TLX fluorescent probe compound and preparation method and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a TLX fluorescent probe compound and a preparation method and application thereof. The compound can be used as a fluorescent probe for screening a TLX ligand, is simple to prepare, stable in structure, high in sensitivity, fast in detection speed, suitable for high-throughput screening of the TLX ligand, and uses a competitive binding TR-FRET screening system (different from a traditional fluorescence polarization system) when used for screening the TLX ligand, thereby providing an effective system for screening more drugs targeting the TLX.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a TLX fluorescent probe compound, its preparation method, and its application. Background Technology

[0002] The nuclear receptor tailless protein (TLX) is a member of the nuclear receptor subfamily 2 group E member 1 (NR2E1). It is a transcription factor, a homolog of the Drosophila tailless gene, and is highly expressed in the human eye and brain, known for regulating neurogenesis and retinal progenitor cell proliferation. TLX plays a crucial role in neural stem cell activation, participating in the self-renewal and division of neural stem cells from the embryonic stage in vertebrates until adulthood, maintaining the differentiation of terminal neurons in the neural stem cell population, and managing the ecological localization of stem cells. In adults, it is a transcriptional repressor, inhibiting neural cell differentiation by recruiting co-repressors to form a complex. As a regulator of neurogenesis, inverse TLX activation can activate neural stem cells, making it a potential target for treating neurodegenerative diseases such as Alzheimer's and Parkinson's. Simultaneously, dysregulation of TLX expression has been confirmed in studies under various pathological conditions; TLX activation can enhance the inhibition of cancer cell spread and migration and regulate prognosis and recurrence, making it a potential target for treating gliomas, neuroblastomas, and other brain tumors. Numerous studies have demonstrated that TLX possesses significant potential application value and drug target characteristics. Furthermore, TLX shares the same DNA-binding domain and ligand-binding domain (LBD) as other nuclear receptors. Figure 1 (As shown in A and B), however, there is currently no very effective TLX modulator, which hinders further verification of it.

[0003] High-throughput drug screening (HTS) is a method for discovering potential compounds or drug candidates and efficiently screening their interactions with protein targets on a large scale. It is a key step in modern drug discovery, aiming to accelerate the process and improve the efficiency and success rate of drug development. However, previously reported TLX ligand discovery and structure optimization detection mechanisms are primarily based on the cellular level, necessitating the development of simpler, more efficient, and reliable molecular-level high-throughput drug screening systems. High-throughput screening at the protein level can rapidly assess the interactions of a large number of compounds with specific proteins within a short time. Common detection methods include fluorescence polarization-based (FPS) screening. Figure 2) and Time-Resolved Fluorescent Resonance Energy Transfer (TR-FRET), Figure 3 Both methods utilize the properties of fluorescent molecules to study intermolecular interactions. Both methods offer high sensitivity and the ability to detect low-concentration samples, making them suitable for high-throughput screening and quantitative analysis.

[0004] FP (Fluorescent Phosphor) measures the polarization direction of fluorescent molecules. When a fluorescent tracer binds to a target protein, it transitions from low polarization to high polarization to form a larger complex. It primarily provides spatial orientation information of the fluorescent molecule, but its drawback is the need to purify large quantities of protein. TR-FRET (Transient-Fluorescent Phosphor) measures the energy transfer process between the donor and acceptor, focusing on measuring this process. It requires a lanthanide donor (such as terbium-Tb) and a known acceptor fluorescently labeled peptide (such as a polypeptide). For an electronic transition energy transfer to occur, the spatial distance between the donor and acceptor must be within a certain range. Within the range, such as Figure 3 As shown, the fluorescent donor Tb first specifically binds to the Histidine (His) tag. Then, the his-TLX-LBD protein binds to the agonist / inhibitor ligand, recruiting co-activating / inhibiting peptides. Next, the donor fluorophore absorbs energy and is excited upon photoexcitation (EX), transferring electron energy to the polypeptide with the fluorescent acceptor, resulting in energy transfer. Before binding, the donor and acceptor are in a directly excited state; the donor emits light within milliseconds, while the acceptor emits light within nanoseconds. With energy transfer between the donor and acceptor, the acceptor's emission (EM) becomes millisecond-level, allowing for time-resolved measurements to understand protein-compound binding. Building upon TR-FRET, the TR-FRET competitive binding assay not only replaces expensive fluorescent peptides with small molecule probes (different names: fluorescent ligands, tracers) but also simplifies the procedure, lowers the experimental threshold, and increases detection accuracy and sensitivity. Figure 4 Similar to TR-FRET, when the target protein binds to the fluorescent ligand probe, the energy of photoexcitation is transferred from the terbium-labeled donor end of the nuclear acceptor TLX-LBD to the acceptor end of the fluorescent probe. If the analyte compound is added, it competitively displaces the fluorescent ligand probe from the drug delivery site of the TLX-LBD, resulting in the loss of the FRET signal between the donor and acceptor, and a decrease in the overall detection signal intensity. At the same time, the gradient relationship between the competing compound and the fluorescence signal can be determined to find the Ki value. Summary of the Invention

[0005] The first aspect of this invention is to provide a compound.

[0006] The second aspect of this invention aims to provide a method for preparing the compound of the first aspect of this invention.

[0007] A third aspect of the present invention is to provide the application of the compounds of the first aspect of the present invention.

[0008] The fourth aspect of this invention is to provide a reagent.

[0009] The fifth aspect of this invention is to provide a reagent kit.

[0010] The sixth aspect of this invention aims to provide a detection system.

[0011] The seventh aspect of this invention aims to provide a method for screening TLX ligands.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a compound having the chemical structural formula shown in formula (I):

[0014]

[0015] In equation (Ⅰ), R 1 Selected from

[0016] R 2 Selected from

[0017] Preferably, R 1 In this context, n is an integer.

