A small molecule compound inhibitor of gremlin-1 and its use in treating colorectal cancer
By using DNA-encoded chemical library screening technology, small molecule compound inhibitors were prepared, which solved the problems of poor penetration and high cost in existing GREM1 treatment strategies, achieved effective inhibition of colorectal cancer, and reduced development costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE SEVENTH AFFILIATED HOSPITAL SUN YAT SEN UNIV SHENZHEN
- Filing Date
- 2026-04-02
- Publication Date
- 2026-06-30
AI Technical Summary
Existing GREM1 treatment strategies, such as neutralizing antibodies, suffer from poor tissue penetration and high costs, and lack systematic small molecule screening and mechanism research.
Using DNA-encoded chemical library (DEL) screening technology, small molecule compound inhibitors were efficiently screened out and combined with GREM1 protein to prepare compounds I and II for antagonizing GREM1 and inhibiting the migration and invasion of colorectal cancer.
The screened small molecule compound inhibitors are small in size, highly permeable, and low in cost. They can significantly inhibit the migration and invasion of colorectal cancer, have good pharmacokinetic properties, and reduce drug development costs.
Smart Images
Figure CN122304037A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of compound technology, specifically to a small molecule compound inhibitor that antagonizes GREM1 and its application in the treatment of colorectal cancer. Background Technology
[0002] Colorectal cancer is a malignant tumor that occurs in the colon and rectum, also known as colorectal cancer. It ranks third in incidence and fifth in mortality among malignant tumors in my country, with a significantly higher incidence in urban areas than in rural areas. Etiology involves environmental factors (high-fat diet, insufficient dietary fiber), genetic factors (familial adenomatous polyposis), and high-risk factors.
[0003] Distant metastasis of colorectal cancer (CRC) remains a leading cause of death among patients, and existing strategies have limited effectiveness. Therefore, the search for new treatment strategies is of significant clinical importance.
[0004] Gremlin-1 (GREM1), a classic antagonist of the bone morphogenetic protein (BMP) family, is widely involved in the formation of various organs, including the limbs and intestines, and regulates the development of diseases such as damage repair and tissue fibrosis. Recent studies have shown that GREM1 also plays an important role in various malignant tumors, especially in colorectal cancer (CRC). For example, GREM1 derived from mesenchymal cells can promote CRC growth by regulating BMP signaling. Similarly, GREM1 expression in epithelial cells can inhibit BMP signaling and, in combination with Wnt, activate the potential for tumor cell self-renewal and malignant transformation, ultimately promoting cancer development. Recent studies have also suggested that GREM1 can drive epithelial-mesenchymal transition (EMT) and distant metastasis in cancer cells. Previous research further demonstrates that GREM1 levels in both the mesenchymal and epithelial systems are significantly associated with poor patient prognosis. Furthermore, the Grem1-CreERT2;Rosa-LSL-DTA genetically engineered mouse metastasis model has demonstrated that GREM1 can promote CRC cell EMT and metastasis both in vivo and in vitro. Therefore, GREM1 is a therapeutic target with significant clinical translational potential.
[0005] Currently, treatment for GREM1 primarily relies on neutralizing antibodies. Although these antibodies have shown some efficacy in preclinical models, their application remains limited. For example, they can only act on extracellular GREM1, their large molecular weight restricts tissue penetration, and their production costs are high. In contrast, small molecule inhibitors offer advantages such as small molecular weight, strong tissue penetration, ease of pharmacokinetic regulation, and reduced production costs. However, no systematic small molecule screening and mechanistic studies targeting GREM1 have been conducted domestically or internationally.
[0006] In drug development, efficiently identifying small-molecule ligands that regulate target function has always been a core challenge. The rise of high-throughput screening (HTS) has made compound library screening routine, but its throughput, cost, and chemical space coverage remain limited. Subsequent advancements such as phage display, mRNA display, and yeast display, while generating massive libraries of peptides or peptide mimics through biological evolution mechanisms, are still confined to the realm of biomacromolecules. The emergence of DNA-encoded chemical libraries (DELs) combines the advantages of both: it integrates synthetic chemistry with DNA encoding, enabling the parallel screening of tens to billions of compounds in a single experiment. Compared to traditional techniques, DELs can achieve synthesis and screening at the nanometer or even picomolar level, significantly reducing experimental costs, and can overcome throughput limitations, enabling the exploration of a larger and richer chemical space. In particular, the continuous expansion of DEL reactions in recent years has demonstrated unprecedented innovation and broad application prospects in drug development. Summary of the Invention
[0007] The purpose of this invention is to provide a small molecule compound inhibitor that antagonizes GREM1 and its application in the treatment of colorectal cancer, so as to solve the problems mentioned in the background art.
