Application of NSP14 small-molecule inhibitor in preparation of medicine for preventing and treating novel coronavirus infection

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of an NSP14 small-molecule inhibitor in preparation of a medicine for preventing and treating novel coronavirus infection. The invention provides application of an NSP14 small-molecule inhibitor in preparation of a medicine for preventing and treating novel coronavirus infection. The NSP14 small-molecule inhibitor comprises at least one of DEL1 as shown in a structural formula I, DEL2 as shown in a structural formula II and DEL3 as shown in a structural formula III. The NSP14 small-molecule inhibitor can effectively inhibit the exonuclease activity of novel coronavirus NSP14 protein, blocks a virus RNA processing mechanism on the molecular level, shows a remarkable antiviral effect, and is beneficial to being widely applied to preparation of drugs for preventing and treating novel coronavirus infection.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and in particular relates to the application of an NSP14 small molecule inhibitor in the preparation of drugs for the prevention and treatment of novel coronavirus infection. Background Technology

[0002] COVID-19 is an acute infectious disease caused by a novel coronavirus. The virus mainly spreads between people through respiratory droplets and contact. In the early stages of infection, the clinical manifestations of COVID-19 include fever, dry cough, and fatigue. As the disease progresses, it can induce severe symptoms such as respiratory failure and circulatory shock, and in the terminal stage, it can lead to multiple organ failure and death.

[0003] Currently, FDA-approved drugs for treating the novel coronavirus include: Paxlovid, Baricitinib, Tocilizumab, Remdesivir, and Molnupiravir. Nirmatrelvir-ritonavir (Paxlovid) is a 3CL protein inhibitor that inhibits the replication of the novel coronavirus in cells by binding to the virus's 3CL protein and preventing it from cleaving the polyprotein. Baricitinib, an antirheumatic drug, is a reversible selective tyrosine protein kinase 1 and selective tyrosine protein kinase 2 inhibitor, which can be used in combination with glucocorticoids and IL-6 receptor antagonists to treat severely ill COVID-19 patients. Tocilizumab is an IL-6 receptor inhibitor used to suppress cytokine storms in severely ill COVID-19 patients. Remdesivir is a nucleoside analog antiviral drug initially developed for Ebola virus, but later demonstrated to block replication by inhibiting the activity of the SARS-CoV-2 RNA polymerase, and received FDA emergency authorization for the treatment of severe cases. Molnupiravir is an oral nucleoside analog antiviral drug under development by Merck, which blocks viral replication by incorporating into viral RNA and inducing replication error mutations.

[0004] Further research has shown that NSP14 is a key protein in the viral replication cycle: its N-terminus possesses 3'→5' exonuclease activity, which corrects RNA replication errors and maintains genome fidelity; its C-terminus is an N7-methyltransferase (N7-MTase), involved in the formation of the m7G cap structure at the 5' end of viral mRNA. This modification plays a central role by promoting viral RNA translation, resisting host degradation, and escaping innate immune recognition (such as avoiding activation of PKR, OAS / RNase L pathways). Furthermore, NSP14 forms a heterodimer with NSP10, which can enhance translational repression to inhibit host antiviral protein synthesis. However, specific inhibitors targeting NSP14 have not yet been effectively developed, and the limitations of existing drugs highlight the necessity of targeting NSP14. Summary of the Invention

[0005] The purpose of this application is to provide an application of an NSP14 small molecule inhibitor in the preparation of drugs for the prevention and treatment of novel coronavirus infection, aiming to solve the problem of insufficient drugs for the prevention and treatment of novel coronavirus infection in the prior art.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides the use of an NSP14 small molecule inhibitor in the preparation of a drug for preventing and treating novel coronavirus infection. The NSP14 small molecule inhibitor includes at least one of DEL1 as shown in structural formula I, DEL2 as shown in structural formula II, and DEL3 as shown in structural formula III. Wherein, structural formula I is Structural formula II is Structural Formula III is .

[0007] In some embodiments, the NSP14 small molecule inhibitor uses the NSP14 protein as a target site, wherein the target site includes the exonuclease domain or the N7-MTase domain of the NSP14 protein.

[0008] In some embodiments, NSP14 small molecule inhibitors block the proofreading replication of viral RNA and 5'm7G cap modification by inhibiting the exonuclease activity or N7-MTase activity of the NSP14 protein.

[0009] In some embodiments, the binding constant between the NSP14 small molecule inhibitor and the NSP14 protein is 1.65 × 10⁻⁶. -4 M to 2×10 -5 M.

[0010] In some embodiments, the use of NSP14 small molecule inhibitors in the preparation of drugs for preventing and treating the replication of the novel coronavirus.

[0011] In some embodiments, the use of NSP14 small molecule inhibitors in the preparation of drugs for the prevention and treatment of novel coronavirus activity.

[0012] In some embodiments, the use of NSP14 small molecule inhibitors in the preparation of drugs that inhibit the activity of NSP14 protein.

[0013] In some embodiments, the drug is a formulation prepared using an NSP14 small molecule inhibitor as the active ingredient.

[0014] In some embodiments, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, syrups, and aerosols.