[0018] Preferably, R 1 In this context, n can be 0, 1, 2, 3, or 4.

[0019] Preferably, R 2 Selected from R 1 Selected from -(CH2)3-, -(CH2)4-, -CH2-CH2-O-CH2-CH2-.

[0020] More preferably, R 2 Selected from R 1 Selected from -(CH2)4- and -CH2-CH2-O-CH2-CH2-.

[0021] More preferably, R 2 Selected from R 1 Selected from -CH2-CH2-O-CH2-CH2-.

[0022] Preferably, R 2 Selected from R 1 Selected from -(CH2)0-, -(CH2)4-, -CH2-CH2-O-CH2-CH2-.

[0023] Preferably, R 2 Selected from R 1 Selected from -(CH2)4- and -(CH2)0-.

[0024] Preferably, R 2 Selected from R 1 Selected from -(CH2)4-.

[0025] Preferably, the compound is selected from compound A, compound B, compound C, compound D, compound E, compound F, compound G, compound H, compound I, compound J, compound K, and compound L (the specific structural formulas of each compound are shown in Table 1); further selected from compound D, compound E, compound F, compound G, compound K, and compound L; even further selected from compound F, compound K, and compound G; and still even further selected from compound F.

[0026] A second aspect of the present invention provides a method for preparing the compound of the first aspect of the present invention, comprising the following steps:

[0027] S1: The ligand is reacted with an organic intermediate via an amide reaction to obtain intermediate 1a, wherein the organic intermediate is selected from mono-Boc hydrazine, mono-Boc methyldiamine, mono-Boc ethylenediamine, mono-Boc propylenediamine, mono-Boc butyldiamine, and mono-Boc-2,2'-oxydiethylamine, and the ligand is selected from 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid and 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid;

[0028] S2: The intermediate 1a is deprotected, and the deprotected intermediate 1a undergoes a nucleophilic addition reaction with fluorescein isothiocyanate to obtain the compound of the first aspect of the present invention.

[0029] Preferably, the ligand is selected from 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid, and the organic intermediate is selected from mono-Boc propylenediamine, mono-Boc butylenediamine, and mono-Boc-2,2'-oxydiethylamine.

[0030] Preferably, the ligand is selected from 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid, and the organic intermediate is selected from mono-Boc butanediamine and mono-Boc-2,2'-oxydiethylamine.

[0031] Preferably, the ligand is selected from 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid, and the organic intermediate is selected from mono-Boc-2,2'-oxydiethylamine.

[0032] Preferably, the ligand is selected from 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid, and the organic intermediate is selected from mono-Boc hydrazine, mono-Boc butanediamine, and mono-Boc-2,2'-oxydiethylamine.

[0033] Preferably, the ligand is selected from 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid, and the organic intermediate is selected from mono-Boc butanediamine and mono-Boc hydrazine.

[0034] Preferably, the ligand is selected from 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid, and the organic intermediate is selected from mono-Boc butanediamine.

[0035] Preferably, the amide reaction described in S1 is carried out under conventional amide reaction conditions, such as overnight at room temperature.

[0036] Preferably, the organic solvent used in the amide reaction in S1 includes at least one of DMF, DHF, and acetonitrile; further comprising DMF.

[0037] Preferably, the condensing agent used in the amide reaction described in S1 includes at least one of HATU, PyBoc, HOAT, HOBT, HBTU, and BOP; and further includes HATU.

[0038] Preferably, the base used in the amide reaction in S1 includes at least one of N,N-diisopropylethylamine and triethylamine; more preferably, it includes N,N-diisopropylethylamine.

[0039] Preferably, the molar ratio of the ligand to the organic intermediate in S1 is 1:1 to 1:4; more preferably, it is 1:1.1 to 1:3.

[0040] Preferably, the reaction in S1 further includes the following steps: distributing the reacted solution in an organic solvent and water, retaining the organic phase, washing, and drying.

[0041] Preferably, the reaction in S1 further includes the following steps: partitioning the reacted solution in an organic solvent and water, retaining the organic phase, extracting once with the aqueous organic solvent, combining the organic phases, washing, and drying.

[0042] Preferably, the organic solvent comprises ethyl acetate.

[0043] Preferably, the washing process includes: washing with an aqueous citric acid solution, washing with saturated sodium bicarbonate solution, and washing with a sodium chloride solution.

[0044] Preferably, the drying process uses sodium sulfate drying.

[0045] Preferably, the deprotection reaction described in S2 is carried out under conventional deprotection reaction conditions, such as overnight at room temperature.

[0046] Preferably, the organic solvent used in the deprotection reaction in S2 includes at least one of DCM, 1,4-dioxane, DMF, and THF; and further includes DCM.

[0047] Preferably, the acid used in the deprotection reaction in S2 includes at least one of TFA, HCl, and acetic acid; more preferably, it includes TFA.

[0048] Preferably, the deprotection reaction in S2 further includes the following step: concentration to remove the organic solvent.

[0049] Preferably, the nucleophilic addition reaction described in S2 is carried out under conventional nucleophilic addition reaction conditions, such as overnight at room temperature in the dark.

[0050] Preferably, the organic solvent used in the nucleophilic addition reaction in S2 includes at least one of 1,4-dioxane, DMF, THF, and ethanol; and further includes THF and ethanol.

[0051] Preferably, the volume ratio of THF to ethanol is (1-3):1.

[0052] Preferably, the base used in the nucleophilic addition reaction in S2 includes at least one of N,N-diisopropylethylamine and triethylamine; further comprising triethylamine.

