Therapeutic diagnostic probes and their applications for targeting and / or labeling EGFR kinases and / or cells expressing EGFR family members

Abstract

This invention relates to therapeutic diagnostic probes and their applications for targeting and / or labeling EGFR kinases and / or cells expressing EGFR family members.

Description

Technical Field

[0001] Applications of probes that target and / or label EGFR kinases and / or express EGFR family members in clinical diagnosis and treatment. Background Technology

[0002] In recent years, technologies for testing patients' blood and bodily fluids have developed rapidly. Compared with traditional tissue biopsies, liquid biopsies have the advantages of being non-invasive and low-cost, and can be performed multiple times to track cancer progression, which is especially important for patients with financial difficulties and serious complications.

[0003] Liquid biopsy relies on the detection of tumor markers, including circulating tumor cells (CTCs) and their DNA, or overexpressed protein markers. Currently, CTC detection requires enrichment using immunoassay or microfluidic technology, followed by comprehensive genomic, transcriptomic, and methylome analysis of the enriched cells to assess and monitor carcinogenesis drivers, progression, and metastasis. This two-step strategy can be applied to the diagnosis of various tumors. However, its specificity and efficiency are not high, especially when carcinogenic markers have been identified. On the other hand, antibodies currently used for CTC immunoclassification have the characteristic of broadly recognizing a variety of tumor cells, but they cannot monitor mutations in intracellular carcinogenic drivers or recognize conformational changes in these drivers. These mutations or conformational changes in carcinogenic drivers are often key to determining the therapeutic window and resistance of targeted drugs.

[0004] For example, approximately 20% of non-small cell lung cancer (NSCLC) is associated with mutations in the intracellular kinase domain of EGFR. EGFR-targeting inhibitors are generally sensitive to certain mutational states of the kinase. However, secondary mutations in the EGFR kinase can lead to resistance to these inhibitors. Therefore, it is necessary to develop new methods to efficiently enrich tumor cells and report the mutational status of oncogenic biomarkers.

[0005] EGFR, along with its homologs HER2, HER3, and HER4, belongs to the receptor tyrosine kinase family. They play crucial roles in cell growth, differentiation, and migration, and are closely related to the occurrence, development, and metastasis of various cancers, making them key targets for cancer treatment. The intracellular kinase activity of EGFR is activated in a concentration-dependent manner through ligand-induced receptor dimerization. Along with changes in kinase activity, the kinase conformation also changes. In the absence of ligand binding, the receptor's kinase domain is in a self-inhibited, inactive state (Figure 3). At this time, the α-C helix of the kinase swings outward, while the activation loop folds inward, blocking substrate binding. However, upon ligand binding, the kinase domains of the two receptors approach each other, forming an asymmetric, activated dimer similar to the Cyclin / CDK binding pattern. In this state, one kinase acts as an activation donor, stabilizing the other kinase in the activated conformation. In the activated conformation, the α-C helix swings inward, and the activation loop extends outward, facilitating substrate binding and phosphorylation. Oncogenic mutations or abnormally high expression of EGFR can dysregulate kinase activity through various mechanisms, such as stabilizing the activated state of the kinase or disrupting its inactive state. EGFR kinase inhibitors, on the other hand, competitively inhibit ATP binding to suppress kinase activity and are widely used in the treatment of various cancers. These kinase inhibitors are conformationally selective, including gefitinib and PD168393, and are highly sensitive to certain EGFR mutants, allowing them to be used to report kinase mutations or conformational changes.

[0006] Prostate cancer has the highest mortality rate among male malignant tumors. Therefore, early diagnosis and timely monitoring of disease progression and treatment response can greatly improve the survival rate and quality of life of these patients.

[0007] Currently, ultrasound- or magnetic resonance imaging (MRI)-guided targeted biopsy is the gold standard for prostate cancer diagnosis and is widely used in clinical practice. During the biopsy, to prevent missed lesions, multiple sites need to be sampled from suspicious areas, followed by histochemical and staining analyses of the collected tissue samples. However, this invasive biopsy can lead to a series of complications, including hematuria, hematochezia, postoperative infection, and vagal reflex. Furthermore, due to the sparse nature of the sampling, it may result in the omission of small lesions and an inability to comprehensively monitor cancer progression. Therefore, various liquid biopsy techniques for cancer have emerged.

[0008] Liquid biopsy for cancer involves collecting bodily fluids or blood from a patient to detect free cancer biomarkers. Currently, biomarkers used for cancer detection include circulating tumor DNA (CTC), circulating tumor cells (CTCs), or overexpressed protein markers. CTCs are detached tumor cells circulating in bodily fluids or blood. The number of these CTCs is directly related to the degree of tumor malignancy and treatment prognosis; however, their specificity and efficiency are low. Antibodies currently used for CTC immune classification can typically target antigens on many different types of cancer. However, these antibodies cannot report intracellular mutations or conformational changes in biomarkers associated with tumorigenesis. These mutations or conformational changes are often critical in the selection of therapeutic windows and the development of drug resistance. Therefore, it is necessary to develop a new method that can efficiently enrich tumor cells and report the mutational status of oncogenic biomarkers for diagnosis and treatment.

[0009] On the other hand, the gene expression or mutation profiles of tumor cells cannot be directly translated into treatment strategies, and clinical trials designed based on these data may fail in practice. For example, EGFR overexpression is observed in one-third of prostate cancer patients, and EGFR overexpression is associated with biochemical recurrence and hormone-resistant status of prostate cancer. Furthermore, EGFR mutations have been reported to be associated with accelerated prostate cancer development. The link between EGFR abnormalities and prostate cancer progression has prompted the clinical use of EGFR inhibitors to treat hormone-resistant patients with EGFR overexpression. However, multiple center-wide clinical treatments have failed, indicating the need for further research into its pathogenic mechanisms and the development of more precise subtyping strategies. Summary of the Invention

[0010] We developed modular therapeutic probes targeting specific conformations of EGFR based on structural design. We demonstrated how to screen inhibitors using competitive assays and how to screen and image cells overexpressing or mutated by EGFR. Interestingly, we found that D168393 is more responsive to an activated dimer, which conformation differs from previously discovered asymmetric activated dimers.

[0011] Here, we also designed a modular fluorescent probe that irreversibly binds to epidermal growth factor receptor (EGFR) kinase. This probe can specifically inhibit EGFR kinase activity in a concentration-dependent manner, selectively label EGFR-overexpressing cells by flow cytometry, is sensitive to binding to EGFR containing oncogenic mutations, and preferentially binds to an intermediate-state kinase dimer, which is distinct from the classic activated asymmetric dimer.

[0012] In the first part, this application presents this diagnostic and therapeutic probe in the form of formula (1):

[0013] Module 2-L-Module 1 (1)

[0015] in,

[0016] L is a link.

[0017] Module 1 is a part that targets EGFR kinase; and

[0018] Module 2 is an optional probe for imaging, including fluorescent probes, MRI and radioactive probes; or salt that can be used for treatment.

[0019] In one example from the first part, L is -C(O)O- or -C(O)NH-.

[0020] In another example in Part 1, Module 1 is an EGFR kinase inhibitor.

[0021] In another example in Part 1, Module 1 is represented by Formula (2):

[0022]

[0023] in

[0024] In formula (2), R1 is H, a halogen (e.g., F, Cl, or Br), or C. 2-6 Alkyne group (e.g., ethynyl group);

[0025] In formula (2), R2 is H, a halogen (e.g., F, Cl, or Br), and C. 6-10 Aromatic C 1-6 Alkoxy (e.g., phenylmethoxy) or 5- to 6-membered heteroaryl C1-6 alkoxy (e.g., pyridinemethoxy, especially pyridine-2-methoxy), wherein C 6-10 The aromatic group and the 5- to 6-membered heteroaromatic group can be substituted with halogens (F, Cl or Br) (e.g., R2 in formula (2) is 3-fluorophenylmethoxy), and the 5- to 6-membered heteroaromatic group contains 1, 2, 3 or 4 heteroatoms selected from O, S or N;

[0026] In formula (2), R3 is H, which is arbitrarily chosen to be controlled by C. 1-6 alkoxy-substituted C 1-6 Alkoxy (e.g., R3 in formula (2) is methoxy, ethoxy or methoxyethoxy), 5 to 6-membered heterocyclic alkyloxy (e.g., tetrahydrofuranoxy, especially tetrahydrofuran-3-yloxy), wherein the 5 to 6-membered heterocyclic alkyl includes 1 to 2 heteroatoms selected from O, S and N;

[0027] In formula (2), R4 is the connection point from module 1 to L, and is -(CH2). n-; and n in formula (2) can be 0, 1, 2, 3, 4, 5 or 6.