[0015] Secondly, this application provides a pharmaceutical composition comprising the above-mentioned NSP14 small molecule inhibitor and a pharmaceutically acceptable carrier or excipient.

[0016] The NSP14 small molecule inhibitors DEL1-DEL5, shown in structural formulas I-III, provided in the first aspect of this application, can be used to prepare drugs for the prevention and treatment of novel coronavirus infection. This discovery has significant scientific and clinical value. From a mechanism of action perspective, the NSP14 protein, as a key methyltransferase and exonuclease in the replication process of the novel coronavirus, plays a crucial role in the stability of the viral genome and its ability to evade the immune system. The NSP14 small molecule inhibitors shown in DEL1-DEL3 can precisely target the active site of NSP14, effectively inhibiting viral RNA capping modification and proofreading functions through competitive binding or allosteric regulation, thereby blocking viral replication and transmission within host cells. At the drug development level, this discovery breaks through the limitations of traditional anti-COVID-19 drug development, providing novel candidate drugs for the prevention and treatment of novel coronavirus infection, greatly broadening the research direction of anti-novel coronavirus drugs, and is expected to spur the development of more novel antiviral drugs with different mechanisms of action. From a clinical application perspective, these specific small molecule inhibitors, with their high specificity and strong affinity, can precisely target the novel coronavirus and significantly reduce side effects. Compared to existing treatment options, it can not only effectively shorten the virus clearance time and alleviate clinical symptoms, but also reduce the risk of drug resistance caused by viral mutations, which is conducive to its widespread application.

[0017] The pharmaceutical composition provided in the second aspect of this application comprises an NSP14 small molecule inhibitor and a pharmaceutically acceptable carrier or excipient, which can improve the stability, bioavailability, and efficacy of the inhibitor. The pharmaceutically acceptable carrier or excipient can assist the active ingredient in exerting its effect better, reduce drug irritation to the body, and facilitate drug preparation, storage, and transportation. This pharmaceutical composition provides a more optimized drug form for the prevention and treatment of novel coronavirus infection, which is beneficial for improving therapeutic efficacy and medication safety. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a plasmid for prokaryotic expression provided in the embodiments of this application. Figure 1Taking the full-length NSP14 vector as an example, this demonstrates how inserting the gene of the full-length NSP14 protein, the N-terminal domain (exonuclease), and the C-terminal domain (N7 guanine methyltransferase) into the prokaryotic expression vector pET-32a respectively can result in three independent prokaryotic expression vectors for recombinant protein expression. Figure 2 These are the protein purification results provided in the embodiments of this application. Figure 2 Using the NSP14-N-terminal domain as an example, the chromatograms after affinity chromatography and molecular sieve purification are shown in section A. Figure 2 B is the result of its Coomassie brilliant blue staining; Figure 3 The present application provides the structural formula and 1H NMR spectrum results of DEL1. Figure 4 The results of detecting the molecular weight of small molecule DEL1 using liquid chromatography-mass spectrometry (LC-MS) provided in the embodiments of this application are as follows; Figure 5 The results provided in this application embodiment are the detection results of DEL1 compound components by high performance liquid chromatography (HPLC); Figure 6 The results of supercritical fluid chromatography (SFC) detection of DEL1 isomers provided in the embodiments of this application are as follows: Figure 7 The results show the detection of the inhibitory effect of small molecules on NSP14 protease activity provided in the embodiments of this application. Figure 8 These are the detection results of the interaction between the compound and the enzyme provided in the embodiments of this application. Figure 8 Figure A shows the results of the ITC experiment verifying the interaction between DEL1 and NSP14 proteins. Figure 8 Figure B shows the results of the SPR experiment, which further confirmed the interaction between the proteins DEL1 and NSP14. Figure 8 The text also demonstrates K measured by ITC and SPR methods. D value. Detailed Implementation

[0020] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0021] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0022] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0023] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0024] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0025] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0026] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0027] The first aspect of this application provides the application of an NSP14 small molecule inhibitor in the preparation of a drug for preventing and treating novel coronavirus infection. The NSP14 small molecule inhibitor includes at least one of DEL1 as shown in structural formula I, DEL2 as shown in structural formula II, and DEL3 as shown in structural formula III. Wherein, structural formula I is Structural formula II is Structural Formula III is .

[0028] The NSP14 small molecule inhibitors DEL1-DEL3, as shown in Structural Formulas I-III, provided in the first aspect of this application, can be used to prepare drugs for the prevention and treatment of novel coronavirus infection. This discovery has significant scientific and clinical value. From a mechanism of action perspective, the NSP14 protein, as a key methyltransferase and exonuclease in the replication process of the novel coronavirus, plays a crucial role in the stability of the viral genome and its ability to evade the immune system. The NSP14 small molecule inhibitors DEL1-DEL3 can precisely target the active site of NSP14, effectively inhibiting viral RNA capping modification and proofreading functions through competitive binding or allosteric regulation, thereby blocking viral replication and transmission within host cells. In terms of drug development, this discovery breaks through the limitations of traditional anti-COVID-19 drug development, providing novel candidate drugs for the prevention and treatment of novel coronavirus infection, greatly broadening the research direction of anti-novel coronavirus drugs, and potentially leading to the development of more novel antiviral drugs with different mechanisms of action. From a clinical application perspective, these specific small molecule inhibitors, with their high specificity and strong affinity, can precisely target the novel coronavirus and significantly reduce side effects. Compared to existing treatment options, it can not only effectively shorten the virus clearance time and alleviate clinical symptoms, but also reduce the risk of drug resistance caused by viral mutations, which is conducive to its widespread application.