[0008] To achieve the above objectives, the present invention provides the following technical solution: a small molecule compound inhibitor that antagonizes GREM1, the specific preparation process of which is as follows:
[0009] Step 1: First, dry the DEL (DNA-encoded chemical library) library under vacuum, then dissolve it in a protein solution containing 5-10 µM human Grem1 protein, and incubate it in the DEL selection buffer system for 30 minutes.
[0010] Step 2: After incubation, fix the Grem1 protein with 20 µl of HisPur™ Ni-NTA Magnetic Beads and continue incubation for 15 minutes. Then wash with 10 × 100 µl of screening buffer under shaking. Finally, remove unbound library molecules by vacuum aspiration.
[0011] Step 3: Before incubation, reserve 1 µl of DEL input sample and determine the input amount by quantitative PCR (Q-PCR). The bound DEL molecules are eluted and collected by centrifugation. Reserve 1 µl of purified DNA material and quantify it again by Q-PCR. All other samples continue to undergo a new round of affinity screening until the required amount of DEL template residue is reached. Finally, perform next-generation DNA sequencing.
[0012] Step 4: The DEL screening data is decoded and analyzed using internally developed software to process the sequence information obtained from the screening. By applying a customized filter, fragment combinations with counts below the noise floor are removed, and the true binding signals are identified. Through combined experiments and functional verification, the following small molecule compounds I and II are finally screened out.
[0013] Preferably, the Grem1 protein has a His tag at its C-terminus.
[0014] Preferably, the DEL screening buffer system comprises 50 mM Tris-HCl pH 8.5, 50 mM NaCl, 1.5 mM MgCl2, 1 mM TCEP, 0.005% Tween 20, and 1 mg / ml herring sperm vector DNA (HS-DNA).
[0015] Preferably, when all other samples undergo a new round of affinity screening, freshly prepared protein mixtures should be used.
[0016] Preferably, compound I is (S)-4-((5-chloropyridin-2-yl)oxy)-N-(3-(2-(dibenzo[b,d]furan-1-yl)phenyl)-1-(methylamino)-1-oxopropyl-2-yl)benzamide; and compound II is (S)-N-(3-(2-(dibenzo[b,d]furan-1-yl)phenyl)-1-(methylamino)-1-oxopropyl-2-yl)-4-methylnicotinamide.
[0017] Application of a small molecule compound inhibitor that antagonizes GREM1 in the treatment of colorectal cancer, which can inhibit the migration and invasion of colorectal cancer.
[0018] Compared with the prior art, the beneficial effects of the present invention are:
[0019] This application describes a small molecule inhibitor screened using DNA-encoded libraries (DEL). This small molecule inhibitor has the advantages of small size, strong permeability, adjustable pharmacokinetics, and low cost. This small molecule compound inhibitor can inhibit the migration and invasion of colorectal cancer. Attached Figure Description
[0020] Figure 1 This is a flowchart of the preparation steps for the small molecule compound inhibitor;
[0021] Figure 2 Structural diagrams of compounds I and II;
[0022] Figure 3This is a schematic diagram showing the binding affinity of compounds I and II with GREM1;
[0023] Figure 4 This is a comparison chart showing the effect of small molecule inhibitors on CRC cell migration detected by scratch assay in Example 2;
[0024] Figure 5 This is a schematic diagram illustrating the inhibition of colorectal cancer cell migration by small molecule compound inhibitors in Example 2.