[0028] In another example in Part 1, Module 2 is represented by Equations (3) and (4):

[0029]

[0030] in

[0031] In formula (3) or formula (4), R1 is the connection point from module 2 to L, which is -(CH2). n -;

[0032] In formula (3) or formula (4), R2 is -(CH2). n CH3; and n in formula (3) or formula (4) can be independent of 0, 1, 2, 3, 4, 5 or 6; or module 2 is represented by formula (5) or formula (6):

[0033]

[0034] in

[0035] In formula (5) or formula (6), the symbol * represents the connection point between module 2 and L; n in formula (5) is 0, 1, 2, 3, 4, 5 or 6 respectively;

[0036] If necessary, a counterion may be present. This could be one of the alkali metal and alkaline earth metal ions, particularly one of the groups consisting of sodium, potassium, calcium, or magnesium ions.

[0037] In another example from Part 1, the treatment probe is represented by formula (7):

[0038]

[0039] Also referred to in this article as PDCy3 or compound 2,

[0040] If necessary, a counterion may be present. This could be one of the alkali metal and alkaline earth metal ions, particularly one of the groups consisting of sodium, potassium, calcium, or magnesium ions.

[0041] In another example in Part 1, the EGFR kinase is wild-type.

[0042] In another example in Part 1, the EGFR kinase contains the L834R mutation.

[0043] In another example in Part 1, the EGFR kinase contains the V924R mutation.

[0044] In another example in Part 1, the EGFR kinase contains the V745M mutation.

[0045] In another example in Part 1, the EGFR kinase contains two or three mutations (L834R, V924R, V745M), such as L834R and V924R.

[0046] In another example from Part 1, the EGFR kinase exhibits a dimerized conformation.

[0047] In another example in Part 1, the EGFR kinase presents a wild-type EGFR kinase dimer-like conformation.

[0048] In another example in Part 1, the EGFR kinase presents as an EGFR kinase dimer-like conformation with the L834R mutation.

[0049] In another example in Part 1, the EGFR kinase presents as an EGFR kinase dimer-like conformation with the V924R mutation.

[0050] In another example in Part 1, the EGFR kinase presents as an EGFR kinase dimer-like conformation with the V745M mutation.

[0051] In another example in Part 1, the EGFR kinase presents a kinase dimer conformation containing two or three mutations among L834R, V924R, and V745M, such as L834R and V924R.

[0052] In another example from Part 1, EGFR kinase is associated with tumorigenesis, particularly metastatic, castration-resistant prostate cancer.

[0053] In another example in Part 1, the conformation of EGFR kinase is associated with tumorigenesis, particularly in metastatic and / or castration-resistant prostate cancer.

[0054] In the second part, this application provides probes for diagnosis or treatment, which are those described in the first part for targeting and / or labeling EGFR kinases and / or cells expressing EGFR family members.

[0055] In another example in Part Two, therapeutic and diagnostic probes, as defined in Part One, are used to target and / or label EGFR kinases. In another example in Part Two, therapeutic and diagnostic probes, as defined in Part One, are used on a high-throughput screening platform to target and / or label EGFR kinases. In another example in Part Two, a high-throughput screening platform is used to develop inhibitors against EGFR.

[0056] In another example in Part Two, therapeutic and diagnostic probes, as defined in Part One, are used to target and / or label cells overexpressing EGFR or its family members. In another example in Part Two, therapeutic and diagnostic probes, as defined in Part One, are used to target and / or label cells overexpressing EGFR or its family members in a body fluid biopsy. In another example in Part Two, the body fluid biopsy is non-invasive. In another example in Part Two, the body fluid biopsy is used to detect tumor cells in blood and / or body fluids. In another example in Part Two, the body fluid biopsy is used to detect EGFR overexpression, EGFR oncogenic mutations, and / or drug resistance caused by EGFR mutations in tumor cells. In another example in Part Two, probes are used to target and / or label samples of cells overexpressing EGFR or its family members. In another example in Part Two, the sample is a body fluid (e.g., blood or urine) or biopsy tissue. In another example in Part Two, the sample is urine.

[0057] In another example in Part Two, targeting and / or marking are performed through the following steps:

[0058] Mixing EGFR kinase and / or cells expressing EGFR or its family members with a therapeutic probe; and

[0059] The binding level between the two was detected.

[0060] In another example in Part 2, the EGFR kinase is wild-type.

[0061] In another example in Part 2, the EGFR kinase has the L834R mutation.

[0062] In another example in Part 2, the EGFR kinase has the V924R mutation.

[0063] In another example in Part 2, the EGFR kinase has the V745M mutation.

[0064] In another example in Part 2, the EGFR kinase has two or three mutations among L834R, V924R, and V745M, such as L834R and V924R.

[0065] In another example in Part 2, the EGFR kinase exhibits a dimerized conformation.

[0066] In another example in Part 2, the EGFR kinase presents a conformation similar to that of the wild-type EGFR kinase dimer.

[0067] In another example in Part 2, the EGFR kinase presents as an EGFR kinase dimer-like conformation with the L834R mutation.

[0068] In another example in Part 2, the EGFR kinase presents as an EGFR kinase dimer-like conformation with the V924R mutation.

[0069] In another example in Part 2, the EGFR kinase presents as an EGFR kinase dimer-like conformation with the V745M mutation.

[0070] In another example in Part 2, the EGFR kinase presents a kinase dimer conformation containing two or three mutations of L834R, V924R, and V745M, such as L834R and V924R.

[0071] In another example in Part 2, EGFR kinase is associated with tumorigenesis, particularly metastatic, castration-resistant prostate cancer.

[0072] In another example in Part 2, the conformation of EGFR kinase is associated with tumorigenesis, particularly in metastatic and / or castration-resistant prostate cancer.

[0073] In the third part, this application provides the application of diagnostic or therapeutic probes in body fluid biopsies, as described in the first part.

[0074] In one example from Part 3, body fluid biopsy is non-invasive.

[0075] In another example in Part 3, body fluid testing is used to detect tumor cells in blood and / or body fluids.

[0076] In another example in Part 3, body fluid biopsies were used to detect tumor cells with EGFR overexpression, EGFR oncogenic mutations, and / or drug-resistant mutations.

[0077] In the fourth part, this application provides the application of diagnostic and therapeutic probes in high-throughput screening platforms, as described in the first part.

[0078] In another example from Part 4, a high-throughput screening platform was used to develop inhibitors targeting EGFR.

[0079] In a fifth aspect, this application provides the use of a therapeutic probe, as defined in the first aspect, for stratifying patients with cancer caused by EGFR overexpression.

[0080] In another example in Part 5, the cancer is prostate cancer. In yet another example in Part 5, the cancer exhibits metastasis and / or castration resistance.

[0081] In another example in Part 5, EGFR has the V745M mutation.

[0082] In another example in Part 5, EGFR exists in a dimer conformation.

[0083] In another example in Part 5, EGFR exists in a dimer conformation as the V745M mutant EGFR.

[0084] In another example from Part 5, the layering is performed in the following steps:

[0085] The patient's sample was mixed with the treatment probe; and the binding level between the two was determined.

[0086] In another example from Part 5, the sample is bodily fluid (such as blood or urine) or biopsy tissue. In yet another example from Part 5, the sample is urine.

[0087] In the sixth section, this application provides diagnostic and therapeutic applications in the area of ​​high-throughput screening platforms as described in the first section.

[0088] In one example from Part VI, a high-throughput screening platform is used to develop inhibitors targeting EGFR.

[0089] In another example in Part VI, the EGFR kinase is wild-type. In yet another example in Part VI, the EGFR kinase has mutations in L834R, V924R, and V745M, or two or three of these mutations simultaneously, such as L834R and V924R mutations.

[0090] In another example in Part VI, EGFR exists in a dimer conformation. In another example in Part VI, the EGFR kinase has the same dimer conformation as the wild type. In another example in Part VI, the EGFR kinase has a dimer conformation formed by single-point mutations L834R, V924R, V745M, or by two or three of these mutations. For example, the L834R, V924R mutation.

[0091] In another example in Part VI, EFGR kinase is associated with tumorigenesis, particularly prostate cancer, or metastatic and / or castration-resistant prostate cancer.

[0092] In another example in Part VI, the conformation of EFGR kinase is associated with tumorigenesis, particularly prostate cancer, or metastatic and / or castration-resistant prostate cancer.

[0093] In another example in Part VI, screening is performed by the following steps: purified EGFR or EGFR-overexpressing cells labeled with a probe, and then screening candidate kinase inhibitors by their competitive binding ability to the purified EGFR or EGFR-overexpressing cells.

[0094] In Part 7, this application provides the application of enriching EGFR-overexpressing cells as described in Part 1.

[0095] In one example in Part VII, cells overexpress wild-type EGFR. In another example in Part VII, cells express EGFR with mutations in L834R, V924R, or V745M, or with two or three of these mutations, such as L834R or V924R mutations.

[0096] In another example from Part VII, EFGR kinase is associated with tumorigenesis, particularly prostate cancer, or metastatic and / or castration-resistant prostate cancer. The conformation of EFGR kinase is associated with tumorigenesis, particularly prostate cancer, or metastatic and / or castration-resistant prostate cancer.