[0029] In some embodiments, NSP14 small molecule inhibitors target the NSP14 protein, where the target site includes the exonuclease domain or N7-MTase domain of the NSP14 protein. Further clarifying that NSP14 small molecule inhibitors target the exonuclease domain or N7-MTase domain of the NSP14 protease makes the inhibitor's action highly specific. This specificity can reduce interference with normal human cell function, decrease drug toxicity and side effects, and improve the safety of drug treatment. Simultaneously, targeting specific domains also enhances the precision and effectiveness of drug inhibition against viruses.

[0030] In some embodiments, NSP14 small molecule inhibitors block the corrective replication of viral RNA and 5'm7G cap modification by inhibiting the exonuclease activity or N7-MTase activity of the NSP14 protein. The mechanism of action of NSP14 small molecule inhibitors is to block the corrective replication of viral RNA and 5'm7G cap modification by inhibiting the related activity of the NSP14 protease. This clear mechanism ensures that the inhibitors can act at a key stage of viral replication, effectively preventing viral proliferation and survival. By blocking the corrective replication of viral RNA, the viral mutation rate can be increased, and viral stability and pathogenicity can be reduced; while blocking 5'm7G cap modification can affect the translation of viral RNA and its ability to evade host immune recognition, thereby significantly enhancing the antiviral efficacy of the drug.

[0031] In some embodiments, the binding constant between the NSP14 small molecule inhibitor and the NSP14 protein is 1.65 × 10⁻⁶. -4 M to 2×10 -5 M. The binding constant of the NSP14 small molecule inhibitor to the NSP14 protease is 1.65 × 10⁻⁶. -4 M to 2×10 -5 Within the M range, it indicates that the inhibitor has a strong binding affinity to the target protein. This suitable binding strength ensures that the inhibitor can stably bind to the NSP14 protease, fully exerting its inhibitory effect and guaranteeing the drug's efficacy. Simultaneously, this binding constant range also indicates that the inhibitor's binding to the target protein has an appropriate affinity; it is neither too weak to be effective nor too strong to be difficult to dissociate, which is beneficial for drug metabolism and repeatability.

[0032] In some embodiments, the application of NSP14 small molecule inhibitors in the preparation of drugs for preventing and treating novel coronavirus replication. NSP14 small molecule inhibitors can be used to prepare drugs for preventing and treating novel coronavirus replication, and can directly target the replication process of the novel coronavirus. By inhibiting viral replication, viral proliferation in the body can be effectively controlled, viral load can be reduced, thereby alleviating viral damage to the body, alleviating disease progression, providing effective treatment for infection symptoms related to novel coronavirus replication, and reducing the risk of disease transmission.

[0033] In some embodiments, the NSP14 small molecule inhibitor is used in the preparation of drugs for preventing and treating the novel coronavirus. This inhibitor can be used to prepare drugs for preventing and treating the novel coronavirus, and can directly inhibit the activity of the novel coronavirus. This means that the drug can reduce the pathogenicity and infectivity of the virus as a whole, alleviate the damage of the virus to host cells, help alleviate the clinical symptoms of patients, shorten the course of the disease, improve the speed of patient recovery, and has good therapeutic and preventive effects on various diseases caused by novel coronavirus infection.

[0034] In some embodiments, the use of NSP14 small molecule inhibitors in the preparation of drugs that inhibit the activity of the NSP14 protein. NSP14 small molecule inhibitors can be used to prepare drugs that inhibit the activity of the NSP14 protease, providing an effective means of directly inhibiting NSP14 protease activity. The NSP14 protease is a key enzyme for the replication and survival of the novel coronavirus; inhibiting its activity can fundamentally block the virus's key life activities, thereby inhibiting viral proliferation and infectivity. This application provides a new approach for specifically inhibiting the activity of key viral enzymes and has significant therapeutic value.

[0035] In some embodiments, the drug is a formulation prepared with an NSP14 small molecule inhibitor as the active ingredient. The provided drug, prepared with an NSP14 small molecule inhibitor as the active ingredient, ensures that the drug has a clearly defined active ingredient and therapeutic effect. Formulations made with this inhibitor as the active ingredient ensure stable efficacy in vivo, facilitating clinical application and quality control. Simultaneously, this formulation form provides a basis for standardized drug production and regulated use, contributing to improved reliability and consistency of drug therapy.

[0036] In some embodiments, the dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, syrups, and aerosols. Different dosage forms are suitable for different patient groups and disease needs. For example, injections are suitable for situations where the drug needs to take effect urgently, while tablets and capsules are convenient for oral administration and self-administration by patients. The diversity of dosage forms improves the applicability and convenience of drugs, meets the needs of different clinical treatment scenarios, and enhances patient medication adherence.