[0025] Figure 6 This is a comparison chart of the effects of small molecule inhibitors on CRC cell migration detected by the Transwell assay in Example 3;
[0026] Figure 7 This is a schematic diagram illustrating the inhibition of colorectal cancer cell invasion by the small molecule compound inhibitor in Example 3;
[0027] Figure 8 This is a synthetic route diagram for compounds I and II;
[0028] Figure 9 This is a data graph for Example 1;
[0029] Figure 10 This is a data graph for Example 2;
[0030] Figure 11 This is a data graph for Example 3. Detailed Implementation
[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0032] Please see Figure 1-10 The present invention provides a technical solution:
[0033] A small molecule inhibitor that antagonizes GREM1, the specific preparation process of which is as follows:
[0034] Step 1: First, dry the DEL (DNA-encoded chemical library) library under vacuum, then dissolve it in a protein solution containing 5-10 µM human Grem1 protein, and incubate it in the DEL selection buffer system for 30 minutes.
[0035] Step 2: After incubation, fix the Grem1 protein with 20 µl of HisPur™ Ni-NTA Magnetic Beads and continue incubation for 15 minutes. Then wash with 10 × 100 µl of screening buffer under shaking. Finally, remove unbound library molecules by vacuum aspiration.
[0036] Step 3: Before incubation, reserve 1 µl of DEL input sample and determine the input amount by quantitative PCR (Q-PCR). The bound DEL molecules are eluted and collected by centrifugation. Reserve 1 µl of purified DNA material and quantify it again by Q-PCR. All other samples continue to undergo a new round of affinity screening until the required amount of DEL template residue is reached. Finally, perform next-generation DNA sequencing.
[0037] Step 4: The DEL screening data is decoded and analyzed using internally developed software to process the sequence information obtained from the screening. By applying a customized filter, fragment combinations with counts below the noise floor are removed, and the true binding signals are identified. Through combined experiments and functional verification, the following small molecule compounds I and II are finally screened out.
[0038] The Grem1 protein has a His tag at its C-terminus.
[0039] The DEL screening buffer system consists of 50 mM Tris-HCl pH 8.5, 50 mM NaCl, 1.5 mM MgCl2, 1 mM MTCEP, 0.005% Tween 20, and 1 mg / ml herring sperm vector DNA (HS-DNA).
[0040] When all the remaining samples undergo a new round of affinity screening, freshly prepared protein mixtures must be used.
[0041] Compound I is (S)-4-((5-chloropyridin-2-yl)oxy)-N-(3-(2-(dibenzo[b,d]furan-1-yl)phenyl)-1-(methylamino)-1-oxopropyl-2-yl)benzamide; Compound II is (S)-N-(3-(2-(dibenzo[b,d]furan-1-yl)phenyl)-1-(methylamino)-1-oxopropyl-2-yl)-4-methylnicotinamide.
[0042] Application of a small molecule compound inhibitor that antagonizes GREM1 in the treatment of colorectal cancer, which can inhibit the migration and invasion of colorectal cancer.
[0043] The benefits mentioned, such as small size, strong permeability, adjustable pharmacokinetics, and low cost, are explained as follows:
[0044] (1) DEL is a chemical library composed of a large number of small molecule compounds, each of which is tagged with a unique DNA sequence. This library is very small in size and can usually be stored in a standard 96-well or 384-well plate, making it easy to store and handle. The screening process is carried out at the microliter (µl) or even nanoliter (nl) level. For example, the HisPur™ Ni-NTA Magnetic Beads used in step two to fix the protein usually only requires 20µl of bead suspension. The volume of the entire screening system is much smaller than that of traditional high-throughput screening (HTS) based on purified proteins and small molecule compounds. From incubation in step one, washing in step two to elution in step three, all operations can be completed in a miniaturized system, which not only saves reagents but also greatly improves the efficiency and throughput of screening.
[0045] (2) The goal of DEL screening is to find small molecules that can bind to target proteins (such as GREM1) with high affinity. These bound compounds must have good physicochemical properties, including suitable lipid-water partition coefficient (LogP), molecular weight, and solubility. In DEL screening, in order to be successfully screened from a large library and bind to the protein, the compound usually needs to have certain drug-like properties. This includes: usually between 200-500 Daltons. If the molecular weight is too large, the compound will have difficulty crossing the cell membrane and entering the cell to exert its effect (i.e., poor permeability). The molecular weight of the compounds screened by DEL generally falls within this range, ensuring that they have basic cell penetration ability. The LogP value is usually between 1-5. Compounds in this range can dissolve in lipid cell membranes and water, thus enabling them to be effectively transported and distributed in organisms (such as blood and intracellular fluid). During the DEL screening process, various filtration steps (such as removing fragment combinations with counts below the background noise in step four) will eliminate some compounds that, although bound, have extremely poor physical properties, thereby increasing the probability that the final compound has good permeability.