[0097] In another example in Part 7, enrichment is performed by the following steps: as described in Part 1, EGFR-overexpressing cells are labeled with a therapeutic probe; then EGFR-overexpressing cells are collected.

[0098] This application may be embodied in any other form without departing from its spirit or scope. This application includes any and all combinations of the foregoing aspects and examples. It should be understood that any example can be combined with any other example. It should also be understood that a single element from any example can be combined with any and all other elements from any other example to describe an additional example.

[0099] Caption

[0100] Figure 1. Modular design of EGFR-targeting probes. A) The non-activated (left, PDB 2GS7) and activated (right, PDB 2GS6) conformations of the EGFR kinase domain are shown in cartoon form. In the non-activated conformation, the α-C helix (cyan) of the kinase is out-rotated, and the activation loop (blue) forms a small helix to stabilize the position of the α-C helix. The oncogenic mutation L834R on the activation loop is highlighted in red. In the active conformation, the two EGFR kinases form a specific asymmetric dimer, one acting as the activator (light blue) to stabilize the other (magenta) in the active conformation. The V924R mutation (green) can break the asymmetric dimer. B) Structural overview (left) and details (right) of PD168393 binding to EGFR kinase. PD168393 (gold) binds to the activation site of the EGFR kinase (light blue). The acrylamide group of PD168393 points outward from the kinase. Amino acid residues that interact with the inhibitor are represented by blue sticks. C) Modular design of diagnostic and therapeutic probes. PD168393 (targeting module) and Cy3 (imaging module) are linked by aminoacyl bonds. Both modules can be modified to meet different application requirements.

[0101] Figure 2. PDCy3 specifically inhibits EGFR kinase activity. A) Fluorescence spectrum of PDCy3 in DMSO. Black, excitation spectrum; red, emission spectrum. B) Dose-dependent inhibition of EGFR kinase activity by PDCy3. HEK293T cells were transfected with plasmids encoding EGFR and starved in serum-free medium for 2 days, followed by treatment with EGF, PDCy3, or gefitinib. EGFR expression and phosphorylation levels were detected using antibodies against protein C and 4G10, respectively. C) Gefitinib completely inhibits the binding of PDCy3 and EGFR. EGFR-transfected cells were treated with 1 μM PDCy3, 10 nM EGF / 1 μM PDCy3, or 5 μM gefitinib / 10 nM MEGF / 1 μM PDCy3. PDCy3 binding and EGFR expression were analyzed by flow cytometry (detected with 528 antibody). D) Relative binding of PDCy3 to EGFR-expressing cells. The relative binding level of PDCy3 is equal to the percentage of PDCy3-positive cells multiplied by the average fluorescence intensity of those cells. The bar graph shows the mean and standard deviation of three experiments.

[0102] Figure 3. Sensitivity of PDCy3 to EGFR expression and binding to mutant cells. A) Comparison of the binding of different concentrations of PDCy3 to HEK293T cells overexpressing EGFR by flow cytometry. B) Comparison of the binding ability of PDCy3 to wild-type and mutant EGFR at a concentration of 1 μM. C) The binding of 1 μM PDCy3 to cells reflects the effect of EGFR mutation on probe binding. The bar graph shows the mean and standard deviation of the three experiments.

[0103] Figure 4. Effect of EGFR dimerization on PDCy3 sensitivity. A) A protein complementary pair was designed by adding split-YFP after EGFR. Refolded YFP structures and restored fluorescence were obtained by dimerizing the receptor on the cell surface. B) The relationship between EGFR dimerization and PDCy3 binding was studied using the receptor pair designed above. Each mutation was introduced into the receptor pair. Two cell populations with different sensitivities to PDCy3 (labeled AB) were detected by flow cytometry at a concentration of 1 μM PDCy3. C) YFP-positive cells were divided into two homogeneous cell subpopulations based on YFP intensity. The mean fluorescence intensity and corresponding standard deviation of PDCy3-binding cells were plotted as a function of YFP intensity in these cells, with YFP correlated with EGFR dimer expression levels. D) Effects of EGFR oncogenic mutations and asymmetric dimerization on PDCy3 binding. The fraction of PDCy3-sensitive cells was calculated based on three experiments.

[0104] Figure 5. Comparison of EGFR kinase structures bound by PD168393. (AC) Comparison of crystal structures of PD168393 and EGFR kinase complexes. Wild-type EGFR kinase structure in activated conformation (shown in light blue cartoon, PDB 2GS6); A) EGFR kinase complex with PD168393 containing the L834R mutation (green, PDB 4LQM); B) D770-D771 Ins NPG mutant kinase complex with PD168393 (light pink, PDB 4LRM); C) EGFR L834R / T766M mutant kinase complex with PD168393 (yellow, PDB 4LL0). D) Comparison of activated EGFR structure with EGFR kinase complex with Mig6, the latter having an essentially disordered aC helix (pink, PDB 2RF9). (EG) Comparison of factor B structures of EGFR kinase. The structures of the EGFR active state (E), the L834R / T766M mutant binding to PD168393 (F), and the Mig6 binding (G) are colored according to their Cα atom B factor. The salt bridge between Lys721 and Glu736 in the active conformation is preserved in the PD168393-L834R / T766M mutant kinase complex (E, F), but is disrupted in the Mig6 kinase complex (G).

[0105] Figure 6. Chemical stratification of urine samples from cancer patients. A) Flow cytometry was used to detect the binding of urine samples from different subjects to the PDCy3 probe. Urine samples from each subject were stained with a specific concentration of the PDCy3 probe. The stained samples were analyzed by flow cytometry. B) The binding of cells collected from the urine of different subjects to PDCy3 was compared under different concentrations of PDCy3 probe treatment. The relative binding of PDCy3 was calculated by multiplying the percentage of PDCy3-positive cells by the average fluorescence intensity of PDCy3. C) The expression levels of EGFR relative to GAPDH were compared among samples from different subjects by RT-qPCR. D) Three mutations in the EGFR kinase domain of subject 4 were detected by forward Sanger sequencing. Using RNA extracted from each sample as a template, cDNA fragments encoding the EGFR kinase domain were amplified by 20 cycles of high-fidelity PCR with Novizan. Mutations on the PCR products of each sample were analyzed by Sanger sequencing. E) The effect of the identified EGFR kinase mutations on PDCy3 binding. 293T cells were transiently transfected with plasmids encoding WT EGFR and its mutants. The binding of PDCy3 to these transfected cells was analyzed by flow cytometry at specified concentrations. F) Comparison of the relative binding of PDCy3 to wild-type EGFR and mutants. The relative binding of PDCy3 to wild-type EGFR and mutants was compared in the presence or absence of 10 nMEGF and with 0.5 μM PDCy3. The relative binding of PDCy3 was calculated using the method described above. Bar graphs show the means and standard deviations of the three experiments. G) Effect of identified EGFR kinase mutants on kinase activity. EGFR expression and phosphorylation levels were detected by Western blot using antibodies against protein C and 4G10, respectively. H) HE staining of biopsy specimen from subject 4. I) Immunohistochemical staining for androgen receptor expression in biopsy specimen from subject 4. Androgen receptor was detected using antibody ab74272.

[0106] Figure 7. EGFR expression and mutations in cancer patients. A) Comparison of relative EGFR expression levels for β-actin in samples from different subjects using Q-PCR. B) Detection of EGFR kinase domain mutations in subject 4 using reverse Sanger sequencing. C) Effect of identified EGFR mutations on PDCy3 binding. 293T cells were transiently transfected with plasmids encoding WT EGFR and its mutants. Flow cytometry was used to analyze the binding of PDCy3 to these transfected cells at specified concentrations.

[0107] Figure 8. Tissue biopsy of specimen from subject 4. A) HE staining of specimen. (B, C, D, E, F, G) Immunohistochemical staining for androgen receptor (B), CK7 (C), CK20 (D), PSA (E), TTF (F) and Villin (G).

[0108] example.

[0109] Example 1: Probe Preparation

[0110] General steps

[0111] The proton NMR spectrum was recorded on a Bruker AVIII 400.

[0112] LCMS measurements were performed on an Agilent 1200 HPLC / 6100 SQ system under the following conditions: mobile phase: A: water (10 mM NHHCO3), B: MeCN; flow rate: 1.8 mL / min; column: 4.6 × 50 mm XBridge C18 column (3.5 μm particles); detection methods included diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0113] Synthesis scheme

[0114]

[0115] In the following synthesis, unless otherwise stated, a counterion, which is a sodium ion, is present if necessary.

[0116] 1. Synthesis of compound a-1:

[0117]

[0118] Dissolve 5.0 g (26.56 mmol) of a-01 in 30 mL of AcOH, and add 4.58 g (53.13 mmol) of 3-methylbutane-2-one. Heat the mixture under reflux for 5 hours. Then cool the solution to room temperature. Add a saturated solution of CH3COOK in 2-propanol, during which a solid is observed. Filter to give 5.5 g (74%) of a-1 as a yellow solid.

[0119] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0120] LC / MS m / z 240.0[m+H]+ ;RT = 1.17 min.

