Application of ridecevir in preparation of medicine for treating NUDT1 high-expression cancer

Abstract

The invention relates to the technical field of medicines, and in particular discloses a novel application of RDV (Registevir, RDV) in preparation of a medicine for treating NUDT1 (NUDT1, also known as MTH1) high-expression cancer, in particular to a novel application of RDV in preparation of a medicine for treating NUDT1 (NUDT1, also known as MTH1) high-expression cancer. According to the application disclosed by the invention, various means such as an ATP competitive binding experiment, a molecular docking experiment, a thermal shift assay (TSA) experiment, a Western blot experiment, a long-chain PCR (Polymerase Chain Reaction) experiment and a cell proliferation experiment are used for verifying that the redefovir can be combined with an ATP binding site of NUDT1 protein, the protein stability is reduced, and the degradation of the NUDT1 protein is promoted, so that the level of 8-oxoguanine (8-oxoG) in DNA (Deoxyribonucleic Acid) under oxidative stress is increased, and the proliferation of cancer cells is obviously inhibited. According to the application, the potential of the ridecevir as the NUDT1 inhibitor is disclosed for the first time, high expression of the NUDT1 is used as an accompanying diagnosis index of treatment by using the ridecevir, and a brand-new scheme is provided for realizing accurate anti-cancer treatment. The application is essentially different from known antiviral effects and mechanisms of the ridecevir.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a new pharmaceutical use of the known antiviral drug remdesivir, particularly its use in the preparation of a drug for treating cancers with high NUDT1 expression. Background Technology

[0002] NUDT1 (Nudix Hydrolase 1, also known as MTH1) is a highly conserved nucleotide pool cleaner. Its core function is to catalyze the hydrolysis of oxidatively modified purine nucleoside triphosphates within cells, with 8-oxo-guanine deoxynucleoside triphosphate (8-oxo-dGTP) being the most critical substrate. During normal cellular physiological processes, endogenous reactive oxygen species (ROS) continuously attack the nucleotide pool, producing abnormal nucleotides such as 8-oxo-dGTP. If these damaged nucleotides are incorporated into the genome by DNA polymerases during DNA replication, it will lead to G:C to T:A transversion mutations, driving genomic instability. NUDT1 effectively prevents this process by hydrolyzing it into its corresponding monophosphate form (8-oxo-dGMP), thus being considered a guardian of the genome and playing a crucial role in maintaining the fidelity of genetic information.

[0003] However, in various human malignant tumors (such as colorectal cancer and lung cancer), significantly elevated intracellular ROS levels lead to the massive production of oxidized nucleotides. To cope with this persistent oxidative stress, cancer cells abnormally increase NUDT1 expression through multiple mechanisms. This high expression state makes cancer cells "addicted" to NUDT1—that is, they rely on its efficient cleansing function to remove large amounts of 8-oxo-dGTP, thereby avoiding the accumulation of oxidatively damaged DNA and maintaining their rapid proliferation and survival advantage. Numerous clinicopathological studies have confirmed that high expression of NUDT1 in various cancer tissues is closely related to tumor malignant progression, resistance to traditional chemotherapy, and poor patient prognosis. It is this cancer cell-specific dependence that makes NUDT1 a highly attractive and ideal anti-cancer target. Selective inhibition of NUDT1 holds promise for specifically causing cancer cells to die due to the explosive accumulation of oxidatively damaged DNA, even when normal cells can tolerate it, thus achieving targeted therapy.

[0004] Based on the aforementioned mechanism, numerous research institutions and pharmaceutical companies worldwide are dedicated to developing small molecule inhibitors of NUDT1. Several inhibitors, such as TH287 and karonudib, have already entered preclinical or early clinical research stages. However, developing a completely new chemical entity typically faces significant challenges, including long development cycles, high costs, potential off-target effects, and unpredictable toxic side effects. Currently, no highly effective and selective NUDT1 inhibitors have been successfully marketed, and standard therapies targeting this target remain lacking in clinical treatment. Therefore, exploring whether existing drugs possess new functions that inhibit NUDT1—a concept known as "drug repurposing"—has become a highly valuable alternative strategy, significantly shortening development time and reducing development risks.

