A screening method of molecular glue drugs and application in screening of degrading drugs

Abstract

The application discloses a screening method of molecular glue drugs and application in degradation drug screening. The screening method of molecular glue drugs comprises the following steps: preparing a first microarray chip from a compound library, screening compounds combined with an effector protein based on an OI-RD image to obtain first candidates; preparing a second microarray chip, measuring binary binding kinetics of the first candidates and the effector protein based on OI-RD optical real-time signals, verifying, and obtaining second candidates; and preparing a third microarray chip, screening compounds for ternary reactions based on OI-RD real-time signals of ternary reactions. The screening method directly detects the formation of a target protein-molecular glue-effector protein ternary complex, significantly improves screening accuracy, can effectively identify molecular glue types that do not directly bind to target proteins, and realizes higher specificity and higher flux molecular glue screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for screening molecular gel drugs and its application in the screening of degradation drugs. Background Technology

[0002] The rapid development of protein degraders (such as PROTACs and molecular gels) is primarily driven by the limitations of traditional inhibitors in terms of target selectivity and drug resistance. Many disease-related proteins have long been considered "undruggable" targets due to the lack of active pockets that traditional small molecules can bind to. The core mechanism of protein degraders lies in inducing the formation of a "target protein-degrader-effect protein" ternary complex, utilizing the cell's natural protein degradation system to directly clear the target protein, rather than merely inhibiting its activity. This effectively overcomes multiple challenges such as catalytic activity inhibition and protein accumulation, demonstrating the therapeutic potential for low-dose, long-lasting, and broad-spectrum targeting.

[0003] Among protein degraders, both PROTACs and molecular glues aim to construct ternary complexes, but their pathways differ. PROTACs, as heterobifunctional molecules, bind to the target protein and E3 ubiquitin ligase via two independent ligands, respectively, and use a linker chain to shorten their spatial distance, promoting the formation of a ternary complex and initiating ubiquitination degradation. These compounds have large molecular weights, offering high flexibility in rational design, but also face challenges in drug development, such as membrane permeability and metabolic stability. In contrast, molecular glues induce or enhance the interaction interface between the target protein and effector proteins (such as E3 ligases or autophagy-related proteins LC3), promoting "molecular adhesion" and thus forming a ternary complex that triggers degradation. Molecular glues have the advantages of small molecular weight and superior pharmacokinetic properties, but due to the complex interfacial reconstruction process involved in their mechanism of action, rational design is challenging, and current discovery strategies are primarily based on serendipitous findings.

[0004] Currently, some technologies have been attempted to screen molecular adhesives. Based on their principles, they can be mainly divided into two categories: labeled technologies and label-free technologies.

[0005] 1. Label-based screening techniques and their limitations

[0006] Techniques such as DEL (DNA-encoded compound library screening), ALPHA Screen (amplified luminescence proximity homogeneous detection), fluorescence polarization (FP), and time-resolved fluorescence resonance energy transfer (TR-FRET) all rely on exogenous labels such as DNA and fluorescent groups to achieve signal detection. The introduction of these labels may alter the physicochemical properties or spatial conformation of the molecules themselves, thereby interfering with the natural interaction mechanisms between the molecules and the target sites. This can easily lead to interference phenomena such as steric hindrance and non-specific binding, affecting the accuracy and reliability of the screening results.

[0007] 2. Label-free verification techniques and their limitations

[0008] While label-free techniques such as surface plasmon resonance (SPR), biomembrane interferometry (BLI), and isothermal titration calorimetry (ITC) can provide real and reliable molecular binding information, their detection throughput is generally low, making them suitable for validation after initial screening rather than for the initial screening of large-scale compound libraries.

[0009] 3. Oblique Incidence Reflection Differential Method (OI-RD) and its Challenges

[0010] This study combines OI-RD technology to develop a label-free, high-throughput bio-interaction detection technique, providing an effective solution to overcome the aforementioned technical bottlenecks. In previous studies, we established a systematic screening method: using the same small molecule chip, we separately detected the binding of effector proteins and target proteins to immobilized small molecules on the chip. By analyzing the binding signals of the two proteins, we screened candidate molecules that can interact with both proteins simultaneously. This strategy has been successfully applied to the discovery of lead compounds in specific molecular gels, validating the practical value of OI-RD technology in the field of molecular gel screening.