[0121] 2. Synthesis of compound a-2:

[0122]

[0123] 2.2 g of 2,3,3-trimethyllindolenium-5-sulfonate (a-1) was suspended in 30 mL of methyl iodide. The reaction mixture was heated to boiling in a sealed tube for 25 hours. After cooling, excess methyl iodide was poured off, and the residue was suspended in 50 mL of acetone. The solution was filtered to give 2.48 g (98% purity) of a-2 as a pink solid.

[0124] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0125] LC / MS m / z 254.0 [m+H] + RT = 1.34 min.

[0126] 3. Synthesis of compound a-3:

[0127]

[0128] α-1 (2.77 g, 10 mmol) and 6-bromohexanoic acid (2.34 g, 12 mmol) were dissolved in sulfolane (2.5 mL) and heated at 130 °C for 3 hours. The reaction mixture was cooled to room temperature, and then DCM was added to the residue. The resulting solid was filtered and dried under reduced pressure to give a pink solid.

[0129] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0130] LC / MS m / z 353.0[M+H].RT=1.19min.

[0131] 4. Synthesis of compound a-4:

[0132]

[0133] β-CD (25.77 g, 21.48 mmol) was dissolved in water (200 mL), heated to a clear solution, and cooled to room temperature. To this clear solution, amine (20.0 g, 214.75 mmol) was added dropwise with stirring, followed by the dropwise addition of triethylene glycol ester (15.91 g, 107.38 mmol), and the mixture was stirred overnight at room temperature. After the reaction was complete (monitored by TLC), the reaction mass was extracted with ethyl acetate. The evaporation of the organic layer resulted in a crude product, which was then purified by simple recrystallization from hexane and ethyl acetate to obtain pure product a-4 as a white solid.

[0134] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0135] LC / MS m / z 196.0 [M+H] + RT = 1.83 min.

[0136] 5. Synthesis of compound A:

[0137]

[0138] A solution of A-3 (2.95 g, 7.52 mmol) and A-4 (1.77 g, 9.03 mmol) in acetic acid (4.5 mL) and acetic anhydride (4.5 mL) was heated at 120 °C for 4 hours. The progress of the reaction was monitored by absorption spectroscopy in methanol. Then, a-2 (2.2 g, 7.52 mmol) and more acetic anhydride (4.5 mL) and pyridine (4.5 mL) were added. The mixture was heated for 30 minutes until the anyl intermediate disappeared (monitored by absorption spectroscopy). The reaction mixture was cooled and poured into ethyl acetate (50 mL). The crude product was collected by centrifugation and washed twice with ethyl acetate. Preparative HPLC purification gave compound A (260 mg) as a deep purple solid.

[0139] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0140] LC / MS m / z 617.0[M+HI.RT=1.15min.

[0141] 'H NMR(400MHz,D20)8 8.43-8.36(t,J=26.8Hz,1H),7.77-7.76(d,J=1.6Hz,2H),7.71-7.68(m,J=12Hz,2H ),7.24-7.19(m,J=20.8Hz,2H),6.27-6.20(m,J=29.6Hz,2H),3.97-3.93(t,J=14.4H z,2H),3.54-3.47(d,J=28Hz,3H),2.08-2.05(t,J=14.8Hz,2H),1.72-1.66(m,J=22 .4Hz,2H),1.61(s,12H),1.53-1.46(t,J=29.6Hz,2H),1.33-1.28(m,J=23.6Hz,2H).

[0142] 6. Synthesis of compound e-1:

[0143]

[0144] 3-Bromoaniline (1 mL, 9.5 mmol) was added to a 2-propanol solution of e-01 (2 g, 9.5 mmol), and stirred overnight at room temperature under argon atmosphere. The precipitate was washed with water and ether, and then dried under vacuum to give 6-nitro-4-(3-bromophenylamino)quinazoline (e-1) (72%) as a yellow solid.

[0145] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0146] LC / MS m / z 344.0[M+H].RT=1.90min.

[0147] 7. Synthesis of compound e-2:

[0148]

[0149] The separated e-1 (1.0 g, 2.80 mmol) was added to an aqueous solution of ethanol (1:2, 58 mL) and acetic acid (2.8 mL), and Fe (1.95 g, 34.77 mmol) was added. The resulting turbid mixture was refluxed and stored for 1 hour. The reaction was cooled to room temperature, alkalized with concentrated ammonia, and extracted with DCM. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain the target compound e-2 as a yellow solid.

[0150] Mobile phase: A: water (0.1% TFA), B: ACN (0.1% TFA); gradient: 5% B, increasing to 95% B over 1.5 min; flow rate: 1.5 mL / min; column: 4.6 × 50 mm XBridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0151] LC / MS m / z 316.0 [M+H] + RT = 1.00 min.

[0152] 8. Synthesis of compound e-3:

[0153]

[0154] Under Dean-Stark conditions, a mixture of 4-amino-1-butanol (SM-1) (5 g, 56.09 mmol) and phthalic anhydride (8.30 g, 56.09 mmol) in toluene (150 mL) was heated under reflux for 3 h. The crude product, obtained by cooling and desolventizing under vacuum, was eluted by flash column chromatography with PE / EA to give a colorless oil, which, after standing, yielded 12.4 g (97%) of colorless crystalline solid.

[0155] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0156] LC / MS m / z 219.0 [M+H] + RT = 1.52 min.

[0157] 9. Synthesis of compound e-4:

[0158]

[0159] At -78°C, a solution of dimethyl sulfoxide (6.45 ml, 22.81 mmol) in dichloromethane was added dropwise to a solution of oxaloyl chloride (5.79 g, 45.61 mmol) in dichloromethane, followed by the addition of 2-(4-hydroxybutyl)-1H-isoindole-1,3(2H)-dione (e-3) (5.0 g, 22.81 mmol) in dichloromethane.

[0160] After stirring for 20 minutes, a dichloromethane solution of triethylamine (9.23 g, 91.22 mmol) was added, the reaction temperature was raised to 0 °C, and stirring was continued for another 30 minutes. After the reaction was complete, a saturated aqueous sodium chloride solution was added, followed by extraction with ethyl acetate. The organic layer was washed with water and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The solvent was distilled under reduced pressure to give the title compound (6.0 g, 82% purity).

[0161] Mobile phase: A: water (0.1% TFA), B: ACN (0.1% TFA); gradient: 5% B, increasing to 95% B over 1.5 min; flow rate: 1.5 mL / min; column: 4.6 × 50 mm XBridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0162] LC / MS m / z 217.0 [M+H] + RT = 1.05 min.

[0163] 10. Synthesis of compound e-5:

[0164]

[0165] 4-Imidaclopridaldehyde (1.0 g, 4.60 mmol) was dissolved in CH₂Cl₂ (10 mL), and CH₂Cl₂ (10 mL) was treated with methyl(triphenylphosphine)imine acetate (1.53 g, 4.60 mmol). After 1 h, the solution was concentrated, inverted onto a silica gel column, and eluted with PE / EA to give a white solid (79%).

[0166] 11. Synthesis of compound e-6:

[0167]

[0168] A solution of e-5 (0.9 g, 3.29 mmol) and a solution of 4N HCl (10 mL) were refluxed in dioxane for 4 hours. The solution was cooled to RT and the solvent was removed. The residue was subjected to column chromatography (SiO2, DCM to ethyl acetate) to give the title compound as a white solid.

[0169] 12. Synthesis of compound e-7:

[0170]

[0171] e-2 (1.0 g, 3.17 mmol), e-1 (0.55 g, 2.11 mmol), and HATU (0.63 g, 1.66 mmol) were dissolved in DCM (10 mL) and DMF (1 mL). N-ethyldiisopropylamine (0.041 mg, 3.17 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The organic layer was extracted with DCM, dried over Na2SO4, and concentrated to obtain the crude product, which was then purified by silica gel column chromatography (eluent: DCM / MeOH).

[0172] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0173] LC / MS m / z 557.0 [M+H] + RT = 1.88 min.

[0174] 13. Synthesis of compound E:

[0175]

[0176] e-7 (1.4 g, 2.52 mmol) was dissolved in ethanol (60 mL) and CHCl3 (20 mL) and heated to 80 °C. H2O (0.7 mL) was added to the solution, and the mixture was heated at 80 °C for 1 hour. The solution was then cooled to room temperature, and the precipitate was removed by filtration and washed with diethyl ether. The filtrate was concentrated to give the crude product. Preparative high-performance liquid chromatography (HPLC) purification yielded compound E (600 mg) as a yellow solid.

[0177] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0178] LC / MS m / z 425.0 [M+H] + RT = 1.60 min.

[0179] 14. Synthesis of PDCy3 (compound 2):

[0180]

[0181] Compound A (50 mg, 0.081 mmol), compound E (51 mg, 0.12 mmol), and HATU (33 mg, 0.088 mmol) were dissolved in DCM (2 mL). Then, n-ethyldiisopropylamine (20.9 mg, 0.16 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Prior purification by high-performance liquid chromatography yielded compound 2 (40 mg) as a pink solid.