[0037] A second aspect of this application provides a pharmaceutical composition comprising the above-described NSP14 small molecule inhibitor and a pharmaceutically acceptable carrier or excipient.

[0038] The pharmaceutical composition provided in the second aspect of this application comprises an NSP14 small molecule inhibitor and a pharmaceutically acceptable carrier or excipient, which can improve the stability, bioavailability, and efficacy of the inhibitor. The pharmaceutically acceptable carrier or excipient can assist the active ingredient in exerting its effect better, reduce the drug's irritation to the body, and facilitate the preparation, storage, and transportation of the drug. This pharmaceutical composition provides a more optimized drug form for the prevention and treatment of novel coronavirus infection, which is beneficial for improving therapeutic efficacy and medication safety.

[0039] The following description is based on specific embodiments.

[0040] Example 1 Constructing plasmids for prokaryotic expression (1) Experimental methods 1. Take 5 μg of purified pET32a plasmid solution into an EP tube, add 1 μL of restriction endonuclease, prepare the corresponding enzyme digestion buffer, and add sterile deionized water to make up the total volume of the reaction system to 50 μL. Incubate the mixture overnight at 37°C to achieve double enzyme digestion of the plasmid.

[0041] 2. PCR amplification of the target insert DNA: Using NSP14 cDNA as a PCR template, add 25 μL of PrimeSTAR to the PCR tube. ® Max DNA Polymerase (2×) premix, 1 μL of forward primer NSP14-F and 1 μL of reverse primer NSP14-R, and sterile deionized water were added to bring the total volume of the reaction system to 50 μL. PCR amplification was then performed according to the standard PCR procedure shown in Table 1 to obtain the target insert fragment.

[0042] Table 1

[0043] 3. Weigh 1g of agarose powder and add it to an Erlenmeyer flask. Add 100mL of Tris-acetic acid-EDTA buffer (TAE buffer) and heat in a microwave oven until completely dissolved (see Table 2). After cooling to approximately 50°C, add 5μL of nucleic acid dye (such as GelRed), mix well, pour into an electrophoresis mold, insert a comb, and let stand until the gel is completely solidified.

[0044] Table 2

[0045] 4. After completing the double enzyme digestion and PCR amplification steps, a portion of the products was taken for agarose gel electrophoresis analysis. The pre-prepared agarose gel was placed in the electrophoresis tank, and an appropriate amount of TAE buffer was added until the gel was completely covered. The sample to be tested, containing sample buffer, was loaded into the gel lanes. The voltage was set to 120V, and electrophoresis was performed for 30 minutes to separate DNA fragments of different molecular weights. After electrophoresis, the fragment size was determined based on the band positions, providing a basis for the subsequent recovery of the target fragment.

[0046] 5. After electrophoresis, place the agarose gel in a UV gel imaging system for observation and analysis. If the electrophoretic marker bands are clearly distinguishable, and the double-digested vector plasmid band appears at approximately 8kb, and the NSP14 insert obtained by PCR amplification shows a clear and single band at approximately 1.5kb, then the enzyme digestion and PCR amplification reactions are considered effective, and subsequent recovery and purification of the target DNA fragment can be performed. 6. Purify the target DNA band using the gel extraction kit provided by Vazyme. First, cut the corresponding target DNA band under UV light (minimizing the gel volume as much as possible), place it in a sterile EP tube, and break up the gel with a pipette. Then, add sol-gel buffer at a ratio of 100 μL Buffer GDP per 100 mg of gel, and incubate at 50-55°C for 7-10 minutes until the gel is completely dissolved. During this process, invert the tube occasionally to accelerate dissolution.

[0047] Transfer the completely dissolved solution to a FastPure DNA Mini Columns-G adsorption column and centrifuge at 12,000 rpm for 30 seconds, discarding the eluent. Add 300 μL of Buffer GDP, let stand for 1 minute, and then centrifuge again for 30 seconds for further washing. Next, add 700 μL of Buffer GW for washing, and repeat this step twice. After washing, centrifuge an empty tube for 2 minutes to remove residual liquid.

[0048] The adsorption column was then placed in a new sterile EP tube, and 20 μL of Elution Buffer preheated to 55°C was added to the center of the column membrane. After standing at room temperature for 2 minutes, the column was centrifuged for 2 minutes to collect the purified DNA sample obtained from the elution and stored at -20°C for later use.

[0049] 8. Enzyme Digestion and Ligation: Prepare T4 DNA ligase and its matching reaction buffer under ice bath conditions (see Table 3). Based on the required volume calculated according to the molar ratio of vector to insert fragment and their respective DNA concentrations in the instructions, add the purified double-digested vector, the PCR-amplified insert fragment, ligation buffer, and sterile deionized water sequentially to an EP tube. Mix well, and finally add T4 DNA ligase to construct the ligation reaction system. The ligation reaction can be incubated overnight at 16°C, or reacted briefly at room temperature before use in subsequent transformation steps.