[0005] Remdesivir (GS-5734) is an adenosine analog that is metabolized intracellularly to its pharmacologically active nucleoside triphosphate analog (RDV-TP). Remdesivir has been approved in the United States and several other countries for the treatment of COVID-19. Its recognized primary mechanism of action is that RDV-TP competitively inhibits viral RNA-dependent RNA polymerase (RdRp), blocking viral RNA synthesis by acting as a chain terminator. Furthermore, several published patent documents (e.g., CN113827603B, CN113827604B) disclose that remdesivir has shown growth-inhibiting effects on certain mouse tumors (specifically mouse glioma, pancreatic cancer, liver cancer, and lymphoma) in animal experiments. However, these existing technologies lack an explanation of the anticancer mechanism of remdesivir. Crucially, the existing technologies have completely failed to reveal any direct or functional link between remdesivir and the specific anticancer target NUDT1. No literature or patents have demonstrated that remdesivir can directly bind to and reduce the stability of NUDT1, nor has any proposed "high NUDT1 expression" as a biomarker for predicting the anticancer efficacy of remdesivir. This mechanistic uncertainty severely limits the precise application and further development of remdesivir in the field of cancer treatment.

[0006] In summary, the core technical problems in this field are: first, the lack of safe and effective NUDT1-targeted therapy strategies; and second, a significant gap in the understanding of the potential anti-cancer mechanism of remdesivir, making it impossible to identify the suitable patient population. This invention aims to solve these problems by revealing for the first time that remdesivir is an effective inhibitor of NUDT1 and creatively proposing its use in treating "NUDT1-highly expressed cancers," providing a novel solution for precision cancer treatment based on a clearly defined molecular mechanism. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and provide a novel, well-defined medical use for remdesivir, thereby offering an efficient precision medicine strategy for treating NUDT1-overexpressing cancers.

[0008] The primary objective of this invention is to provide the use of remdesivir or its pharmaceutically acceptable salts, esters, solvates or prodrugs in the preparation of medicaments for the treatment of NUDT1-overexpressing cancers.

[0009] Another objective of this invention is to provide a precision treatment method for cancers with high NUDT1 expression, which includes first detecting the expression level of NUDT1 in the patient's tumor tissue, and then administering remdesivir to those with high expression.

[0010] Another object of the present invention is to provide a pharmaceutical composition for treating cancers with high NUDT1 expression, comprising remdesivir and a reagent for detecting NUDT1 expression levels.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] (1) Novel applications based on new targets

[0013] This invention reveals and confirms for the first time that NUDT1 is a key new target for remdesivir in cancer cells. The remdesivir and its active triphosphate form (RDV-TP) produced during intracellular metabolism can bind to the NUDT1 protein, thereby reducing its stability and promoting its degradation.

[0014] (2) A novel mechanism of action

[0015] Based on the above target discovery, this invention establishes a novel mechanism of action for the anticancer activity of remdesivir: by inhibiting NUDT1, remdesivir disrupts the cancer cells' ability to clear oxidized nucleotides, leading to the large-scale incorporation of 8-oxo-dGTP into the newly synthesized DNA strand, resulting in a significant accumulation of 8-oxoguanine (8-oxoG) damage, which in turn triggers fatal gene mutations, induces cancer cell apoptosis or inhibits their proliferation.

[0016] (3) Precise treatment plan

[0017] The core innovation of this invention lies in establishing "high NUDT1 expression" as a biomarker and prerequisite for remdesivir treatment. "High NUDT1 expression" refers to a significantly higher level of NUDT1 protein or mRNA in cancer tissue than that in corresponding normal tissue or a predetermined threshold level, as detected by methods such as immunohistochemistry, Western blotting, or quantitative PCR. This approach achieves a paradigm shift from "broad-spectrum anti-cancer" to "precision targeting."

[0018] (4) Specific implementation plan

[0019] In a preferred embodiment, the cancers with high NUDT1 expression include, but are not limited to, colorectal cancer, cervical cancer, lung cancer, kidney cancer, breast cancer, and melanoma. The drug can be used in combination with one or more other anticancer therapies (such as chemotherapy, radiotherapy, and immunotherapy) to enhance efficacy.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) Repurposing an existing drug with significant development advantages: This study reveals for the first time a novel target and anticancer mechanism of remdesivir that inhibits NUDT1, opening up a completely new therapeutic field. Utilizing an already marketed drug for development ensures a clear safety profile and can significantly shorten the research and development cycle, reduce costs and risks.