[0053] Preferably, the nucleophilic addition reaction described in S2 further includes the following steps: drying and purification.

[0054] A third aspect of the present invention provides the application of the compounds of the first aspect of the present invention, as detailed below:

[0055] a1) The application of the compound of the first aspect of the present invention as a fluorescent probe in screening TLX ligands;

[0056] a2) The use of the compound of the first aspect of the present invention in the preparation of products for screening TLX ligands.

[0057] Preferably, the product includes at least one of reagents, reagent kits, and detection systems.

[0058] Preferably, the TLX ligand includes at least one of a TLX agonist and a TLX antagonist; further comprising a TLX agonist.

[0059] A fourth aspect of the present invention provides a reagent comprising a compound of the first aspect of the present invention.

[0060] Preferably, the reagent is used to screen TLX ligands.

[0061] Preferably, the TLX ligand includes at least one of a TLX agonist and a TLX antagonist; further comprising a TLX agonist.

[0062] A fifth aspect of the present invention provides a reagent kit comprising at least one of b1) to b2):

[0063] b1) The compound of the first aspect of the present invention;

[0064] b2) The reagent of the fourth aspect of the present invention.

[0065] Preferably, the kit further comprises a protein containing a TLX ligand-binding domain (TLX-LBD) and said protein being linked to a tagged protein.

[0066] Preferably, the kit further comprises: a lanthanide-labeled antibody against the tagged protein.

[0067] Preferably, the tag protein comprises at least one of c-Myc, His, GST, HA, and Flag; more preferably, it comprises His.

[0068] Preferably, the lanthanide element comprises at least one of La, Sc, Y, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu; and further comprises Tb.

[0069] Preferably, the kit further comprises: a buffer solution.

[0070] Preferably, the buffer solution comprises at least one of PBS buffer, Tris buffer, and phosphate buffer.

[0071] Preferably, the buffer solution is a buffer solution containing Tween20.

[0072] Preferably, the kit is used to screen TLX ligands.

[0073] Preferably, the TLX ligand includes at least one of a TLX agonist and a TLX antagonist; further comprising a TLX agonist.

[0074] A sixth aspect of the present invention provides a detection system comprising: at least one of c1) to c3) and an enzyme-linked immunosorbent assay (ELISA) reader.

[0075] c1) The compound of the first aspect of the present invention;

[0076] c2) The reagent of the fourth aspect of the present invention;

[0077] c3) The reagent kit of the fifth aspect of the present invention.

[0078] Preferably, the detection system is used to screen TLX ligands.

[0079] Preferably, the TLX ligand includes at least one of a TLX agonist and a TLX antagonist; further comprising a TLX agonist.

[0080] A seventh aspect of the present invention provides a method for screening TLX ligands, comprising the step of using at least one of d1) to d4);

[0081] d1) The compound of the first aspect of the present invention;

[0082] d2) The reagent of the fourth aspect of the present invention;

[0083] d3) The reagent kit of the fifth aspect of the present invention;

[0084] d4) The detection system of the sixth aspect of the present invention.

[0085] Preferably, the method includes the following steps:

[0086] S1: A mixture is prepared by mixing a protein containing a TLX ligand-binding domain (TLX-LBD), the protein being linked to a tag protein, a lanthanide-labeled anti-tag protein antibody, a compound of the first aspect of the present invention, and the analyte.

[0087] S2: The signal value of the mixture is determined using fluorescence resonance energy transfer technology, and the analyte is confirmed to be a TLX ligand based on the signal value.

[0088] Preferably, the amino acid sequence of the TLX-LBD contains SEQ ID NO.1.

[0089] Preferably, the concentration of the protein in the mixture is 30–50 nM; more preferably 35–45 nM.

[0090] Preferably, the concentration of the antibody in the mixture is 0.5–1.5 nM; more preferably 0.8–1.2 nM.

[0091] Preferably, the concentration of the compound in the mixture is 1–8 μM; more preferably 2–6 μM; and even more preferably 2–4 μM.

[0092] Preferably, the TLX ligand includes at least one of a TLX agonist and a TLX antagonist; further comprising a TLX agonist.

[0093] The beneficial effects of this invention are:

[0094] This invention provides a compound that can be used as a fluorescent probe for screening TLX ligands. It is simple to prepare, structurally stable, highly sensitive, and fast in detection, making it suitable for high-throughput screening of TLX ligands. When used to screen TLX ligands, it employs a competitive binding TR-FRET screening system (different from the traditional fluorescence polarization system), providing an effective system for subsequent screening of more drugs targeting TLX. Attached Figure Description

[0095] Figure 1 Here is a structural diagram of TLX-FL: A is the full-length TLX-FL PDB structural diagram generated by AlphaFold; B is a schematic diagram of the composition of TLX-FL (the ligand binding domain is a specific structure that has been clearly identified).

[0096] Figure 2 This is a schematic diagram illustrating the principle of a fluorescence polarization experiment.

[0097] Figure 3 This is a schematic diagram illustrating the principle of time-resolved fluorescence resonance energy transfer experiments.

[0098] Figure 4 This is a schematic diagram illustrating the principle of a time-resolved fluorescence resonance energy transfer competitive binding experiment.

[0099] Figure 5 The diagram shows the chemical structure of the TLX ligand and the calculated docking results with TLX: A is the chemical structure of 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphthyl-2-yl)vinyl]benzoic acid; B is the chemical structure of 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthyl-2-yl]vinyl]benzoic acid; C is the calculated docking result of 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphthyl-2-yl)vinyl]benzoic acid with TLX; D is the calculated docking result of 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthyl-2-yl]vinyl]benzoic acid with TLX.