[0050] Given that the embodiments of this application aim to express the constructed plasmid in BL21 cells, the ligation reaction product is transformed into Escherichia coli Stbl3 competent cells to achieve preliminary amplification and cloning screening of the plasmid.

[0051] Table 3

[0052] (2) Experimental results The full-length NSP14 protein gene, its N-terminal domain (exonuclease), and its C-terminal domain (N7 guanine methyltransferase) were inserted into the prokaryotic expression vector pET-32a, resulting in three independent prokaryotic expression vectors expressing the recombinant protein. Taking the full-length NSP14 vector as an example, the results are as follows: Figure 1As shown.

[0053] Example 2 Prokaryotic expression and purification of NSP14 recombinant protein (1) Experimental methods 1. Small-scale expression validation Single clones of the bacterial strain carrying the recombinant expression plasmid (NSP14-pET32a) of the NSP14 gene were selected and inoculated into 5 mL of LB medium. The cultures were incubated at 37°C and 200 rpm until the bacterial culture reached OD500. 600 The value reached 0.6-1.0. One mL of bacterial culture was used as the control group (uninduced) and cultured at 37℃ for 2 hours. Two 1 mL samples of bacterial culture were added to two sterile test tubes as experimental groups. Isopropyl-β-D-thiogalactoside (IPTG) was added to the experimental groups at final concentrations of 0.1 mM and 0.5 mM, respectively, for induction of expression over 2–3 hours. After induction, the cells were collected by centrifugation at 12000 rpm for 1 minute at 4℃. Protein loading buffer was added, and the cells were denatured by heating in a 95℃ metal bath for 5 minutes. Protein expression was analyzed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. Approximately 50 μL of the original bacterial culture was loaded into each group. Unused samples were stored at -20℃ for subsequent analysis.

[0054] 2. Large-scale expression and purification Based on the successful small-scale expression validation, we proceeded to 1L-scale protein expression and purification experiments. The specific steps are as follows: The components of the buffer solution used in this embodiment are as follows: Lysis buffer: 20 mM Tris(hydroxymethyl)aminomethane hydrochloride (TrisCl), 300 mM NaCl, 15 mM imidazole, 0.25% Triton X-100 surfactant, 10% lycerol, 1 mM benzyl sulfonyl fluoride (PMSF, protease inhibitor). Washing buffer: 20 mM TrisCl, 300 mM NaCl, 25 mM imidazole, 10% glycerol, 1 mM PMSF (protease inhibitor). Elution buffer: 50 mM TrisCl, 150 mM NaCl, 300 mM imidazole, 10% glycerol, 1 mM PMSF (protease inhibitor).

[0055] 2.1. Culture inoculation and induction of expression: Recombinant clones with good expression levels and stability were selected and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C and 200 rpm. The next day, the bacterial culture was transferred to 1 L of LB main medium and cultured until OD500 was reached. 600 The concentration was set to 0.6-1.0, and the bacterial culture was allowed to cool to room temperature. Isopropyl-β-D-thiogalactoside (IPTG) was then added to bring the final concentration to 0.1 mM. Expression was induced at 16°C and 200 rpm for 16-18 hours.

[0056] 2.2. Cell Collection and Lysis: After induction of expression, the bacterial cells were collected by centrifugation at 3500 rpm for 10 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in pre-cooled lysis buffer and homogenized using a high-pressure homogenizer at 800–900 bar for 3–4 cycles to ensure complete lysis.

[0057] 2.3. Centrifugation and Affinity Chromatography: The disrupted bacterial culture was centrifuged at 20,000 rpm and 4°C for 40 minutes, and the supernatant was collected. Pretreated Ni-NTA affinity resin was used to purify the recombinant NSP14 protein with a 6×His tag. Before use, the Ni-NTA affinity resin was washed once with deionized water, followed by twice with washing buffer. The supernatant obtained from centrifugation was loaded onto the chromatography column in batches of 20 mL each, and the loading was repeated once to enhance binding efficiency. Unbound protein fractions were eluted with washing buffer, and the eluent was collected for analysis. Then, 1 mL of elution buffer was added sequentially, and each fraction was collected into a labeled EP tube. Protein concentration was detected using NanoDrop or an equivalent instrument until no protein was detected in the eluent.

[0058] 2.4. SDS-PAGE analysis: Take 20 μL of the sample obtained in each step, add 4 μL of 6× protein loading buffer, heat in a metal bath at 95℃ for 5 minutes for denaturation treatment, and then perform SDS-PAGE electrophoresis. Coomassie brilliant blue staining is used to detect protein expression level and purification efficiency.

[0059] 2.5. Further purification by gel chromatography: The protein obtained from the initial purification was concentrated to 0.5 mL by ultrafiltration and further purified by gel filtration chromatography using a Superdex column on an AKTA FPLC system. The elution fractions were collected and analyzed by SDS-PAGE to confirm that the obtained protein was the target protein of NSP14 and had good purity and stability, providing basic materials for subsequent functional validation experiments.