[0022] (2) Clear mechanism and strong targeting: This invention overcomes the shortcomings of the existing technology in understanding the anti-cancer mechanism of remdesivir, and provides a complete chain of evidence from molecular target to cellular effect. The mechanism of action is clear and well-defined, laying a solid scientific foundation for precise clinical application.

[0023] (3) Breaking through the bottleneck of precision medicine: The companion diagnostic strategy of “NUDT1 high expression” as a biomarker was creatively proposed, which can accurately screen out the advantageous patient group that is sensitive to remdesivir treatment, significantly improve the treatment response rate, avoid ineffective treatment, and realize true individualized precision treatment.

[0024] (4) Strong intellectual property barriers: The core of the protection of this invention lies in the specific combination of "new target + new biomarker", which has not been disclosed by any existing technology, thus building an intellectual property barrier that is difficult to bypass and has huge commercial value. Attached Figure Description

[0025] Figure 1 Western blot results showed that remdesivir (RDV) competitively binds to the ATP binding site of NUDT1. Quantitative data (right figure) are the mean ± standard error (SEM) of four independent experiments. "***" indicates p < 0.001. The p-value was calculated using an unpaired two-tailed Student's t-test.

[0026] Figure 2 The molecular docking diagram shows the binding patterns of ATP, remdesivir, remdesivir triphosphate (RDV-TP), and NUDT1 protein. The binding free energies of NUDT1 with ATP, RDV, and RDV-TP are -8.6, -9.1, and -8.7 kcal / mol, respectively.

[0027] Figure 3The thermal shift assay (TSA) results showed that remdesivir significantly reduced the thermal stability of NUDT1 protein. Quantitative data (below) are the mean ± standard error (SEM) of four independent experiments. "*" indicates p < 0.05, and "**" indicates p < 0.01. The p-values ​​were calculated using an unpaired two-tailed Student's t-test.

[0028] Figure 4 Western blot results showed a significant decrease in intracellular NUDT1 protein levels after remdesivir treatment. Quantitative data (right figure) are the mean ± standard error (SEM) of three independent experiments. "**" indicates p < 0.01, and p-values ​​were calculated using an unpaired two-tailed Student's t-test.

[0029] Figure 5 Long-chain PCR results showed that the level of 8-oxoguanine in DNA was significantly increased after remdesivir treatment.

[0030] Figure 6 The CCK-8 assay results showed that remdesivir could inhibit the proliferation of human colon cancer cells HCT116 and human cervical cancer cells HeLa. Quantitative data are the mean ± standard error (SEM) of three independent experiments. "**" indicates p < 0.01, and "***" indicates p < 0.001. The p-value was calculated using an unpaired two-tailed Student's t-test. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0032] Example 1: Remdesivir competitively binds to the ATP binding site of NUDT1 protein

[0033] To verify the direct interaction between remdesivir and the NUDT1 protein, we conducted an in vitro competitive binding experiment.

[0034] (1) Protein expression and purification: The human NUDT1 gene was cloned into the pGEX-4T-1 prokaryotic expression vector with GST tag, transformed into Escherichia coli Rosetta (DE3) strain, and after IPTG induction, the high-purity recombinant GST-NUDT1 fusion protein was obtained by purification by GST affinity chromatography column.

[0035] (2) Competitive binding assay: Equal amounts of purified NUDT1 protein were incubated with 1 mM remdesivir (dissolved in DMSO) or an equal amount of DMSO (as a control) in binding buffer (50 mM HEPES, pH 7.4, 75 mM NaCl, 5% glycerol) at room temperature for 1.5 hours. Subsequently, MgCl2, MnCl2, and CaCl2 were added to final concentrations of 50 mM, 5 mM, and 5 mM, respectively. Then, an equal amount of biotin-labeled ATP affinity probe was added, and incubation was continued at room temperature for another 1.5 hours.

[0036] (3) Pull-down and detection: The above mixture was incubated with streptavidin agarose beads for 2 hours. The beads were collected by centrifugation and washed thoroughly with PBS and ultrapure water to remove unbound proteins. Finally, the proteins bound to the beads were eluted and analyzed by Western blot, and detected using a NUDT1-specific antibody.

[0037] Result: As Figure 1 As shown, compared with the DMSO control group, the binding signal of NUDT1 protein to the ATP probe was significantly weakened in the remdesivir treatment group. This result indicates that remdesivir can competitively bind to the ATP binding site of NUDT1, thereby interfering with the normal binding of NUDT1 to ATP.