[0182] Mobile phase: A: water (10 mm NH4HCO3), B: MeCN. Flow rate: 1.8 mL / min. Column: 4.6 × 50 mm Bridge C18 column (3.5 μm particles). Detection methods include diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0183] Compound 2:

[0184] 1-(6-((E)-6-(4-(3-bromophenylamino)quinazolin-6-ylamino)-6-oxohex-4-enylamino)-6oxohexyl)-3,3-dimet hyl-2-((1E,3E)-3-(1,3,3-trimethyl-5-sulfonatoindolin-2-ylidene)prop-1-enyl)-3H-indolium-5-sulfonate

[0185] Chemical formula: C 50 H 53 BrN7O8S2 -

[0186] Appearance: Pink solid

[0187] LC / MS m / z 1024.0 [M+H] + [M+H] + / 2.RT=1.46min.

[0188] 'H NMR (400MHz, DMSO) δ10.37(s,1H),8.83(d,1H),8.68(s,1H),8.33(t,1H),8.13(s,1H),8.02-8.01(s,J=2Hz,1H),7.80-7.99(d,J=2H z,1H),7.82-7.79(m,J=11.2Hz,4H),7.72-7.66(m,J=21.6Hz,2H),7.44-7.37(m,J=28Hz,4H),7.22(s,1H),7.09(s,1H),7.96(s,1H), 6.51-6.46(d,J=17.6Hz,2H),4.30(s,1H),4.12(s,2H),3.65-3.63(d,J=7.6Hz,3H),2.75-2.71(t,J=17.6Hz,1H),2.33(s,1H),2.21- 2.15(m,J=24.4Hz,2H),1.90-1.85(m,J=21.2Hz,2H),1.75-1.73(d,J=8Hz,2H),1.68(s,12H),1.67-1.65(d,J=10Hz,1H),1.5(s,2H).

[0189] Example 2. Bioanalysis

[0190] Materials and methods

[0191] Material. The plasmids encoding EGFR (1-988) and the split YFP fragment were obtained from Addgene (#11011, #27097, and #22010), donated by Dr. Timothy A. Springer and Dr. Chang-Deng Hu. Gefitinib was obtained from Med-Chem Express (NJ, USA). Recombinant human EGF was obtained from Sinocare (China) and recombined and stored according to recommendations. Restriction endonucleases and the Gibson recombination kit were purchased from NEB (MA, USA); DNA polymerase was purchased from Novizum (China); anti-protein C antibody was purchased from Genscript (China); and anti-phosphorylation antibody 4G10 was purchased from Merck Millipore (USA).

[0192] Synthesis of PDCy3 probe.Details of the PDCy3 probe synthesis are provided in the Supplementary Methods. The synthesized material was characterized by IH NMR spectra recorded on a Bruker AVIII 400 and LCMS measurements performed on an Agilent 1200 HPLC / 6100 SQ system. In the LCMS experiments, mobile phase A was water (10 mM NH4HCO3), and mobile phase B was MeCN. A 4.6 x 50 mm Bridge C18 column (particle size 3.5 μm) was eluted with a linear gradient of 5% B–95% B over 1.3 min at a flow rate of 1.8 mL / min. Detection methods included diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0193] Inhibits the activity of EGFR kinase. HEK293T cells cultured in 24-well plates were transfected as described. Transfected cells were starved for 44 hours in EX-Cell 293 serum-free medium (SIGMA) containing 6 mM glutamine, followed by treatment with EGF, an inhibitor, or a probe. Cells were lysed with 40 μL / well lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, 1 mM Na3VO4, 2 mM PMSF). Lysates were centrifuged at 13200 g for 20 min. The supernatant was mixed with 6x SDS sample buffer for SDS-PAGE and Western blotting analysis. EGFR expression and phosphorylation levels were detected using antibodies against protein C and 4G10, respectively.

[0194] Cell screening. After starving the cells in EX-Cell 293 serum-free medium for 44 hours, the cells were transfected and then treated with gefitinib, PDCy3, or EGF at 37°C for 4 hours. Cells in each well were washed twice with 500 μL of pre-chilled PBS and then resuspended in 400 μL. The final cell density was estimated to be 5 x 10⁻⁶ cells / well. 5 -1X10 6 Cells / mL. 3 μL of anti-EGFR 528 antibody (Santa Cruz Biotechnology Inc., USA), at a concentration of 200 μg / mL, was added to each well to detect EGFR expression. After incubating on ice for 30 min, the cells were washed twice with cold PBS and then resuspended in 400 μL of PBS. Next, 0.3 μL of FITC-labeled secondary antibody (2 mg / mL) was added to each sample. After incubating on ice for 30 min, the cells were washed three times with PBS and resuspended in 400 μL / well of PBS. Flow cytometry analysis was performed using a BD FACSCalibur system.

[0195] Protein complementarity analysis. For protein complementarity analysis, we designed two complementary constructs: one encoding human EGFR (1-998) fused with a split-YFP-N (1-172) fragment, and the other encoding human hEGFR (1-998) fused with a split-YFP-C (155-238) fragment. Kinase mutations were introduced into both complementary constructs to form complementary pairs. All constructs were validated by sequencing. The paired constructs encoding the same receptor were co-transfected into HEK293T cells. Transfected cells were starved in EX-Cell 293 serum-free medium for 44 hours, followed by treatment with 1 μM PDCy3 for 4 hours. Flow cytometry was used to analyze EGFR dimerization and the binding of the dimer to PDCy3.

[0196] result

[0197] This fluorescent probe can competitively inhibit the kinase activity of EGFR.

[0198] The functions of each module of the probe were analyzed. In general, the fluorescence spectrum of this probe is very similar to that of Cy3, except for an additional small peak at 674 nm in the emission spectrum (Figure 2A). In imaging applications, this additional small peak can extend the detection range of the PDCy3 probe to the visible red region, such as in FRET experiments.

[0199] Next, we analyzed the inhibitory effect of the PDCy3 probe on EGFR kinase activity (Figure 2B). In this experiment, HEK293T cells transfected with EGFR were treated with different concentrations of PDCy3 probe for 10 min with or without 10 nM EGF. Western blotting was used to detect EGF receptor expression and phosphorylation levels. As shown in Figure 2B, the probe inhibited EGFR kinase activity in a dose-dependent manner. At a probe concentration of 20 μM, EGFR kinase activity was completely inhibited. In contrast, 5 μM gefitinib completely inhibited EGFR kinase activity (Figure 2B).

[0200] We used gefitinib as a competitive inhibitor to demonstrate the specificity of the PDCy3 probe in inhibiting EGFR kinase activity (Figure 2C). EGFR-positive or control cells were simultaneously labeled with the EGFR-specific antibody 528 and the PDCy3 probe in the presence or absence of 5 μM gefitinib. The binding ability of the PDCy3 probe to EGFR-expressing cells under different conditions was compared by flow cytometry. Treatment with 10 nM EGF did not enhance the binding of the PDCy3 probe to EGFR-expressing cells (Figure 2C). However, in the presence of EGF, 5 μM gefitinib reduced the number of EGFR-expressing PDCy3-positive cells from 45.4% to 5.3% (Figure 2C). These results indicate that the PDCy3 probe can specifically bind to the receptor on the cell surface and competitively inhibit EGFR kinase activity. From another perspective, this experiment also demonstrates the practicality of this PDCy3 probe in competitively screening for EGFR inhibitors.

[0201] The PDCy3 probe is sensitive to EGFR and its oncogenic mutations.

[0202] To expand the application of the PDCy3 probe in diagnostic-related settings, we optimized the labeling conditions to enable more specific labeling of EGFR-overexpressing cells. We compared the efficiency of labeling EGFR-overexpressing and non-overexpressing cells with different concentrations of PDCy3 (Figure 3A). We found that at a concentration of 1 μM, EGFR-positive cells could be specifically labeled by PDCy3. Under this condition, the probe could label more than 84.9% of EGFR-positive cells. Under the same conditions, 6.7% of EGFR-negative cells and 1.4% of untransfected cells were labeled with the PDCy3 probe. However, when the PDCy3 concentration was increased to 5 μM or higher, untransfected or transfected EGFR-negative cells also began to be labeled nonspecifically.

[0203] The PDCy3 probe, derived from PD168393, is compatible with both activated and inactive conformations of the kinase and promotes conformational dimerization of the epidermal growth factor receptor in the absence of EGF on the cell surface. We investigated whether this probe is more sensitive to EGFR asymmetric dimers or oncogenic activating mutations, the latter being required for allosteric activation of the kinase (Fig. 3B). The introduction of the L834R mutation disrupted the inactive conformation of the EGFR kinase, increasing the number of EGFR-positive cells binding to PDCy3 from 52.4% to 65.9% (Fig. 3B, 3C). Introducing the V924R mutation to disrupt the asymmetric kinase dimer did not weaken the binding of PDCy3 to transfected EGFR-positive cells; instead, it enhanced it by 8% (Fig. 3B, 3C).