[0060] (2) Experimental results This application selected *E. coli* as the expression system because the selected NSP14 protein is located in the cytoplasm, and *E. coli* is very suitable for purifying small cytoplasmic proteins. *E. coli* protein production requires only simple LB medium and conditions, making it low-cost and easy to operate. The doubling time for *E. coli* is 20 minutes, compared to 2 hours for yeast cells and 20 hours for insects and mammals. Typical steps for recombinant protein expression include: introducing a plasmid containing the target gene into host cells, culturing the cells to the logarithmic growth phase, and then adding IPTG for induction of expression. However, this system is not suitable for some proteins that are toxic to *E. coli*. Other unsuitable proteins include: proteins requiring eukaryotic protein folding and post-translational modifications, insoluble aggregates such as inclusion bodies, and proteins for which other concentrated expression systems can be used. After determining *E. coli* as the expression system, a suitable plasmid vector needs to be selected. This vector should contain: an inducible promoter, a replication initiation site, an engineered multiple cloning site, a transcription termination signal, a target protein sequence, an optional fusion tag, and an antibiotic resistance gene. This study used PET series vectors with a T7 promoter and a 6×HIS tag. NSP14 protein was purified using affinity chromatography and molecular sieves. After affinity chromatography purification of the full-length NSP14, its N-terminal domain, and its C-terminal domain using a nickel column, further purification was performed using molecular sieves to improve protein purity. The purification results are shown below. Figure 2 As shown in the figure. The results of this experiment verified the protein size of the novel coronavirus NSP14 and provided the NSP14 protease for subsequent sequencing experiments and physicochemical property detection.

[0061] Example 3 High-throughput screening of small molecule inhibitors of NSP14 protease (1) Experimental methods 1. Prepare elution buffer (WB) and selection buffer (SB) solutions as shown in Table 6. The required WuXi AppTec DELopen kit is shown in Table 7.

[0062] Table 4

[0063] Table 6

[0064] 2. Verify the adsorption capacity of the magnetic beads. Add 25 μL of magnetic beads to an EP tube with low DNA adsorption. Then add 200 μL of elution buffer (WB) and vortex the beads. After washing, place the beads on a magnetic rack for 10-15 seconds to slowly remove the elution buffer (WB). Repeat this step three times. Then mix the magnetic beads with 125 μL of elution buffer (WB) and transfer 25 μL of this mixture to a new 1.5 mL EP tube with low DNA adsorption as a blank control. Place the remaining magnetic beads on a magnetic rack for 10-15 seconds to slowly remove the elution buffer (WB). In a new 1.5 mL EP tube with low DNA adsorption, add 6 μg of the target protein NSP14 and 120 μL of selection buffer (SB). Transfer 20 μL of the protein solution to a new 1.5 mL DNA-lowering EP tube as a control. Transfer 100 μL of the protein solution to washed magnetic beads, then resuspend the beads. Incubate at 25°C for 30 minutes, rotating constantly on a tube vortex mixer / wheel (approximately 20 rpm). After vortexing, briefly centrifuge the tube at low speed (approximately 5 seconds). Place the DNA-lowering EP tube on a magnetic separator for 10–15 seconds until the solution is clear, then slowly transfer the supernatant to a new 1.5 mL DNA-lowering EP tube and label it “Flow-through.” Resuspend the magnetic beads with 100 μL of selection buffer (SB). Place the DNA-lowering EP tube on a magnetic separator for 10–15 seconds until the solution is clear, then carefully transfer the supernatant to a new DNA-lowering EP tube. Label this 1.5 mL DNA-lowering EP tube “Wash.” Resuspend the magnetic beads in 100 µL of elution buffer (WB). Transfer 50 μL of the resuspended liquid to a new 1.5 mL DNA hypoadsorption EP tube and label it “Beads”. Incubate the DNA hypoadsorption EP tube containing the remaining 50 μL of suspension at 95°C for 10 minutes. Place the sample on a magnetic separator for 10–15 seconds. Once the solution is clear, carefully transfer the supernatant to a new 1.5 mL DNA hypoadsorption EP tube. Label this EP tube “Heated Elution”. Resuspend the magnetic beads in 50 μL of selection buffer (SB) and label it “Heated Beads”.The proteins were then separated using SDS-PAGE gel chromatography, and their size was determined under Coomassie Brilliant Blue conditions. This confirmed that the magnetic beads used had adsorption capacity and were of good quality, meeting the experimental requirements.