[0038] Example 2: Molecular docking simulations reveal high-affinity binding of remdesivir to NUDT1

[0039] To elucidate the binding modes and affinities at the molecular level, we performed molecular docking analysis.

[0040] (1) Structural preparation: The crystal structure of NUDT1 protein (PDBID: 5FSK) was downloaded from the RCSB PDB database. The molecular structures of ATP (CID: 5957), remdesivir (RDV, CID: 121304016) and its active triphosphate form (RDV-TP, CID: 56832906) were obtained from the PubChem database.

[0041] (2) Docking process: Using AutoDock Vina 1.1.2 software, the ATP binding pocket of NUDT1 protein is defined as the docking region, and ATP, RDV and RDV-TP are docked into the pocket respectively.

[0042] Result: As Figure 2As shown, the docking results indicate that both remdesivir and its triphosphate form (RDV-TP) can stably bind within the ATP-binding pocket of NUDT1, forming hydrogen bonds and hydrophobic interactions with key amino acid residues. Binding energy calculations show that the binding energy of NUDT1 to ATP is -8.6 kcal / mol, while the binding energies to remdesivir (RDV) and RDV-TP are -9.1 kcal / mol and -8.7 kcal / mol, respectively. This suggests that remdesivir has a stronger binding affinity to NUDT1 than its natural substrate ATP, theoretically predicting its potential as a highly effective inhibitor.

[0043] Example 3: Protein thermal migration experiment confirms that remdesivir binding reduces NUDT1 protein stability. To verify the effect of binding on protein conformational stability, we conducted a protein thermal migration experiment.

[0044] (1) Experimental procedure: The purified NUDT1 protein was incubated with 1 mM remdesivir or DMSO control at room temperature for 2 hours. The mixture was then divided into equal parts and heat-shocked at 50°C, 55°C, 60°C and 65°C for 3 minutes, respectively, followed by centrifugation at 17,000g for 20 minutes.

[0045] (2) Detection: The supernatant was subjected to Western blot analysis to detect the amount of NUDT1 protein that did not precipitate (i.e., soluble).

[0046] Result: As Figure 3 As shown, with increasing temperature, the NUDT1 protein in the DMSO control group gradually denatured and precipitated, while the protein content in the supernatant decreased. However, in the remdesivir-treated group, at the same temperature, the amount of soluble NUDT1 protein remaining in the supernatant was significantly less than that in the control group. This suggests that the binding of remdesivir may alter the conformation of NUDT1, reducing its thermal stability and making it more prone to denaturation and aggregation at lower temperatures.

[0047] Example 4: Remdesivir treatment reduced intracellular NUDT1 protein levels.

[0048] To investigate the intracellular effects of remdesivir, we examined its influence on endogenous NUDT1 expression.

[0049] (1) Cell treatment: HEK293T cells were treated with 5 μM remdesivir for 24 hours, while a DMSO solvent control group was set up.

[0050] (2) Protein level detection: Total cell protein was extracted and analyzed by Western blot. NUDT1 antibody and Tubulin antibody (as internal control) were used for detection.

[0051] Result: As Figure 4As shown, compared with the control group, the level of endogenous NUDT1 protein in cells was significantly decreased in the remdesivir treatment group, while its mRNA level did not change significantly (data not shown). This result is consistent with the findings of Example 3, indicating that remdesivir promotes the degradation of NUDT1 protein by inducing its instability through binding, thereby leading to a decrease in intracellular protein levels.

[0052] Example 5: Long-chain PCR confirms that remdesivir treatment leads to the accumulation of DNA oxidative damage.

[0053] To verify the biological effects of remdesivir on NUDT1 function, we examined the accumulation of intracellular 8-oxoG.

[0054] (1) Cell treatment and DNA extraction: HEK293T cells were treated with 5 μM remdesivir or DMSO for 48 hours. To simulate oxidative stress, some cells were also exposed to a low concentration of H2O2 at the end of the treatment. Total DNA was then extracted from the cells using a genomic DNA extraction kit.

[0055] (2) DNA damage treatment and PCR amplification: The extracted DNA was treated with Fpg glycosidase (which specifically recognizes and cleaves the 8-oxoG site). Subsequently, a mitochondrial DNA fragment of 8.9 kb was amplified using long-chain PCR technology, and a 220 bp fragment was simultaneously amplified as an internal control. The higher the DNA integrity, the higher the amplification efficiency of the long fragment; if there is more 8-oxoG damage, the DNA strand breaks after Fpg cleavage will increase, and the amplification amount of the long fragment will decrease.