[0204] To further investigate how EGFR kinase dimerization affects PDCy3 binding, we performed a protein complementation experiment. In this experiment, we recombined a split-YFP fragment into the C-terminus of EGFR and then reflected the level of EGFR dimerization by co-transfecting the receptor-assembled intact YFP (Fig. 4A). As shown in Fig. 4B, cells expressing EGFR dimers were divided into two cell populations. In one population, the amount of PDCy3 binding increased linearly with the amount of EGFR dimer formation (Fig. 4C). In the other population, the amount of PDCy3 binding was independent of the amount of EGFR dimer formation. The L858R mutation sensitized both receptor-dimerized cell populations to PDCy3 binding (Fig. 4C). However, the cell population equivalent to its wild-type receptor was 5-fold more sensitive to PDCy3 binding than the other population. Furthermore, the percentage of cells highly sensitive to PDCy3 decreased from 38.4% to 27.4% compared to the wild-type receptor (Fig. 4C, 4D). On the other hand, the V924R mutation introduced on L834R did not block receptor dimerization (Fig. 4C, 4D). Instead, it altered the cell population less sensitive to PDCy3, making it independent of or resistant to the EGFR dimer. Furthermore, the V924R mutation increased the percentage of cells highly sensitive to PDCy3 from 27.4% to 49.2% (Fig. 4C, 4D). These results indicate that there are two distinct forms of EGFR dimer on the cell surface: one highly sensitive to PDCy3 binding and the other insensitive. Since the V924R mutation can disrupt the asymmetric kinase dimer, the dimer highly sensitive to PDCy3 should be different from this form of dimer.

[0205] discuss

[0206] In this paper, we present our concept for the modular design and development of probes targeting oncogenic receptors. With the dramatic increase in the cost of new drug development and the significant reduction in therapeutic targets, the re-evaluation of drugs in the market is receiving increasing attention from industry and academia. Based on structural design, we explore the potential of reprogrammed EGFR kinase inhibitors for drug screening, cell sorting, and protein-protein interaction imaging. The benefits of our modular design include, but are not limited to, interchangeable imaging and targeting modules. Interchangeable imaging modules can be used for different biological and / or therapeutic situations with different imaging properties. On the other hand, interchangeable targeting modules can be used to detect different targets or different states of specific targets. In our case, PDCy3 is selective for EGFR-overexpressing cells and is more sensitive to oncogenic mutations of L834R. Future research will address whether this probe can be used for clinical diagnostics and how EGF receptors dynamically interact on the cell surface.

[0207] Based on sensitivity to PDCy3 probe binding, we identified two distinct cell populations with different states of EGFR dimers on the cell surface. One population was highly sensitive to PDCy3 binding, while the other was insensitive. The L834R mutation promoted the formation of dimers with low PDCy3 sensitivity, while the V924R mutation promoted the formation of dimers with high PDCy3 sensitivity. Considering that the V924R mutation can disrupt the active conformation of the asymmetric EGFR dimer, while L834R favors the active conformation of the kinase, we believe that the EGFR dimer with low PDCy3 sensitivity represents an asymmetric active kinase dimer. On the other hand, we believe that the EGFR dimer with high PDCy3 sensitivity actually represents another kinase binding state. Early structural studies have suggested that the EGFR kinase domain can form both asymmetric active and symmetric inactive dimers in vitro. Intuitively, the PDCy3-sensitive dimer might be equivalent to or associated with the symmetric inactive kinase dimer. However, much evidence opposes this possibility. Studies have shown that gefitinib and PD168393 are compatible with both the active and inactive conformations of EGFR kinase, driving homodimerization or heterodimerization of unbound EGFR on the cell surface. In contrast, lapatinib and HKI-272 stabilize the kinase in the inactive conformation rather than the other. This difference is attributed to the conformational and dynamic properties of the kinase. In fact, the existence of intermediate transition states for EGFR kinase has been demonstrated in molecular dynamics simulations. Interestingly, the structure of PD168393 after binding to EGFR kinase varies greatly in the positions of its N-lobes and α-C-helices (rmsd from 1.8 Å to 2.7 Å, when their C-lobes are stacked together) (Fig. 5A). One of the structures (PDB 4LLO) is closer to the intermediate state (PDB 2RF9) with an inherently disordered α-C-helix found in molecular dynamics simulations (Fig. 5B). However, we provide a tool to investigate EGFR dimerization and drug sensitivity. The results indicate the existence of two different states of dimerized kinase receptors on the cell surface. These two different dimerization states exhibit different sensitivities to kinase inhibitors. Identifying these differences in kinase dimerization and drug sensitivity could inform the development of next-generation therapies.

[0208] Example 3. Bioanalysis

[0209] Inspired by the unresolved challenges described in the "Background" section, we designed a modular therapeutic diagnostic probe targeting specific conformations of EGFR kinases associated with tumorigenesis and applied probe-based cell detection technology to the chemical stratification of prostate cancer. We demonstrate examples of using this probe to competitively screen inhibitors, screen cells overexpressing or mutated with epidermal growth factor receptor, and image EGFR interactions on cell surfaces. Surprisingly, we found that the EGFR kinase inhibitor PD168393 is more sensitive to an active intermediate state different from the asymmetric dimer. Furthermore, we tested eight urine samples from seven prostate cancer patients and one suspected prostate cancer patient using our probe-based cell detection technology. These samples responded similarly to the probe: one was highly sensitive, while the others were not. EGFR mutations were detected only in samples with high sensitivity to the probe, but not in other wild-type (WT) EGFR overexpression samples. Clinical analysis confirmed that subjects whose samples were highly sensitive to the probe progressed to castration resistance and metastasis.

[0210] Materials and methods

[0211] Material. The plasmids encoding EGFR (1-988) and the split YFP fragment were obtained from Addgene (#11011, #27097, and #22010), donated by Dr. Timothy A. Springer and Dr. Chang-Deng Hu. Gefitinib was obtained from Med-Chem Express (NJ, USA). Recombinant human EGF was obtained from Sinocare (China) and recombined and stored according to recommendations. Restriction endonucleases and the Gibson recombination kit were purchased from NEB (MA, USA); DNA polymerase was purchased from Novizum (China); anti-protein C antibody was purchased from Genscript (China); and anti-phosphorylation antibody 4G10 was purchased from Merck Millipore (USA). Anti-CK7 (ab181598), anti-CK20 (ab76126), anti-androgen receptor (ab74272), anti-chorionic villi (ab130751), and anti-TTF-1 (ab72876) antibodies were obtained from Abcam (Cambridge, MA, USA); anti-PSA (31-1210-00) antibody was obtained from RevMAbBiosciences (San Francisco, CA, USA).

[0212] Synthesis of PDCy3 probe.Details of the PDCy3 probe synthesis are provided in the Supplementary Methods. The synthesized material was characterized by IH NMR spectra recorded on a Bruker AVIII 400 and LCMS measurements performed on an Agilent 1200 HPLC / 6100SQ system. In the LCMS experiments, mobile phase A was water (10 mM NH4HCO3), and mobile phase B was MeCN. A 4.6 x 50 mm Bridge C18 column (particle size 3.5 μm) was eluted with a linear gradient of 5% B–95% B over 1.3 min at a flow rate of 1.8 mL / min. Detection methods included diode array (DAD), evaporative light scattering (ELSD), and positive / negative electrospray ionization.

[0213] Inhibits the activity of EGFR kinase. HEK293T cells cultured in 24-well plates were transfected as described. Transfected cells were starved for 44 hours in EX-Cell 293 serum-free medium (SIGMA) containing 6 mM glutamine, followed by treatment with EGF, an inhibitor, or a probe. Cells were lysed with 40 μL / well lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, 1 mM Na3VO4, 2 mM PMSF). Lysates were centrifuged at 13200 g for 20 min. The supernatant was mixed with 6x SDS sample buffer for SDS-PAGE and Western blotting analysis. EGFR expression and phosphorylation levels were detected using antibodies against protein C and 4G10, respectively.

[0214] Cell screening. After starving the cells in EX-Cell 293 serum-free medium for 44 hours, the cells were transfected and then treated with gefitinib, PDCy3, or EGF at 37°C for 4 hours. Cells in each well were washed twice with 500 μL of pre-chilled PBS and then resuspended in 400 μL. The final cell density was estimated to be 5 x 10⁻⁶ cells / well. 5 -1X10 6 Cells / mL. 3 μL of anti-EGFR 528 antibody (Santa Cruz Biotechnology Inc., USA), at a concentration of 200 μg / mL, was added to each well to detect EGFR expression. After incubating on ice for 30 min, the cells were washed twice with cold PBS and then resuspended in 400 μL of PBS. Next, 0.3 μL of FITC-labeled secondary antibody (2 mg / mL) was added to each sample. After incubating on ice for 30 min, the cells were washed three times with PBS and resuspended in 400 μL / well of PBS. Flow cytometry analysis was performed using a BD FACSCalibur system.