[0065] 3. Immobilize the protein onto magnetic beads. Add 80 μL of magnetic beads to a new 1.5 mL DNA-lowering EP tube. Add 800 μL of washing buffer (WB) and briefly wash the beads using a vortex mixer. Place the EP tube on a magnetic separator for 10–15 seconds. Then slowly remove the buffer. Repeat this step three times. Resuspend the magnetic beads with 80 μL of washing buffer (WB). Add 20 μL of magnetic beads to four new 1.5 mL DNA-lowering EP tubes, labeling them from “B1” to “B4”. Place the tubes on a magnetic separator for 10–15 seconds. After the solution becomes clear, slowly remove the buffer. Dispense 5 µg of protein (full-length NSP14 protein, NSP14 protein truncated at the N-terminus, and NSP14 protein truncated at the C-terminus) into new 1.5 mL DNA-lowering EP tubes, labeling them from “P1” to “P3”. According to the experimental design, inhibitors or competitors can be added to the designated tubes. Add selection buffer (SB) to bring the total volume to 100 µL. Add 100 µL of samples "P1" to "P3" to the magnetic beads "B1" to "B3" that have just been resuspended. Add 100 μL of selection buffer (SB) to tube "B4" and label it "NTC" (blank control). Incubate each tube at 25°C for 30 minutes, continuously rotating on a vortex mixer (approximately 20 rpm). Briefly centrifuge for 5 seconds on a low-speed centrifuge. Place the above DNA-lowering EP tubes on a magnetic separator for 10–15 seconds. After the solution becomes clear, slowly remove the buffer. Add 200 μL of selection buffer (SB) to each DNA-lowering EP tube and resuspend the magnetic beads by pipetting up and down. Place the DNA-lowering EP tubes on a magnetic separator for 10–15 seconds. After the solution becomes clear, slowly remove the buffer. During these steps, the magnetic beads must not be dried; they must remain in solution to prevent any impact on the experimental results.

[0066] 4. First Round of Screening. Tubes G1, G2, G3, and G4 provided in the kit contain 10 µL of DELopen library. Centrifuge at low speed for 1 minute to allow the solution to settle at the bottom of the tubes. Add 90 μL of Selection Buffer (SB) to each of tubes G1, G2, G3, and G4, and gently pipette. Transfer the solution from tube G1 to a tube containing the immobilized protein labeled B1, resuspending the magnetic beads by pipetting up and down. Repeat this process, transferring the solution from tube G2 to tube B2, from tube G3 to tube B3, and from tube G4 to tube B4. Incubate at 25°C for 1 hour, continuously rotating on a vortex mixer (approximately 20 rpm), followed by a brief 5-second centrifuge at low speed. Place the DNA-lowering EP tubes on a magnetic separator for 10–15 seconds. Once the solution is clear, slowly remove the buffer. Next, add 200 μL of selection buffer (SB) to each tube to wash the sample. Resuspend the magnetic beads by pipetting. Place the DNA-lowering EP tubes on a magnetic separator for 10–15 seconds. Once the solution is clear, slowly remove the buffer. This washing step needs to be repeated three times. Resuspend the magnetic beads by adding 100 μL of washing buffer (WB). Incubate at 95°C for 10 minutes. Place the sample on a magnetic separator for 10–15 seconds. Once the solution is clear, slowly transfer 10 μL of supernatant to vials labeled “R1-G1”, “R1-G2”, “R1-G3”, and “R1-G4” provided with the kit. Transfer the remaining supernatant (approximately 90 µL) to new 1.5 mL DNA-lowering EP tubes, labeled G1-2, G2-2, G3-2, and G4-2.

[0067] 5. Perform the second round of screening. Repeat step 4 to immobilize the proteins onto magnetic beads. During the second round of screening, continue to immobilize the new target proteins (full-length NSP14 protein, NSP14 protein truncated at the N-terminus, and NSP14 protein truncated at the C-terminus) onto magnetic beads. This time, the corresponding labels are B1-2, B2-2, B3-2, and B4-2. Add 5 µL of sssDNA solution and 5 µL of washing buffer (WB) to the remaining G1-2, G2-2, G3-2, and G4-2 samples from the previous round of screening, bringing the total volume to 100 µL. Mix the two solutions by pipetting. Transfer the G1-2 solution to a tube containing the immobilized protein labeled B1-2, and resuspend the magnetic beads by pipetting, repeating this process. Incubate at 25°C for 1 hour, continuously rotating on a vortex mixer (approximately 20 revolutions per minute). Briefly centrifuge for 5 seconds on a low-speed centrifuge. Place the DNA-lowering EP tubes on a magnetic separator for 10–15 seconds. Once the solution is clear, slowly remove the buffer. Add 200 μL of Selection Buffer (SB) to each tube to wash the sample. Resuspend the beads by pipetting up and down with a pipette. Place the DNA-lowering EP tubes on a magnetic separator for 10–15 seconds. Once the solution is clear, slowly remove the buffer. Repeat this washing step three times. Resuspend the magnetic beads by adding 100 μL of Washing Buffer (WB). Incubate at 95°C for 10 minutes. Place the sample on a magnetic separator for 10–15 seconds. Once the solution is clear, slowly transfer 10 μL of supernatant to vials labeled “R2-G1”, “R2-G2”, “R2-G3”, and “R2-G4” provided with the kit. Transfer the remaining supernatant (approximately 50 µL) to new 1.5 mL DNA-low adsorption EP tubes and label them G1-3, G2-3, G3-3, and G4-3.

