID NO: 16 and the second handle comprises SEQ ID NO: 18. 7. The system according to any one of claims 1 to 6, wherein the first handle is connected to the N-terminus of the bait protein.
  12. 8. The system according to any one of claims 1 to 6, wherein the first handle is connected to the C-terminus of the bait protein.
  13. 9. The system according to any one of claims 1 to 8, wherein the second handle is connected to the N-terminus of the prey protein.
  14. 10. The system according to any one of claims 1 to 8, wherein the second handle is connected to the C-terminus of the prey protein.
  15. 11. The system according to any one of claims 1 to 10, wherein the first construct and the second construct further comprise a tag, wherein the tag is one or more of a V5 tag, an HA tag, and a 3xFLAG tag.
  16. 12. A method for detecting the interaction between a first protein or part thereof (bait protein) and a second protein or part thereof (prey protein) comprising: (a) providing the system according to any one of claims 1 to 11;
  17. (b) incubating the first construct and the second construct in a homogenous liquid phase under conditions that allow the formation of the GP41-1; and
  18. (c) adding to the incubate of (b) a Δ11S peptide of NanoLuc and a substrate that generates a luminescence signal in the presence of NanoLuc, whereby detection of the luminescence signal being indicative that the bait protein or part thereof and the prey protein or part thereof interact.
  19. 13. The method of claim 12, wherein the method further comprises comparing the luminescence signal with the luminescence signal of a control to determine a binding strength between the bait protein and the prey protein.
  20. 14. A method for screening a binding inhibitor between two proteins, the method comprising:

Description

SPLIT LUCIFERASE-COUPLED SYSTEM FOR DETECTING PROTEIN-PROTEIN INTERACTIONS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to United States Provisional Patent Application No. 63/713,170, filed October 29, 2024, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on October 23, 2025, is named “180354.0098. xml” and is 27,448 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. FIELD OF THE DISCLOSURE The present disclosure relates to novel split luciferase-coupled systems and methods for detecting protein-protein interactions with an in vivo genetic system. BACKGROUND OF THE DISCLOSURE Protein-protein interactions (PPIs) are fundamental biochemical steps in cellular processes1-6. Their alterations are involved in various diseases such as cancer, making PPIs attractive therapeutic targets78. However, it is notoriously difficult to target PPIs directly due to the common characteristics of interacting surfaces, which are usually large and flat. Accordingly, PPIs were long considered as “undruggable”. This scenario has been subject to change, however, due to an evolving understanding of protein interaction interfaces and the recent development of new targeting strategies9-11. Indeed, the milestone PPI inhibitor, venetoclax12, which targets the BCL2/BAX interaction, has been approved by the FDA for treating chronic lymphocytic leukemia, small lymphocytic leukemia, and acute myeloid leukemia. Numerous PPI inhibitors are also currently under development, with many undergoing clinical trials13. In line with inhibitors, therapeutic potential was also demonstrated for other types of small molecule PPI modulators such as molecular glues that rewire cellular functions through artificially enhancing or enforcing target PPIs14, Proteolysis Targeting Chimeras (PROTACs) that direct targets to the ubiquitin proteasome system for degradation15’16, or other chemicals operating through the modality of enhanced proximity17 Comprehensive examination of PPIs, both in-depth mechanistic investigation and proteome-scale exploration, is essential for driving biological understanding and drug discovery. However, PPI research heavily depends on technology development. Although numerous techniques for the study of PPIs are available, each one is accompanied by certain limitations2’18. Moreover, many methods, whether biophysical or biochemical, are not ideal for applications in PPI-targeted drug discovery. Thus, continued technology advancement is needed to maximize our capabilities in exploring PPIs. To meet this demand, we developed a method called Split-Intein Medicated Protein Ligation (SIMPL)19 For this, we first engineered the GP41-1 split intein and created a version with markedly reduced intrinsic affinity between its two fragments - N-terminal intein (IN) and C-terminal intein (IC) - but without deterioration of its intein activity. Next, we fused the engineered IN and IC, along with two different peptide tags, respectively to two proteins of interest (which we refer to generally as ‘B’ and ‘P’ for convenience). If a PPI occurs between them, spatial proximity allows the IN and IC to reconstitute into a functional enzyme, which splices the B and P proteins into an intact polypeptide or transfers an associated tag from one protein to its partner (depending on the configuration of the constructs). SIMPL demonstrates marked sensitivity and specificity and can be applied to different cellular compartments and various organisms. As part of our initial development of the technology, we demonstrated that SIMPL is compatible with high-throughput screening (HTS) via coupling to the enzyme-linked immunosorbent assay (ELISA). This SIMPL-ELISA platform can also be used for characterizing PPI inhibitors. However, the cumbersome procedures involved in ELISA and the associated expense do not make SIMPL-ELISA an ideal choice for use as an HTS assay. Alternatively, SIMPL can be coupled with homogeneous time-resolved fluorescence (HTRF)20, a platform with significantly improved operability. Unfortunately, the cost of reagents and equipment for HTRF limits its widespread application. A recently developed tri-part strategy splits NanoLuc® (NLuc), the brightest luciferase identified so far, into three fragments: two short peptides (β9 and β10 each containing 11 amino acids) and one 16 kDa fragment (Δ11S)21’22. When β9 and β10 are close, these peptides rapidly bind to Δ11S, and NanoLuc assembles and achieves the catalytic activity of luciferase. To obtain a better luciferase signal, it would be advantageous to present β9 and β10 as one peptide fragment instead o