[0011] However, this "separate screening and intersection taking" strategy may have the following problems: First, although the candidate molecules obtained by this method can bind to effector proteins and target proteins respectively, if the two binding sites are the same or there is a spatial conflict, the small molecule will be unable to bridge the two proteins simultaneously due to competitive binding, thus making it difficult to form the required ternary complex; Second, this method also has another blind spot: for molecular gels that only bind to effector proteins and indirectly promote binding to target proteins by changing the structure of effector proteins, they will be directly ignored when "intersection taking" because they have no signal in target protein screening, resulting in the missed screening of such important molecules.

[0012] In summary, although OI-RD technology has the advantages of high throughput and label-free operation, previous screening methods have obvious shortcomings: on the one hand, they cannot guarantee that all screened candidate molecules can effectively form ternary complexes, and on the other hand, they may miss important molecular gel types that do not directly bind to target proteins. Summary of the Invention

[0013] In view of this, the purpose of this invention is to provide a method and application for screening molecular glue drugs based on label-free high-throughput technology, which can directly detect drug formation, thereby overcoming the limitations of existing technology and providing a more effective tool for the development of molecular glue drugs.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] A method for screening molecular adhesive drugs includes the following steps:

[0016] (1) The compound library was prepared into a first microarray chip, and an effector protein solution was flowed through the chip surface. Based on the OI-RD image, compounds that bind to the effector protein were screened to obtain the first candidate.

[0017] (2) The first candidate is prepared into a second microarray chip, and the effector protein solution flows through the chip surface. The binary binding dynamics of the first candidate and the effector protein are measured based on the OI-RD optical real-time signal to verify whether the first candidate can bind to the effector protein and obtain the second candidate.

[0018] (3) The target protein is prepared into a third microarray chip, and a mixture of the effector protein and the second candidate flows through the chip surface. Based on the real-time OI-RD signal of the ternary reaction, compounds that can undergo ternary reactions with the effector protein and the target protein are screened from the second candidate verified by binary binding kinetics.

[0019] Furthermore, the concentration of the compound library is 10 mM.

[0020] Furthermore, in step (1), the first microarray chip contains functional groups of a fixed compound, which are fixed on the chip surface in the form of a microarray.

[0021] Further, in step (1), the first microarray chip is placed in a fluid system, a protein buffer is introduced, the compound microarray is scanned and imaged using OI-RD, the effector protein solution is introduced into the fluid system and incubated, the OI-RD image of the microarray is acquired again, and the difference is made with the image before the effector protein solution is introduced to find positive points where the signal changes. Based on the position of the positive points on the microarray, the compound represented by them is traced back as the first candidate.

[0022] Furthermore, in step (2), the second microarray chip is placed into a fluid system, a protein buffer is introduced, a sampling grid is set on the compound matrix, the effector protein solution is introduced, and then the effector protein solution is replaced with a buffer solution. The changes in the OI-RD signal on the matrix are monitored in real time, and the compounds corresponding to the verified molecular points are traced.

[0023] Furthermore, in step (3), the third microarray chip contains functional groups for immobilizing proteins, and different target proteins are immobilized on the chip surface in the form of a microarray.

[0024] Furthermore, in step (3), the third microarray chip is loaded onto a multichannel parallel fluid system, the effector protein and the second candidate verified by kinetics are pre-mixed and incubated, a sampling grid is set on the protein array, and a different mixture of the second candidate and the effector protein flows through each channel. The mixture is then replaced with buffer, and the changes in the OI-RD signal on the array are monitored in real time. Based on the position of the positive spot on the array, compounds that can form an effector protein-compound-target protein ternary complex are screened.

[0025] Application of any of the above-mentioned screening methods for molecular gel drugs in the screening of degradation drugs.

[0026] The screening method of this invention has the following outstanding advantages: First, by directly detecting the formation of the target protein-molecular glue-effect protein ternary complex, it effectively avoids the false positive problem caused by binding site competition in the traditional "separate screening-intersection" method, significantly improving screening accuracy; Second, this method can effectively identify molecular glue types that do not directly bind to the target protein, solving the problem of systematically missing such important molecules due to the lack of signal response in traditional screening; Furthermore, this method breaks through the throughput bottleneck of existing screening methods, achieving molecular glue screening with higher specificity, higher throughput, and lower cost, and is particularly suitable for the rapid discovery of novel ternary complex active molecular glues in large-scale compound libraries.