[0100] Figure 6 Here is the general formula diagram for compounds A through L: Orange represents the original ligand group, green represents the linking chain group, blue represents the fluorescent group, and R... 1 Selected from R 2 Selected from

[0101] Figure 7 These are the RMSD and RMSF diagrams of compounds A through F.

[0102] Figure 8 These are the RMSD and RMSF diagrams of compounds G to L.

[0103] Figure 9 The following diagram shows the simulation results of the molecular motion of compound F and His-TLX-LBD: A is a schematic diagram of the binding of compound F and His-TLX-LBD; B is a schematic diagram of the His end distance between compound F and His-TLX-LBD; and C is a schematic diagram of the contact between compound F and His-TLX-LBD.

[0104] Figure 10 The following diagram shows the simulation results of molecular motion of compound K and His-TLX-LBD: A is a schematic diagram of the binding of compound K and His-TLX-LBD; B is a schematic diagram of the His end distance of compound K and His-TLX-LBD; C is a schematic diagram of the contact between compound K and His-TLX-LBD.

[0105] Figure 11 This is the synthetic route diagram for compound F.

[0106] Figure 12 This is the NMR spectrum of compound F.

[0107] Figure 13 This is the synthetic route diagram for compound G.

[0108] Figure 14 This is the NMR spectrum of compound G.

[0109] Figure 15 This is the synthetic route diagram for compound K.

[0110] Figure 16 This is the NMR spectrum of compound K.

[0111] Figure 17 This is a graph showing the saturation analysis results of the fluorescence polarization FP of compound F.

[0112] Figure 18 This is a graph showing the saturation analysis results of the fluorescence polarization FP of compound G.

[0113] Figure 19 This is a graph showing the saturation analysis results of the fluorescence polarization FP of compound K.

[0114] Figure 20 This is a graph showing the experimental results of time-resolved fluorescence resonance energy transfer (TR-FRET) competitive binding of compound F.

[0115] Figure 21 This is a graph showing the experimental results of time-resolved fluorescence resonance energy transfer (TR-FRET) competitive binding of compound G.

[0116] Figure 22 This is a graph showing the experimental results of time-resolved fluorescence resonance energy transfer (TR-FRET) competitive binding of compound K. Detailed Implementation

[0117] The present invention will be further described in detail below through specific embodiments.

[0118] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0119] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0120] Example 1: Computational docking and molecular dynamics simulations for designing novel tailless protein ligand-binding domain (TLX-LBD) fluorescent probes for nuclear receptors.

[0121] In existing publicly available literature, Schrödinger (Maestro) is used. The Glide docking algorithm was used to calculate the docking of known ligand compounds that can bind to TLX-LBD. Molecular dynamics simulations (Schrödinger Maestro Desmond's OPLS4 and Gromacs' charmm36) yielded the following two compounds: 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid ( Figure 5 (A and C) and 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid ( Figure 5(B and D), and suitable linker chains to make fluorescent probes (Reference: Griffett, Kristine, et al. "The orphan nuclear receptor TLX is a receptor for synthetic and natural retinoids." Cell Chemical Biology 27.10(2020):1272-1284.).

[0122] The following calculation steps are for illustrative purposes only and are not intended to limit the scope of the invention. They are generally performed under conventional conditions in the art and help optimize compound structures from virtual conformations, explore protein and compound binding modes, and selectively synthesize compounds. The following are the steps for virtual calculations:

[0123] The virtual docking was performed using Schrödinger's algorithm to virtually dock TLX-LBD, followed by molecular dynamics simulation. First, the structure of the TLX-LBD protein PDB (PDB: 4XAJ) was preprocessed using chainA to remove water and add hydrogen, generating a virtual docking conformation (Receptor Grid Generation) for TLX-LBD re-docking calculations. Chemdraw was used to construct small molecule structures for docking calculations. In addition to generating 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphthyl-2-yl)vinyl]benzoic acid-NXN-FITC and 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthyl-2-yl]vinyl]benzoic acid-NXN-FITC, a topological force field was added to the compounds to calculate the interatomic interaction forces and topological isomers, generating possible three-dimensional folded shapes (Schrödinger's LigPrep module, see: Harder, Edward, et al. "OPLS3: a forcefield providing broad coverage of drug-like small molecules and proteins." Journal of chemical theory and computation 12.1 (2016): 281-296.). After generating the specific structure, the protein docking conformation with TLX-LBD was repeatedly docked 100 times for each molecule using Schrödinger's Extra Precision Glide Docking (Glide XPdocking) (Reference: Friesner, Richard A., et al. "Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy." Journal of medicinal chemistry 47.7(2004):1739-1749.), and then the energy force field was minimized using the MMGBSA module. The optimal molecular docking conformation was selected from the resulting conformations for molecular motion simulation (Gromacs / Desmond) to calculate the atomic motion trajectories, and TLX-LBD and the compound ( Figure 6 Generate composite coordinate systems and their external force parameters for the added aqueous solution; perform classical energy minimization, heating, and equilibrium processing on the system; finally, run molecular motion simulation to calculate Gibbs free binding energy, protein residue-ligand contact, and ligand fluorescent group distance (Table 1). Figures 7-10 , Reference: Páll, Szilárd, etal. "Heterogeneous parallelization and acceleration of molecular dynamics simulations in GROMACS." The Journal of Chemical Physics 153.13 (2020).).

[0124] Table 1 shows the molecular structure diagrams of compounds A to L, their Gibbs binding free energies, and the distance between the fluorophore and the His terminus of TLX-LBD.