[0215] Protein complementarity analysis. For protein complementarity analysis, we designed two complementary constructs: one encoding human EGFR (1-998) fused with a split-YFP-N (1-172) fragment, and the other encoding human hEGFR (1-998) fused with a split-YFP-C (155-238) fragment. Kinase mutations were introduced into both complementary constructs to form complementary pairs. All constructs were validated by sequencing. The paired constructs encoding the same receptor were co-transfected into HEK293T cells. Transfected cells were starved in EX-Cell 293 serum-free medium for 44 hours, followed by treatment with 1 μM PDCy3 for 4 hours. Flow cytometry was used to analyze EGFR dimerization and the binding of the dimer to PDCy3.

[0216] Cell analysis of urine samples from cancer patients. Collect 10-15 mL urine samples from each cancer patient. Centrifuge at 300 g for 10 minutes at 4°C to collect cells, then resuspend in PBS. Cells from each sample are then divided into three aliquots: one for cell assays; another for RT-qPCR; and the remainder mixed with a cryoprotectant (80% fetal bovine serum and 20% DMSO) at a 1:1 ratio and stored in liquid nitrogen. Cells used for assays are co-incubated with 0, 0.5, 1, and 5 μM PDCy3, respectively, in the dark on ice for 20 minutes before analysis by flow cytometry.

[0217] RT-qPCR analysis. Cells were collected from urine samples by centrifugation at 300g for 10 minutes at 4°C and resuspended in 1 ml Trizol. After standing for 5 minutes, 0.25 mL of chloroform was added to each sample. After incubation at room temperature for 5 minutes, the samples were centrifuged at 13,700g for 15 minutes at 4°C. The aqueous phase of each sample was collected and mixed with isopropanol at a 1:1 (v / v) ratio. After standing at -20°C for 30 minutes, the samples were centrifuged at 13,700g for 15 minutes at 4°C. The supernatant was discarded, and the precipitate was washed with 75% ethanol. The samples were then air-dried for half an hour and dissolved in 20 μL of DEPC-treated H2O.

[0218] As described by the manufacturer, reverse transcription of extracted RNA and q-PCR quantification of gene expression were performed using the Transcriptor First Strand cDNA Synthesis Kit and the FastStart Essential DNA Green Master Kit (Roche, USA), respectively. Primers used in the experiments are listed in Table 1. Expression of each gene from each subject was determined by q-PCR analysis using a LightCycler-96 instrument. Experimental statistics were determined using triplicate of extracted RNA samples.

[0219] Table 1: Primers for q-PCR analysis

[0220]

[0221] HE staining. After dewaxing and rehydration, 5 μm long sections were stained with hematoxylin solution for 5 minutes, then immersed five times in 1% acidic ethanol (1% HCl, 70% ethanol). The sections were rinsed with distilled water, stained with eosin solution for 3 minutes, dehydrated with a gradient of alcohols, and cleared with xylene. The mounted slides were examined and photographed using a Leica DM2500 microscope.

[0222] Immunohistochemistry. Formalin-fixed, paraffin-embedded (FFPE) sections were dewaxed and blocked with 3% H2O2. Antigen retrieval was performed twice using microwave in 0.01M citrate for 10 minutes each time, followed by cooling for 60 minutes. Slides were blocked with 3% goat serum and labeled with various primary antibodies. Slides were then labeled with secondary antibodies conjugated to Envision-plus HRP-labeled polymers and stained with DAB staining solution (Zhongshan Jinqiao, Beijing). Counterstaining was performed using Mayer-hematoxylin.

[0223] A statement of morality. Informed consent was obtained from each enrolled patient. This study was approved by the Ethics Committee of the Second Hospital of Tianjin Medical University (Certificate #KY2017K010).

[0224] result

[0225] Competitive inhibition of EGFR kinase activity

[0226] The functions of each module of the probe were analyzed. In general, the fluorescence spectrum of this probe is very similar to that of Cy3, except for an additional small peak at 674 nm in the emission spectrum (Figure 2A). In imaging applications, this additional small peak can extend the detection range of the PDCy3 probe to the visible red region, such as in FRET experiments.

[0227] Next, we analyzed the inhibitory effect of the PDCy3 probe on EGFR kinase activity (Figure 2B). In this experiment, HEK293T cells transfected with EGFR were treated with different concentrations of PDCy3 probe for 10 min with or without 10 nM EGF. Western blotting was used to detect EGF receptor expression and phosphorylation levels. As shown in Figure 2B, the probe inhibited EGFR kinase activity in a dose-dependent manner. At a probe concentration of 20 μM, EGFR kinase activity was completely inhibited. In contrast, 5 μM gefitinib completely inhibited EGFR kinase activity (Figure 2B).

[0228] We used gefitinib as a competitive inhibitor to demonstrate the specificity of the PDCy3 probe in inhibiting EGFR kinase activity (Figure 2C). EGFR-positive or control cells were simultaneously labeled with the EGFR-specific antibody 528 and the PDCy3 probe in the presence or absence of 5 μM gefitinib. The binding ability of the PDCy3 probe to EGFR-expressing cells under different conditions was compared by flow cytometry. Treatment with 10 nM EGF did not enhance the binding of the PDCy3 probe to EGFR-expressing cells (Figure 2C). However, in the presence of EGF, 5 μM gefitinib reduced the number of EGFR-expressing PDCy3-positive cells from 45.4% to 5.3% (Figure 2C). These results indicate that the PDCy3 probe can specifically bind to the receptor on the cell surface and competitively inhibit EGFR kinase activity. From another perspective, this experiment also demonstrates the practicality of this PDCy3 probe in competitively screening for EGFR inhibitors.

[0229] Sensitive to tumorigenic EGFR mutations, but insensitive to asymmetric kinase dimerization.

[0230] To characterize the specificity of PDCy3, we compared the labeling efficiency of EGFR-overexpressing and non-overexpressing cells at different PDCy3 concentrations (Figure 3A). We found that at a concentration of 1 μM, EGFR-positive cells could be specifically labeled by PDCy3. Under this condition, the probe could label more than 84.9% of EGFR-positive cells. Under the same conditions, 6.7% of EGFR-negative cells and 1.4% of untransfected cells were labeled with the PDCy3 probe. However, when the PDCy3 concentration was increased to 5 μM or higher, untransfected or transfected EGFR-negative cells also began to be labeled nonspecifically.

[0231] The PDCy3 probe, derived from PD168393, is compatible with both activated and inactive conformations of the kinase and promotes conformational dimerization of the epidermal growth factor receptor in the absence of EGF on the cell surface. We investigated whether this probe is more sensitive to EGFR asymmetric dimers or oncogenic activating mutations, the latter being required for allosteric activation of the kinase (Fig. 3B). The introduction of the L834R mutation disrupted the inactive conformation of the EGFR kinase, increasing the number of EGFR-positive cells binding to PDCy3 from 52.4% to 65.9% (Fig. 3B, 3C). Introducing the V924R mutation to disrupt the asymmetric kinase dimer did not weaken the binding of PDCy3 to transfected EGFR-positive cells; instead, it enhanced it by 8% (Fig. 3B, 3C).

[0232] To further investigate how EGFR kinase dimerization affects PDCy3 binding, we performed a protein complementation experiment. In this experiment, we recombined a split-YFP fragment into the C-terminus of EGFR and then reflected the level of EGFR dimerization by co-transfecting the receptor-assembled intact YFP (Fig. 4A). As shown in Fig. 4B, cells expressing EGFR dimers were divided into two cell populations. In one population, the amount of PDCy3 binding increased linearly with the amount of EGFR dimer formation (Fig. 4C). In the other population, the amount of PDCy3 binding was independent of the amount of EGFR dimer formation. The L858R mutation sensitized both receptor-dimerized cell populations to PDCy3 binding (Fig. 4C). However, the cell population equivalent to its wild-type receptor was 5-fold more sensitive to PDCy3 binding than the other population. Furthermore, the percentage of cells highly sensitive to PDCy3 decreased from 38.4% to 27.4% compared to the wild-type receptor (Fig. 4C, 4D). On the other hand, the V924R mutation introduced on L834R did not block receptor dimerization (Fig. 4C, 4D). Instead, it altered the cell population less sensitive to PDCy3, making it independent of or resistant to the EGFR dimer. Furthermore, the V924R mutation increased the percentage of cells highly sensitive to PDCy3 from 27.4% to 49.2% (Fig. 4C, 4D). These results indicate that there are two distinct forms of EGFR dimer on the cell surface: one highly sensitive to PDCy3 binding and the other insensitive. Since the V924R mutation can disrupt the asymmetric kinase dimer, the dimer highly sensitive to PDCy3 should be different from this form of dimer.

[0233] Probe-based cytology for prostate cancer stratification

[0234] In two small cohort studies, it was estimated that over 30% of prostate cancer patients were associated with EGFR overexpression, and approximately 10-15% were associated with EGFR mutations. Do these expression or gene alterations truly relate to EGFR dysfunction? How can we effectively identify patients with aberrant EGFR activation? How is EGFR dysfunction associated with the pathogenesis of prostate cancer? Which subtypes of patients can benefit from EGFR inhibitor therapy? To further understand these questions, we established a probe-based cytology approach and categorized prostate cancer patients based on urine samples. A total of seven prostate cancer patients were included in our small cohort study (Table 2).