[0027] Furthermore, the application of this invention fully leverages the high-throughput and label-free advantages of OI-RD technology, enabling large-scale screening in solution environments close to physiological conditions, and providing an efficient and reliable technical platform for the systematic discovery of molecular gel drugs. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 This is a flowchart of the method in Example 1;

[0030] Figure 2 This is a schematic diagram of the compound chip in Example 1 and the high-throughput screening of effector protein binding candidates from the compound library;

[0031] Figure 3 This is a schematic diagram illustrating the kinetic verification of whether the candidate and the effector protein can bind in Example 1;

[0032] Figure 4 This is a schematic diagram of Example 1, which uses a ternary reaction and multi-channel parallel screening method to degrade molecular adhesives. Detailed Implementation

[0033] To further understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0034] A method for screening molecular gel drugs based on label-free high-throughput technology includes the following steps:

[0035] (1) The compound library is prepared into a first microarray chip. The first microarray chip contains functional groups of fixed compounds, and the compounds are fixed on the chip surface in the form of a microarray. The first microarray chip is placed in a fluid system, and protein buffer is introduced. The compound microarray is scanned and imaged using OI-RD. Effector protein solution is introduced into the fluid system and incubated. The OI-RD image of the microarray is acquired again. The difference between the image and the image before the introduction of effector protein solution is calculated to find positive points where the signal changes. Based on the position of the positive points on the microarray, the compounds they represent are identified as the first candidates. The throughput of step (1) can reach 10 3 ~10 4 Type / time.

[0036] (2) The first candidate is prepared into a second microarray chip. The second microarray chip is placed in a fluid system and a protein buffer is introduced. A sampling grid is set on the compound matrix using a computer. An effector protein solution is introduced, and then the effector protein solution is replaced with a buffer. The changes in the OI-RD signal on the matrix are monitored in real time. If the real-time curve shows obvious binding-dissociation behavior, it means that the test has been passed. This process can directly determine the "stability" of the compound binding. However, ELISA technology cannot obtain the dissociation process and may mistakenly identify compounds that bind transiently as positive. The second candidate is obtained by tracing the compound corresponding to the molecular point that has passed the test. The throughput of step (2) can still be maintained at 10 2 ~10 3 Type / time.

[0037] (3) The target proteins are prepared into a third microarray chip containing functional groups for immobilizing the proteins. Different target proteins are immobilized on the chip surface in the form of a microarray. The third microarray chip is loaded onto a multichannel parallel fluid system. The effector protein and the second candidate that has been verified by kinetics are pre-mixed and incubated. A sampling grid is set on the protein matrix using a computer. Each channel flows through a mixture of different second candidates and effector proteins. The mixture is then replaced with buffer. The changes in the OI-RD signal on the matrix are monitored in real time to find positive points with binding-dissociation curves. Based on the position of the positive points on the array, compounds that can form ternary complexes of effector protein-compound-target protein are screened. Step (3) can simultaneously verify the ternary binding activity of multiple candidates with multiple target proteins. The throughput can reach 50-200 types / time, which is significantly higher than the existing technology.

[0038] Example 1:

[0039] A screening method for molecular glue ternary complexes based on label-free high-throughput technology, such as Figure 1 As shown, it includes the following steps:

[0040] (1) The compound library was prepared into a compound chip containing functional groups of immobilized compounds. The compounds were immobilized on the chip surface in the form of a microarray using a bio-spotting instrument. The compound chip was placed in a fluid system, and protein buffer was introduced. The microarray was scanned and imaged using OI-RD. Effector protein solution was introduced into the fluid system and incubated. The OI-RD image of the microarray was acquired again, and the difference between this image and the image before introducing the effector protein solution was calculated. Figure 2 As shown, different colored dots on the chip represent different compounds. The black dot matrix images are the OI-RD images and difference maps before and after the reaction, corresponding to the reaction points on the chip matrix. Positive points where the signal changes are found, and based on the position of the positive points on the microarray, the compounds they represent are identified as first candidates for further verification.

[0041] (2) The first candidate compound was prepared into a first candidate chip. The first candidate chip was placed in a fluid system, and a protein buffer was introduced. A sampling grid was set on the compound matrix using a computer. An effector protein solution was introduced, and then the effector protein solution was replaced with buffer. The changes in the OI-RD signal on the matrix were monitored in real time, such as... Figure 3 As shown, K on The binding rate constant, K off The dissociation rate constant is K d To represent the equilibrium dissociation constant, the curves in the figure depict the kinetics of reaction binding and dissociation, indicating that the candidate and effector protein bind to each other, as shown in the figure. Figure 3The binding-dissociation curves shown indicate that the assay has passed verification. Tracing the compounds corresponding to the verified molecular sites yielded the second candidate.