[0125]

[0126]

[0127] RMSD (Root Mean Square Deviation) is a commonly used metric in molecular dynamics simulations and structural biology. It represents the average positional deviation of a set of atoms during a simulation or when comparing different protein structures. By calculating the root mean square value of the positional differences of all matching atoms between the simulated state and the initial state, RMSD provides a quantitative indicator of the degree of structural change, stability, and conformational changes of a protein during dynamic processes. Low RMSD values ​​generally indicate high structural similarity, while high RMSD values ​​may indicate significant conformational changes. This makes RMSD an important reference measure for analyzing protein folding, complex formation, or other structural changes. RMSF (Root Mean Square Deviation) is a metric used in molecular dynamics simulations to describe the degree of positional fluctuation of atoms, residues, or molecular fragments during the simulation. By analyzing the RMSF of proteins, researchers can better understand their dynamic characteristics and predict functionally relevant regions. It measures the dynamic behavior and flexibility of specific atoms by calculating the average variance of their positions over the simulation time. A larger RMSF value indicates a greater movement or conformational change of the corresponding atom or residue during the simulation, and is usually related to the flexibility of the protein's active site, ligand-binding region, flexible links, or other functionally relevant regions. In summary, RMSD observes the protein-ligand complex from a time dimension, while RMSF starts from the protein residue dimension. Both aim to demonstrate the credibility and stability of molecular dynamics simulations. Our simulation results show that the binding of compounds to TLX-LBD exhibits good continuity and credibility.

[0128] Gibbs binding energy calculations assess the energy released or absorbed when a ligand binds to a target protein in the energy field dimension, typically performed through energy analysis following molecular dynamics simulations. Binding energy calculations generally include van der Waals forces, charge interactions, and possible polar and nonpolar solvation effects. A negative binding energy value indicates that energy is released during ligand binding, suggesting a spontaneous process, and the magnitude of this value can serve as a quantitative indicator of ligand binding affinity. Results show that compounds D, E, F, I, K, and L possess superior binding energies, predicting good binding to proteins. Compounds with better binding energies will have higher time-resolved fluorescence resonance energy transfer (FRET) onset values; however, this can reduce the window size for competitive experiments, as the analyte may struggle to compete with the probe for protein binding, while low binding strength can also result in an undesirable window due to weak binding. Furthermore, since fluorescence polarization (FP) is fixed at protein concentration, its signal originates from the rapid rotation of a low-polarization signal after binding to the protein, transforming into a low-polarization, high-polarization signal after protein binding. Therefore, predicting the distance and its positive / negative difference between the fluorophore FITC and His can also predict the stability of the fluorophore, and the results can be used as a reference to predict the approximate ranking of compounds FP. For example, compound F has a shorter distance and a stable positive / negative deviation value. Figure 9 In summary, to further validate our predictions, compounds F, G, and K were selected for further verification and comparison. The final results also confirmed our predictions: the FP results showed that compound F > compound G > compound K, and the TR-FRET results also showed that compound F had a larger window value. Therefore, compound F is a relatively ideal compound probe for TLX-LBD.

[0129] Example 2 Preparation of compound F

[0130] The synthetic route of compound F is as follows: Figure 11As shown, the specific steps are as follows: 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid (Taoshu T21714, compound 1, 50 mg, 0.132 mmol) was dissolved in 1 mL of dry N,N-dimethylformamide (DMF), and mono-Boc-2,2'-oxydiethylamine (compound 2, 80.5 mg, 0.395 mmol) was added at room temperature, followed by 2-(7-azabenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (HATU, 55 mg, 0.145 mmol), and then N,N-diisopropylethylamine (DIPEA, 56 mg, 0.395 mmol). The mixture was left to stand overnight at room temperature. The reaction mixture was then partitioned in ethyl acetate (EA) and water, retaining the organic phase. The aqueous phase was extracted once with EA, and the organic phases were combined, washed with 0.1 g / mL citric acid aqueous solution, saturated sodium bicarbonate solution, sodium chloride solution, and dried over sodium sulfate. The dried product (compound 3) was dissolved in about 1 mL of dichloromethane (DCM), and 0.25 mL of trifluoroacetic acid (TFA) was added at room temperature. The reaction was allowed to proceed overnight, and the solvent was removed by concentration for the next step. The product (compound 4) after solvent removal was mixed with an appropriate amount of triethylamine (TEA) to adjust the pH to 11, and then dissolved in 1 mL of a tetrahydrofuran / ethanol mixed solvent (vTHF:vEtOH = 2:1). Fluorescein isothiocyanate (FITC, compound 5, 51 mg, 0.132 mmol) was added, and the mixture was incubated overnight at room temperature in the dark. After concentration and drying, a semi-preparative purification was performed to obtain compound F((E)-N-(2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthine]-5-yl)thiourea)ethoxy)ethyl)-4-(2-(5,5-dimethyl-8-phenyl-56-dihydronaphthyl-2-yl)vinyl)benzamide); The semi-preparative conditions were as follows: column type: XBridge Prep C185μm OBDTM 19*250mm Column; Eluent: Mobile phase A, ACN / MeOH = 1:1 (V / V), Mobile phase B, water (with 0.1% TFA); Gradient: 0 min 25% A - 55 min 60% A; Flow rate: 10 mL / min. The NMR spectrum of compound F is shown below. Figure 12 As shown: 1 ¹H NMR (600MHz, solvent DMSO-d6).