[0056] Result: As Figure 5 As shown, under normal conditions without oxidative stress, remdesivir treatment had little effect on PCR amplification efficiency. However, under H2O2-induced oxidative stress, the 8.9 kb PCR product band intensity in the remdesivir-treated group was significantly weaker than that in the control group. This indicates that when oxidative damage is present, remdesivir inhibits NUDT1 function, leading to the ineffective removal of 8-oxo-dGTP, which is then incorporated into DNA in large quantities, resulting in a significant accumulation of 8-oxoG damage.

[0057] Example 6: Remdesivir inhibits the proliferation of HCT116 human colorectal cancer cells and HeLa human cervical cancer cells.

[0058] To verify the anticancer effect of remdesivir, we conducted cell proliferation experiments on cancer cell lines with high NUDT1 expression.

[0059] (1) Cell culture and treatment: Human colon cancer cells HCT116 and human cervical cancer cells HeLa, which are known to highly express NUDT1, were selected. The cells were seeded into 96-well plates and, after adhesion, were treated with different concentrations (e.g., 0, 1, 5, 10 μM) of remdesivir for 24 and 48 hours, respectively.

[0060] (2) CCK-8 assay for cell viability: At each time point, CCK-8 reagent was added to each well, and after incubation for 1-2 hours, the absorbance at 450 nm was measured using a microplate reader. Cell viability is directly proportional to absorbance.

[0061] Result: As Figure 6 As shown, remdesivir significantly inhibited the proliferation of HCT116 and HeLa cells, and the inhibitory effect exhibited clear concentration- and time-dependent characteristics. Cell viability gradually decreased with increasing drug concentration and treatment time. This directly demonstrates that remdesivir has significant growth-inhibiting activity against NUDT1-overexpressing cancer cells.

[0062] The above series of examples comprehensively and powerfully demonstrate, from multiple levels including in vitro binding, computer simulation, protein stability, cellular protein expression, functional effects (DNA damage), and phenotype (inhibition of cancer cell proliferation), that remdesivir can act as an effective inhibitor of NUDT1 and kill cancer cells through this mechanism. Therefore, remdesivir can be used to prepare drugs for treating cancers with high NUDT1 expression.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of treating cancer in a subject, comprising, comprising the steps of: a) detecting the expression level of NUDT1 protein or mRNA in a sample obtained from the subject; b) comparing the expression level with a reference level, wherein the expression level is higher than the reference level indicates that the cancer is a NUDT1 high expression cancer; c) administering a therapeutically effective amount of remdesivir or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof to the subject determined to have a NUDT1 high expression cancer.

2. The method of claim 1, wherein, The detection in step a) is performed by one or more of immunohistochemistry, western blot, enzyme-linked immunoassay or quantitative PCR.

3. Use of remdesivir or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof in the manufacture of a medicament for treating a patient determined to have a NUDT1 high expression cancer via the method of claim 1.

4. The method according to claim 1 or the use according to claim 3, characterized in that, Remdesivir reduces the stability of NUDT1 protein by competitively binding to the ATP binding site of NUDT1 protein, and promotes its degradation.

5. The method or use according to claim 4, characterized in that, The degradation of NUDT1 protein and its inhibition of enzymatic activity leads to the accumulation of 8-oxoguanine in cancer cells and its incorporation into genomic DNA, resulting in specific accumulation of 8-oxoguanine damage.

6. The method or use of any one of claims 1-3, wherein, The NUDT1 high expression cancer is selected from one or more of colorectal cancer, cervical cancer, lung cancer, kidney cancer, breast cancer, melanoma and metastases thereof.

7. A pharmaceutical composition, characterized by, comprising: a) a therapeutically effective amount of remdesivir or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof; b) a kit for performing steps a) to c) of claim 1 to detect and determine a NUDT1 high expression cancer; c) a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is used together with the kit to treat a NUDT1 high expression cancer.

8. A pharmaceutical composition, characterized by, a therapeutically effective amount of remdesivir and one or more other anticancer agents selected from DNA damage repair inhibitors, chemotherapeutic drugs or immune checkpoint inhibitors, for treating a NUDT1 high expression cancer.

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