[0042] (3) Multiple target proteins are prepared into target protein chips containing functional groups for immobilizing proteins. Different target proteins are immobilized on the chip surface in the form of a microarray using a bio-spotting instrument. The target protein chips are loaded onto a multi-channel parallel fluid system. Effector proteins and kinetically validated second candidates are pre-mixed and incubated. A sampling grid is set on the protein array using a computer. Each channel flows through a mixture of different second candidates and effector proteins, such as... Figure 4 As shown, each channel contains the same target protein array, and each array contains different target proteins. Channels one and two flow through mixtures of different compounds and effector proteins; the types of compounds vary, and the target proteins involved in the ternary reaction may also differ. The mixture is then replaced with buffer, and the OI-RD signal changes on the array are monitored in real time. Positive points appear on the binding-dissociation curve. Based on the position of the positive points on the array, compounds that can form effector protein-compound-target protein ternary complexes are screened.

[0043] The screening method described in this embodiment does not rely on known structural or activity information of molecular gels, is free from label interference, and is suitable for studying novel effector proteins or novel target proteins, with a throughput of up to 10. 3 ~10 4 High screening efficiency per species / time.

[0044] Any equivalent transformations and improvements made based on the technical solutions of this invention do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of this invention.

Claims

1. A method for screening of molecular glue drugs, characterized in that, The method comprises the following steps: (1) preparing a first microarray chip with a compound library, flowing an effector protein solution on the chip surface, screening and binding compounds of the effector protein based on OI-RD images to obtain first candidates; (2) preparing a second microarray chip with the first candidates, flowing the effector protein solution on the chip surface, measuring the binary binding kinetics of the first candidates and the effector protein based on OI-RD optical real-time signals, verifying whether the first candidates can bind to the effector protein to obtain second candidates; (3) preparing a third microarray chip with a target protein, flowing a mixed solution of the effector protein solution and the second candidates on the chip surface, screening compounds capable of forming ternary complexes of the effector protein, the compound and the target protein from the second candidates verified by binary binding kinetics based on OI-RD real-time signals of ternary reactions.

2. The method for screening molecular gel drugs according to claim 1, characterized in that, The concentration of the compound library is 10 mM.

3. The method for screening molecular gel drugs according to claim 1, characterized in that, In step (1), the first microarray chip comprises functional groups of immobilized compounds, which are immobilized on the chip surface in the form of a microarray.

4. The method for screening molecular gel drugs according to claim 1, characterized in that, In step (1), the first microarray chip is placed in a fluid system, a protein buffer is introduced, OI-RD is used to scan the compound microarray and imaging is performed, the effector protein solution is introduced into the fluid system and incubated, the OI-RD image of the microarray is acquired again, the image before the effector protein solution is introduced is subtracted, the positive points with signal changes are found, and the compounds represented by the positive points on the microarray are traced back to the first candidates.

5. The method for screening molecular gel drugs according to claim 1, characterized in that, In step (2), the second microarray chip is placed in a fluid system, a protein buffer is introduced, a sampling grid is set on the compound array, the effector protein solution is introduced, and then the effector protein solution is replaced with a buffer, the OI-RD signal changes on the array are monitored in real time, and the compounds corresponding to the verified molecular points are traced.

6. The method for screening molecular gel drugs according to claim 1, characterized in that, In step (3), the third microarray chip comprises functional groups of immobilized proteins, and different target proteins are immobilized on the chip surface in the form of a microarray.

7. The method for screening molecular gel drugs according to claim 1, characterized in that, In step (3), the third microarray chip is loaded on a multi-channel parallel fluid system, the effector protein and the second candidates verified by kinetics are pre-mixed and incubated, a sampling grid is set on the protein array, different mixtures of the second candidates and the effector protein flow through each channel, and then the mixtures are replaced with a buffer, the OI-RD signal changes on the array are monitored in real time, and the compounds capable of forming effector protein-compound-target protein ternary complexes are screened according to the positions of the positive points on the array.

8. The application of the method for screening molecular adhesives in the screening of degradable drugs.

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