[0131] Example 3 Preparation of compound G

[0132] The synthetic route of compound G is as follows: Figure 13As shown, the specific steps are as follows: 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid (compound 1, 10.0 mg, 0.025 mmol) was dissolved in 1 mL of dry N,N-dimethylformamide (DMF), and tert-butoxyformylhydrazine (compound 2, mono-Boc hydrazine, 3.7 mg, 0.0275 mmol) was added at room temperature, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 10.5 mg, 0.0275 mmol), and N,N-diisopropylethylamine (DIPEA, 7.1 mg, 0.055 mmol). The mixture was left to stand overnight at room temperature. The reaction mixture was then partitioned in ethyl acetate (EA) and water, retaining the organic phase. The aqueous phase was extracted once with EA, and the organic phases were combined, washed with 0.1 g / mL citric acid aqueous solution, saturated sodium bicarbonate solution, sodium chloride solution, and dried over sodium sulfate. The dried product (compound 3) was dissolved in about 1 mL of dichloromethane (DCM), and 0.25 mL of trifluoroacetic acid (TFA) was added at room temperature. The reaction was allowed to proceed overnight, and the solvent was removed by concentration for the next step. The product (compound 4) after solvent removal was mixed with an appropriate amount of triethanolamine (TEA) to adjust the pH to 11, and then dissolved in 1 mL of a mixed solvent of tetrahydrofuran (THF) / ethanol (EtOH) (vTHF:vEtOH = 2:1). Fluorescein isothiocyanate (FITC, compound 5, 14.6 mg, 0.0375 mmol) was added, and the mixture was incubated overnight at room temperature in the dark. The solution was concentrated and dried, and semi-preparatively purified to obtain compound G. The semi-preparative conditions were as follows: column type: XBridge Prep C185μm OBDTM 19*250mm Column; eluent: mobile phase A: ACN / MeOH = 1:1 (V / V), mobile phase B: water (with 0.1% TFA); gradient: 0 min 25% A - 55 min 60% A; flow rate: 10 mL / min. The NMR spectrum of compound G is shown below. Figure 14 As shown: 1H NMR (600MHz, solvent DMSO-d6).

[0133] Example 4: Preparation of compound K

[0134] Synthetic route of compound K Figure 15As shown, the specific steps are as follows: 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid (compound 1, 10.0 mg, 0.025 mmol) was dissolved in 1 mL of dry N,N-dimethylformamide (DMF), and tert-butyl (4-aminobutyl)carbamate (compound 2, mono-Boc butanediamine, 5.2 mg, 0.0275 mmol) was added at room temperature, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 10.5 mg, 0.0275 mmol), and N,N-diisopropylethylamine (DIPEA, 7.1 mg, 0.055 mmol). The mixture was left to stand overnight at room temperature. The reaction mixture was then partitioned in ethyl acetate (EA) and water, retaining the organic phase. The aqueous phase was extracted once with EA, and the organic phases were combined. The mixture was washed with 0.1 g / mL citric acid aqueous solution, saturated sodium bicarbonate solution, sodium chloride solution, and dried over sodium sulfate. The dried product (compound 3) was dissolved in about 1 mL of dichloromethane (DCM), and 0.25 mL of trifluoroacetic acid (TFA) was added at room temperature. The reaction was allowed to proceed overnight, and the solvent was removed by concentration for the next step. The solvent-removed product (compound 4) was mixed with an appropriate amount of triethanolamine (TEA) to adjust the pH to 11, and then dissolved in 1 mL of a mixed solvent of tetrahydrofuran (THF) / ethanol (EtOH) (vTHF:vEtOH = 2:1). Fluorescein isothiocyanate (FITC, compound 5, 14.6 mg, 0.0375 mmol) was added, and the mixture was allowed to proceed overnight in the dark at room temperature. The mixture was then concentrated and dried, and semi-preparatively purified to obtain compound A. Semi-preparative conditions: Column type: XBridge Prep C185μm OBDTM 19*250mm Column; Eluent: Mobile phase A: ACN / MeOH = 1:1 (V / V), Mobile phase B: water (with 0.1% TFA); Gradient: 0 min 25% A - 55 min 60% A; Flow rate: 10 ml / min. The NMR spectrum of compound K is shown below. Figure 16 As shown: 1H NMR (600MHz, solvent DMSO-d6).

[0135] Effect Example

[0136] I. Saturation Analysis Experiment of Fluorescence Polarization FP of Compound F

[0137] 1) Dilute 30 μM of His-TLX-LBD protein (amino acid sequence: TESVCESAARLLFMSIKWAKSVPAFSTLSLQDQ LMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGSELRSFRNAAAIAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKKTIGNVPITRLLSDMY, SEQ ID NO.1) in a 1:1 serial dilution with 10% polysorbate-20 test buffer (20 mM potassium phosphate, 50 mM potassium chloride, 5 mM tris(2-carbonylethyl)phosphohydrochloride, pH 7.4) and place 72.8 μL of each solution in a 96-well plate.

[0138] 2) Use a multichannel pipette to pipette 7.2 μL of 2 μM fluorescent probe solution (compound F) into the protein solution of the 96-well plate above, mix well, and then transfer to a 384-well plate, with 22.5 μL in each of the three replicates;

[0139] 3) After incubating the mixture at room temperature for 60 minutes, read the values ​​using a microplate reader. Use a multi-functional microplate reader to read the plate. The emission wavelength and excitation wavelength are 495 nm and 538 nm, respectively.

[0140] 4) Use Graphpad Prism software to perform calculations and graphs on the data. The results are as follows: Figure 17 As shown, the dissociation constant of compound F binding to TLX-LBD is 9.278 μM, indicating that compound F has good binding activity with TLX-LBD.

[0141] II. Saturation Analysis Experiment of Fluorescence Polarization FP of Compound G

[0142] 1) Dilute 60 μM of His-TLX-LBD protein (amino acid sequence: TESVCESAARLLFMSIKWAKSVPAFSTLSLQDQ LMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGSELRSFRNAAAIAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKKTIGNVPITRLLSDMY, SEQ ID NO.1) with 10% polysorbate-20 test buffer (20 mM potassium phosphate, 50 mM potassium chloride, 5 mM tris(2-carbonylethyl)phosphohydrochloride, pH 7.4) at a gradient of 1:0.667 (first 8 spots) and 1:1 (last 8 spots), and place 72.8 μL of each solution in a 96-well plate.