[0046] (3) Pharmacokinetic (PK) properties determine the absorption, distribution, metabolism, and excretion (ADME) processes of drugs in the body. DEL technology provides the possibility of optimizing these properties. Once one or more hit compounds that can bind to the target protein are obtained through DEL screening, DEL technology can be used to rapidly modify and optimize the structure of these compounds. Step four mentions "until the desired DEL template residue is reached," meaning the screening process can be iterative. Each round of screening can be based on the results of the previous round, further optimizing the compound library to improve its binding affinity, selectivity, and ADME properties. The screened compounds can be chemically modified (e.g., by introducing specific functional groups or altering ring structures) to adjust PK parameters such as lipid-water partition coefficient, solubility, and metabolic stability, thereby obtaining candidate drug molecules with superior pharmacokinetic properties. The modular and miniaturized nature of DEL technology allows for compound optimization on a very small scale, significantly reducing optimization costs and time. DEL technology not only rapidly identifies potential drug molecules but also serves as a powerful platform for high-throughput, low-cost optimization and adjustment of the pharmacokinetic properties of these molecules to meet various requirements in preclinical and clinical stages.
[0047] (4) Although the synthesis of DEL libraries requires specialized techniques, once successful, the subsequent amplification and preservation costs are relatively low. In contrast, synthesizing a large number of structurally diverse small molecule compound libraries for traditional HTS screening is extremely expensive. The buffers used in the screening process (such as 50 mM Tris-HCl and 50 mM NaCl in step three) and consumables (such as magnetic beads and PCR tubes) are all conventional laboratory reagents, making the cost relatively controllable. DEL screening can process hundreds or thousands of compounds simultaneously, which is far more efficient than traditional methods that test each compound individually. Due to the miniaturization of the screening system, the operation steps are relatively simple and easy to automate, greatly saving manpower and time. The entire process, from screening to obtaining preliminary candidate compounds, can be completed within a few weeks. Since compounds screened by DEL usually have good drug-like properties, their subsequent optimization and development failure rate is relatively low, which also reduces the cost of drug development in the long run.
[0048] Example 1:
[0049] Verification of the binding affinity of GREM1 to the inhibitor of this small molecule compound:
[0050] Affinity verification was performed using microthermophoresis (MST) on a Monolith NT.115 instrument (NanoTemper Technologies GmbH) to assess the binding affinity between small molecule compounds and the GREM1 protein. 50 nM red fluorescently labeled GREM1-His protein (R&D Systems, 5190-GR) was mixed with solutions of small molecule compounds at different concentrations (incrementally diluted in a 1:2 gradient). The mixtures were incubated in PBS buffer at pH 7.4 for 10 minutes before being measured in NanoTemper premium capillaries. All experiments were performed at room temperature. Fluorescence signals were acquired using NanoTemper MO.Control software, and the data were used for curve fitting and dissociation constant (KD value) calculation using Prism software.
[0051] MST experimental results showed that small molecule inhibitors I and II could bind to GREM1 with affinities of 478.7 nM and 582.1 nM, respectively.
[0052] Example 2:
[0053] This small molecule compound inhibitor can inhibit the migration of colorectal cancer:
[0054] The effect of small molecule inhibitors on CRC cell migration was detected by scratch assay. Procedure:
[0055] ① Plating: Graft 2.0–2.5 × 10^5 cells / well into 6-well plates and incubate overnight in 10% serum medium until 95-100% confluence.
[0056] ② Scratching: A straight scratch was made with a sterile 20μL pipette tip, and the cells were washed twice with PBS to remove cell debris;
[0057] ③ Treatment: Add serum-free culture medium and treat with carrier (DMSO) and different concentrations of small molecule compound inhibitors I and II respectively;
[0058] ④ Photo capture time: 0, 48 h (same field of view positioning marker);
[0059] ⑤ Quantification: ImageJ calculates the change in wound area or width; index = (A0−At) / A0×100%, and each group is tested three times independently.
[0060] ⑥ Judgment: A wound healing rate decrease of ≥30% compared to the DMSO group is considered effective.