[0235] Table 2: Clinical data of enrolled subjects

[0236]

[0237]

[0238]

[0239] Urine samples were labeled with 0.0, 0.5, 1.0, and 5.0 μM probes at 4°C for half an hour, and then analyzed by flow cytometry. Different cell populations were distinguished by the fluorescence intensity and SSC of the probe (Figure 6A). Urine samples from different subjects showed different responses to the probes, expressed as a function of probe concentration (Figures 6A, 6B). In particular, the sensitivity of sample from subject 4 was significantly higher than that of other subjects (Figure 6B). At a probe concentration of 0.5 μM, its relative binding to the sample was 13.6 times higher than the average of other probes. The probe fractions at 0.5 μM and 1.0 μM concentrations were 0.28 and 0.30, respectively, relative to the probe fraction at 5.0 μM concentration. Other samples showed the highest relative binding fractions at 0.5 μM and 1.0 μM probes, at 0.1 and 0.16, respectively.

[0240] To further understand the different response profiles of these samples in probe-based cytometry, we analyzed their expression and mutation status using RT-qPCR and Sanger sequencing (Figs. 6C, 6D, 7A, 7B). In RT-qPCR, EGFR overexpression was detected in all samples except for those in samples 1 and 4 (Figs. 6C, 7A). Furthermore, in the sample from subject 4, bidirectional Sanger sequencing revealed three single nucleotide polymorphisms (SNPs) (V745M, G800D, and P770T) on the EGFR kinase domain, which were not found in other samples (Figs. 6D, 7B). The V745M mutation was found in lung cancer patients treated with gefitinib, but its significance is uncertain, while the G800D mutation was found in head and neck squamous cell carcinoma, suggesting a drug response; no criteria were provided. https: / / www.ncbi.nlm.nih.gov / clinvar / The P770T mutation has not been reported in cancer research (https: / / www.ncbi.nlm.nih.gov / clinvar / ). Therefore, we tested the kinase activity and probe sensitivity of these mutations (Figs. 6E-G, 7C). In transiently transfected 293T cells, the V745M mutant showed higher kinase activity than the WT receptor, but the kinase activity of combined mutants such as G800D and P770T was impaired to some extent (Fig. 6G). Consistent with their kinase activity, cells transfected with the V745M mutant and the WT receptor showed high sensitivity to PDCy3 in flow cytometry, but cells transfected with G800D, P770T, and their combinations did not respond to the probe (Figs. 6E, 6F, 7C). In summary, the activating mutation V745M on EGFR can explain the enhanced probe sensitivity from sample 4.

[0241] Examination of the biopsy specimen from subject 4 confirmed that the patient had metastatic prostate cancer with androgen receptor overexpression (Figures 6H, 6I, 8). Subsequent retrospective examination revealed that the case was androgen resistance (Table 2).

[0242] discuss

[0243] Effective patient stratification is crucial for the diagnosis and treatment of prostate cancer. Currently, tissue specimen testing and genomic profiling are widely used in clinical research. However, abnormal variations in genetic, immune, or metabolic biomarkers detected by these techniques do not necessarily correspond to potential drug responses to personalized treatment strategies. Furthermore, the vast genetic and epigenetic variations associated with tumor heterogeneity and mutational pressure remain largely unrecognized. These limitations hinder the development of new treatment strategies and complicate our understanding of how to link genetic analysis to clinical application. Our chemostratification strategy utilizes a patient's bodily fluids to detect and classify individualized drug responses. Using this liquid biopsy technique in a small cohort study, we demonstrated high sensitivity to our designed therapeutic probe in a prostate cancer patient with an EGFR activating mutation (V745M) and metastatic tumor. Two additional concomitant inactivating mutations (G800D and P770T) were also found in the same patient, indicating an increased mutation frequency or heterogeneity associated with cancer progression. Moreover, we found that patients overexpressing wild-type EGFR did not show high sensitivity to the probe, consistent with reports from earlier multicenter clinical studies. We envision that in future clinical studies, chemical stratification combined with liquid biopsy could supplement genomic mapping.

[0244] Our chemical stratification is built upon the modular design of therapeutic diagnostic probes targeting specific conformations of EGFR kinases associated with tumorigenesis. In our structure-based design, we explored the potential of reprogramming validated EGFR kinase inhibitors for application in drug screening, cell sorting, and protein-protein interaction imaging. The benefits of our modular design include, but are not limited to, interchangeable imaging and targeting modules. Interchangeable imaging modules can be used in diverse biological and / or therapeutic settings with varying imaging properties. On the other hand, interchangeable targeting modules can be used to detect other targets or different states of specific targets. In our case, PDCy3 is selective for EGFR-overexpressing cells and more sensitive to EGFR-activating mutations, including L834R (a well-characterized mutation) and V745M (a mutation identified in this study).

[0245] Based on sensitivity to PDCy3 probe binding, we identified two distinct cell populations with different states of EGFR dimers on the cell surface. One population was highly sensitive to PDCy3 binding, while the other was insensitive. The L834R mutation promoted the formation of dimers with low PDCy3 sensitivity, while the V924R mutation promoted the formation of dimers with high PDCy3 sensitivity. Considering that the V924R mutation can disrupt the active conformation of the asymmetric EGFR dimer, while L834R favors the active conformation of the kinase, we believe that the EGFR dimer with low PDCy3 sensitivity represents an asymmetric active kinase dimer. On the other hand, we believe that the EGFR dimer with high PDCy3 sensitivity actually represents another kinase binding state. Early structural studies have suggested that the EGFR kinase domain can form both asymmetric active and symmetric inactive dimers in vitro. Intuitively, the PDCy3-sensitive dimer might be equivalent to or associated with the symmetric inactive kinase dimer. However, much evidence opposes this possibility. Studies have shown that gefitinib and PD168393 are compatible with both the active and inactive conformations of EGFR kinase, driving homodimerization or heterodimerization of unbound EGFR on the cell surface. In contrast, lapatinib and HKI-272 stabilize the kinase in the inactive conformation rather than the other. This difference is attributed to the conformational and dynamic properties of the kinase. In fact, the existence of intermediate transition states for EGFR kinase has been demonstrated in molecular dynamics simulations. Interestingly, the structure of PD168393 after binding to EGFR kinase varies greatly in the positions of its N-lobes and α-C-helices (rmsd from 1.8 Å to 2.7 Å, when their C-lobes are stacked together) (Fig. 5A). One of the structures (PDB 4LLO) is closer to the intermediate state (PDB 2RF9) with an inherently disordered α-C-helix found in molecular dynamics simulations (Fig. 5B). However, we provide a tool to investigate EGFR dimerization and drug sensitivity. The results indicate the existence of two different states of dimerized kinase receptors on the cell surface. These two different dimerization states have different sensitivities to kinase inhibitors.

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Claims

1. A probe, represented by formula (1): Module 2-L-Module 1 (1) in, L is -C(O)NH-; Module 1 is a part that targets EGFR kinase; and Module 2 is the probe used for imaging, which is represented by equation (3) or equation (4): in R1 in equation (3) or equation (4) is the connection point from module 2 to L, and is -(CH2). n -; In equation (3) or equation (4), R2 is -(CH2). n CH3; and In equation (3) or equation (4), n is an independent 0, 1, 2, 3, 4, 5 or 6; Module 1 is represented by equation (2): in In equation (2), R1 is H or a halogen; In equation (2), R2 is H; In equation (2), R3 is H; In equation (2), R4 is the connection point from module 1 to L, and is -(CH2). n -;and In equation (2), n is 1, 2, 3, 4, 5, or 6; Or it could be a salt of formula (1) that can be used for treatment.

2. The probe of claim 1, wherein a counterion is present, said counterion being selected from alkali metal and alkaline earth metal ions.

3. The probe of claim 1, wherein the probe is represented by formula (7):

4. The probe of claim 3, wherein a counterion is present, said counterion being selected from alkali metal and alkaline earth metal ions.

5. Use of the probe of any one of claims 1-4 in cells that target and / or label EGFR kinase and / or express EGFR kinase for non-diagnostic and non-therapeutic purposes, wherein said EGFR kinase is wild-type or contains only the following mutations: L834R; or V745M; or A combination of L834R and V924R.

6. Use of the probe of any one of claims 1-4 in cells enriched to express EGFR kinase for non-diagnostic and non-therapeutic purposes, wherein the EGFR kinase expressed by said cells is wild-type or has only the following mutations: L834R; or V745M; or A combination of L834R and V924R.

7. The use of claim 6, wherein the enrichment of cells is performed by the following steps: labeling cells overexpressing EGFR kinase with a probe of any one of claims 1-4; collecting the labeled cells overexpressing EGFR kinase.

Images