[0143] 2) Use a multichannel pipette to pipette 7.2 μL of 2 μM fluorescent probe solution (compound G) into the protein solution of the 96-well plate above, mix well, and then transfer to a 384-well plate, with 22.5 μL in each of the three replicates;

[0144] 3) After incubating the mixture at room temperature for 60 minutes, read the values ​​using a microplate reader. Use a multi-functional microplate reader to read the plate. The emission wavelength and excitation wavelength are 495 nm and 538 nm, respectively.

[0145] 4) Use Graphpad Prism software to perform calculations and graphs on the data. The results are as follows: Figure 18 As shown, the dissociation constant of compound G bound to TLX-LBD is 31.12 μM.

[0146] III. Saturation Analysis Experiment of Fluorescence Polarization FP of Compound K

[0147] 1) Dilute 60 μM of His-TLX-LBD protein (amino acid sequence: TESVCESAARLLFMSIKWAKSVPAFSTLSLQDQ LMLLEDAWRELFVLGIAQWAIPVDANTLLAVSGMNGDNTDSQKLNKIISEIQALQEVVARFRQLRLDATEFACLKCIVTFKAVPTHSGSELRSFRNAAAIAALQDEAQLTLNSYIHTRYPTQPCRFGKLLLLLPALRSISPSTIEEVFFKKTIGNVPITRLLSDMY, SEQ ID NO.1) in a 1:1 serial dilution with 10% polysorbate-20 test buffer (20 mM potassium phosphate, 50 mM potassium chloride, 5 mM tris(2-carbonylethyl)phosphohydrochloride, pH 7.4) and place 72.8 μL of each solution in a 96-well plate.

[0148] 2) Use a multichannel pipette to pipette 7.2 μL of 2 μM fluorescent probe solution (compound K) into the protein solution of the 96-well plate above, mix well, and then transfer to a 384-well plate, with 22.5 μL in each of the three replicates;

[0149] 3) After incubating the mixture at room temperature for 60 minutes, read the values ​​using a microplate reader. Use a multi-functional microplate reader to read the plate. The emission wavelength and excitation wavelength are 495 nm and 538 nm, respectively.

[0150] 4) Use Graphpad Prism software to perform calculations and graphs on the data. The results are as follows: Figure 19 As shown, the dissociation constant of compound K bound to TLX-LBD is 45.12 μM.

[0151] IV. Time-resolved fluorescence resonance energy transfer (TR-FRET) competitive binding experiment of compound F

[0152] 1) Take 79.2 μL of test buffer (20 mM potassium phosphate, 10% polysorbate-20, 50 mM potassium chloride, 5 mM tris(2-carbonylethyl)phosphohydrochloride, pH 7.4) containing 40 nM His-TLX-LBD protein solution (SEQ ID NO.1), 1 nM terbium Anti-His-Tb (ThermoFibre, catalog number PV5863), and 4 μM fluorescent probe (compound F) solution, and place it in a 96-well plate;

[0153] 2) Prepare 5 mM of the competing sample (ligand, TLX-LBD reverse agonist: (2E, 4E, 6E, 8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)non-2,4,6,8-tetraenal) (Taoshu T5256), and serially dilute it at 1:1 for 11 spots. Use a multichannel pipette to transfer 0.8 μL of the competing sample to the protein solution in the 96-well plate of step (1) above, mix well, and then transfer it to a 384-well plate, with 22.5 μL in each of the 3 replicate wells;

[0154] 3) After incubating the mixture at room temperature for 120 minutes, read the values ​​using a microplate reader. Use a multi-functional microplate reader to read the plate. The excitation wavelength is 340 nm, and the emission wavelengths are 495 nm and 520 nm.

[0155] 4) Use Graphpad Prism software to perform calculations and graphs on the data. The results are as follows: Figure 20 As shown, the dissociation constant of the competing sample is 4.560 μM, and compound F has a large detection window and high sensitivity.

[0156] V. Time-resolved fluorescence resonance energy transfer (TR-FRET) competitive binding experiment of compound G

[0157] 1) Take 79.2 μL of test buffer (20 mM potassium phosphate, 10% polysorbate-20, 50 mM potassium chloride, 5 mM tris(2-carbonylethyl)phosphohydrochloride, pH 7.4) containing 40 nM His-TLX-LBD protein solution (SEQ ID NO.1), 1 nM terbium Anti-His-Tb (ThermoFibre, catalog number PV5863), and 4 μM fluorescent probe (compound G) solution and place it in a 96-well plate;

[0158] 2) Prepare 5 mM of the competing sample (ligand, TLX-LBD reverse agonist: (2E, 4E, 6E, 8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)non-2,4,6,8-tetraenal) (Taoshu T5256), and serially dilute it at 1:1 for 11 spots. Use a multichannel pipette to transfer 0.8 μL of the competing sample to the protein solution in the 96-well plate of step (1) above, mix well, and then transfer it to a 384-well plate, with 22.5 μL in each of the 3 replicate wells;

[0159] 3) After incubating the mixture at room temperature for 120 minutes, read the values ​​using a microplate reader. Use a multi-functional microplate reader to read the plate. The excitation wavelength is 340 nm, and the emission wavelengths are 495 nm and 520 nm.

[0160] 4) Use Graphpad Prism software to perform calculations and graphs on the data. The results are as follows: Figure 21As shown: the dissociation constant of the competing samples is 0.17 μM. Compound G has higher sensitivity than compound F, but its detection window is smaller.