[0061] Cell migration assays showed that small molecule inhibitors significantly inhibited the migration of colorectal cancer cells.
[0062] Example 3:
[0063] This small molecule compound inhibitor can inhibit the invasion of colorectal cancer:
[0064] The Transwell assay was used to detect the effect of small molecule inhibitors on CRC cell migration. The procedure was as follows:
[0065] ① Prepare the chamber: 8μm pore size.
[0066] ② Plating: Add 200 μL of serum-free suspension to the upper chamber (plant GREM1-OE HCT116 and SW480 cell lines into 6-well plates at 1.0 × 10^5 cells / well), and treat with a carrier (DMSO) and different concentrations of small molecule compound inhibitors, respectively; add 800 μL of culture medium containing 10% FBS to the lower chamber as chemokines.
[0067] ③Incubation: 37 °C, 24 h (adjust according to cell growth rate).
[0068] ④ Staining: Wash with PBS → Fix with 4% PFA for 15 min → Stain with 0.5% crystal violet for 20 min → Remove unmigrated cells (wipe the membrane surface with a cotton swab).
[0069] ⑤ Counting: Randomly select 5 fields of view per well and count the number of cells that have penetrated the membrane. Perform three independent replicates for each group.
[0070] ⑥ Judgment: Compared with the DMSO group, a decrease of ≥30% in the number of cells that have permeated the membrane is considered effective.
[0071] Cell invasion assays showed that small molecule inhibitors significantly inhibited the invasion of colorectal cancer cells.
[0072] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.
[0073] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A small molecule compound inhibitor that antagonizes GREM1, characterized in that, The specific preparation process of this small molecule compound inhibitor is as follows: Step 1: First, dry the DEL (DNA-encoded chemical library) library under vacuum, then dissolve it in a protein solution containing 5-10 µM human Grem1 protein, and incubate it in the DEL selection buffer system for 30 minutes. Step 2: After incubation, fix the Grem1 protein with 20 µl of HisPur™ Ni-NTA Magnetic Beads and continue incubation for 15 minutes. Then wash with 10 × 100 µl of screening buffer under shaking. Finally, remove unbound library molecules by vacuum aspiration. Step 3: Before incubation, reserve 1 µl of DEL input sample and determine the input amount by quantitative PCR (Q-PCR). The bound DEL molecules are eluted and collected by centrifugation. Reserve 1 µl of purified DNA material and quantify it again by Q-PCR. All other samples continue to undergo a new round of affinity screening until the required amount of DEL template residue is reached. Finally, perform next-generation DNA sequencing. Step 4: The DEL screening data is decoded and analyzed using internally developed software to process the sequence information obtained from the screening. By applying a customized filter, fragment combinations with counts below the noise floor are removed, and the true binding signals are identified. Through combined experiments and functional verification, the following small molecule compounds I and II are finally screened out.
2. The small molecule compound inhibitor for antagonizing GREM1 according to claim 1, characterized in that: The Grem1 protein has a His tag at its C-terminus.
3. The small molecule compound inhibitor for antagonizing GREM1 according to claim 1, characterized in that: The DEL screening buffer system consists of 50 mM Tris-HCl pH 8.5, 50 mM NaCl, 1.5 mM MgCl2, 1 mM TCEP, 0.005% Tween 20, and 1 mg / ml herring sperm vector DNA (HS-DNA).
4. The small molecule compound inhibitor for antagonizing GREM1 according to claim 1, characterized in that: When all the remaining samples undergo a new round of affinity screening, freshly prepared protein mixtures must be used.
5. The small molecule compound inhibitor antagonizing GREM1 according to claim 1, characterized in that: Compound I is (S)-4-((5-chloropyridin-2-yl)oxy)-N-(3-(2-(dibenzo[b,d]furan-1-yl)phenyl)-1-(methylamino)-1-oxopropyl-2-yl)benzamide; Compound II is (S)-N-(3-(2-(dibenzo[b,d]furan-1-yl)phenyl)-1-(methylamino)-1-oxopropyl-2-yl)-4-methylnicotinamide.
6. The application of a small molecule compound inhibitor antagonizing GREM1 according to any one of claims 1-5 in the treatment of colorectal cancer, characterized in that: This small molecule compound inhibitor can suppress the migration and invasion of colorectal cancer.