[0161] VI. Time-resolved fluorescence resonance energy transfer (TR-FRET) competitive binding experiment of compound K

[0162] 1) Take 79.2 μL of test buffer (20 mM potassium phosphate, 10% polysorbate-20, 50 mM potassium chloride, 5 mM tris(2-carbonylethyl)phosphohydrochloride, pH 7.4) containing 40 nM His-TLX-LBD protein solution (SEQ ID NO.1), 1 nM terbium Anti-His-Tb (ThermoFibre, catalog number PV5863), and 4 μM fluorescent probe (compound K) solution, and place it in a 96-well plate;

[0163] 2) Prepare 5 mM of the competing sample (ligand, TLX-LBD reverse agonist: (2E, 4E, 6E, 8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)non-2,4,6,8-tetraenal) (Taoshu T5256), and serially dilute it at 1:1 for 11 spots. Use a multichannel pipette to transfer 0.8 μL of the competing sample to the protein solution in the 96-well plate of step (1) above, mix well, and then transfer it to a 384-well plate, with 22.5 μL in each of the 3 replicate wells;

[0164] 3) After incubating the mixture at room temperature for 120 minutes, read the values ​​using a microplate reader. Use a multi-functional microplate reader to read the plate. The excitation wavelength is 340 nm, and the emission wavelengths are 495 nm and 520 nm.

[0165] 4) Use Graphpad Prism software to perform calculations and graphs on the data. The results are as follows: Figure 22 As shown: the dissociation constant of the competing samples is 2.190 μM, and the detection window of compound K is larger than that of compound G but smaller than that of compound F.

[0166] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound having the chemical structural formula shown in formula (Ⅰ): Equation (Ⅰ), In equation (Ⅰ), R 1 Selected from , ; R 2 Selected from , .

2. The compound according to claim 1, characterized in that: R 2 Selected from R 1 Selected from -(CH2)3-, -(CH2)4-, -CH2-CH2-O-CH2-CH2-; or R 2 Selected from R 1 Selected from -(CH2)0-, -(CH2)4-, -CH2-CH2-O-CH2-CH2-.

3. The compound according to claim 2, characterized in that: R 2 Selected from R 1 Selected from -(CH2)4-, -CH2-CH2-O-CH2-CH2-; or R 2 Selected from R 1 Selected from -(CH2)4- and -(CH2)0-.

4. A method for preparing the compound according to claim 1, 2, or 3, comprising the following steps: S1: The ligand is reacted with an organic intermediate via an amide reaction to obtain intermediate 1a, wherein the organic intermediate is selected from mono-Boc hydrazine, mono-Boc methyldiamine, mono-Boc ethylenediamine, mono-Boc propylenediamine, mono-Boc butyldiamine, and mono-Boc-2,2'-oxydiethylamine, and the ligand is selected from 4-[(E)-2-(5,5-dimethyl-8-phenyl-6H-naphth-2-yl)vinyl]benzoic acid and 4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphth-2-yl]vinyl]benzoic acid; S2: Deprotection reaction is carried out on intermediate 1a, and the deprotected intermediate 1a undergoes nucleophilic addition reaction with fluorescein isothiocyanate to obtain the compound described in claim 1, 2 or 3.

5. The preparation method according to claim 4, characterized in that: The organic solvent used in the amide reaction described in S1 includes at least one of DMF, DHF, and acetonitrile.

6. The preparation method according to claim 4, characterized in that: The condensing agent used in the amide reaction described in S1 includes at least one of HATU, PyBoc, HOAT, HOBT, HBTU, and BOP.

7. The preparation method according to claim 4, characterized in that: The base used in the amide reaction described in S1 includes at least one of N,N-diisopropylethylamine and triethylamine.

8. The preparation method according to claim 4, characterized in that: The organic solvent used in the deprotection reaction described in S2 includes at least one of DCM, 1,4-dioxane, DMF, and THF.

9. The preparation method according to claim 4, characterized in that: The acid used in the deprotection reaction described in S2 includes at least one of TFA, HCl, and acetic acid.

10. The preparation method according to claim 4, characterized in that: The organic solvent used in the nucleophilic addition reaction described in S2 includes at least one of 1,4-dioxane, DMF, THF, and ethanol.

11. The preparation method according to claim 4, characterized in that: The base used in the nucleophilic addition reaction described in S2 includes at least one of N,N-diisopropylethylamine and triethylamine.

12. Use of the compound according to claim 1, 2, or 3 in at least one of a1) to a2): a1) as a fluorescent probe for screening TLX ligands; a2) Prepare products for screening TLX ligands.

13. A reagent comprising the compound of claim 1, 2 or 3.

14. A kit comprising at least one of b1) to b2): b1) The compound according to claim 1, 2 or 3; b2) The reagent as described in claim 13.

15. The kit according to claim 14, characterized in that: The kit also includes a protein containing a TLX ligand-binding domain, the protein being linked to a tagged protein.

16. The kit according to claim 14, characterized in that: The kit also contains: a lanthanide-labeled antibody against the tagged protein.

17. The kit according to claim 15, characterized in that: The tagged protein includes at least one of c-Myc, His, GST, HA, and Flag.

18. The kit according to claim 16, characterized in that: The lanthanide elements include at least one of the following: La, Sc, Y, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

19. A detection system comprising: at least one of c1) to c3) and an enzyme-linked immunosorbent assay (ELISA) reader: c1) The compound according to claim 1, 2 or 3; c2) The reagent according to claim 13; c3) The kit according to any one of claims 14 to 18.

20. A method for screening TLX ligands, comprising: using at least one of d1) to d4): d1) The compound according to claim 1, 2 or 3; d2) The reagent according to claim 13; d3) The kit according to any one of claims 14 to 18; d4) The detection system as described in claim 19.

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