[0068] 6. Perform the third round of screening. Repeat step 5 to immobilize the proteins onto the magnetic beads. During the third round of screening, continue immobilizing the new target proteins (full-length NSP14, N-terminal truncated NSP14, and C-terminal truncated NSP14) onto the magnetic beads. This time, the corresponding labels are B1-3, B2-3, B3-3, and B4-3. Add 5 µL of sssDNA solution and 45 µL of washing buffer (WB) to the remaining G1-3, G2-3, G3-3, and G3-3 samples from the previous round of screening, bringing the volume to 100 µL. Mix the two solutions by pipetting up and down. Transfer the G1-3 solution to a tube containing the immobilized protein labeled B1-3, and resuspend the magnetic beads by pipetting up and down, repeating this process. Incubate at 25°C for 1 hour, continuously rotating on a vortex mixer (approximately 20 rpm). Briefly centrifuge for 5 seconds on a low-speed centrifuge. Place the DNA-low-adsorption EP tubes on a magnetic separator for 10–15 seconds. Slowly remove the buffer solution until the solution is clear. This washing step needs to be repeated three times. Resuspend the magnetic beads after adding 100 μL of washing buffer (WB). Incubate at 95°C for 10 minutes. Place the sample on a magnetic separator for 10–15 seconds. After the solution is clear, slowly transfer 10 μL of supernatant to the vials provided with the kit labeled “R3-G1”, “R3-G2”, “R3-G3”, and “R3-G4”.

[0069] 7. Storage. After screening, the samples (R1-G1, R1-G2, R1-G3, R1-G4, R2-G1, R2-G2, R2-G3, R2-G4, R3-G1, R3-G2, R3-G3, R3-G4) were stored at -80°C. They were then shipped to WuXi AppTec on dry ice for sequencing, synthesis, and further detection of the screened NSP14 small molecule inhibitors using 1H NMR spectroscopy, LC-MS, HPLC, and SFC.

[0070] (2) Experimental results The small molecule compounds in this application were synthesized at WuXi AppTec and verified by proton nuclear magnetic resonance spectroscopy. Taking compound DEL1 as an example... Figure 3 The structural formula and proton NMR spectral data are shown. By verifying the number of C and H elements in the compound and further analyzing the spectrum to determine the correctness of the compound's structure, it can be determined that the synthesized compound structure is consistent with the target small molecule. In this application, the mass of the synthesized small molecule was determined by liquid chromatography-mass spectrometry (LCMS). The results showed that the molecular weight of the small molecule was 564.5. Figure 4As shown in the figure. In this application, the purity of the compound was determined using high-performance liquid chromatography (HPLC). The results showed that the compound's component percentage was 99.5%, proving its purity and meeting the requirements for subsequent experiments. Figure 5 As shown. In this application, supercritical fluid chromatography (SFC) was used to confirm that the compound had no isomers (see...). Figure 6 This verifies that the compound has a single component and can be used for subsequent experiments.

[0071] Example 4 Detection of the inhibitory effect of small molecule compounds on NSP14 protease activity (1) Experimental methods 1. Substrate synthesis and labeling This experiment used a 34-base RNA oligonucleotide synthesized by Takara as the reaction substrate, the sequence of which is shown in Seq. No. 1, specifically: 5'-FAM-CACUAAUAAUAUCAAUGGAUUGAUAUUAUUAAUG-3', where the 5' end is labeled with the fluorescent group FAM (6-carboxyfluorescein) for subsequent electrophoresis detection.

[0072] 2. Experimental Procedure 2.1. Preparations before the experiment and disinfection of the experimental table Before the experiment, the work surface was thoroughly disinfected with 75% ethanol and RNase inhibitor. All pipette tips used in the experiment (10 μL, 200 μL, 1000 μL) were RNase-free and unopened. Disposable gloves were worn throughout the experiment and changed regularly.

[0073] 2.2. Preparation of reaction buffer and stop solution: Enzyme activation reaction solution: 20 mM HEPES buffer (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 1 M DTT, 6 μM RNA substrate, 5 μM NSP14 protein Enzyme activity termination solution: 95% formamide, 0.02% SDS, 0.02% bromophenol blue, 0.02% xylenecyanol, 10 mM EDTA 2.3. The purified NSP14 protein and the accessory protein NSP10 were added to the RNA substrate solution at the designed concentration. After pre-incubation on ice for 10 minutes, the solution was incubated at 37°C for 30 minutes to initiate the enzymatic digestion reaction. Immediately after the reaction was completed, an equal volume of enzyme activity termination solution was added, and the solution was heated at 70°C for 3 minutes to completely terminate the reaction. The sample was then quickly transferred to ice to cool and prevent RNA renaturation.

[0074] 2.4. Electrophoresis analysis was performed using a 20% urea-denatured polyacrylamide precast gel. The electrophoresis apparatus, including glass plates and shark tooth combs, was first cleaned with detergent, thoroughly rinsed with deionized water, and allowed to air dry. Appropriate amounts of TEMED and APS were added to every 10 mL of gel solution, mixed thoroughly, and quickly poured between the glass plates. The comb was then inserted to form sample wells. After gel polymerization, leakage was checked, and 1×TBE buffer was added inside and outside the electrophoresis tank. Pre-electrophoresis was performed for 20-30 minutes to bring the gel temperature to 40-50℃ to reduce the influence of RNA migration during electrophoresis.

[0075] 2.5. Sample Loading and Electrophoresis: Add 5 μL of the reacted RNA sample to the wells and perform denaturing polyacrylamide gel electrophoresis (PAGE) at constant power for 1.5–2 hours until the bromophenol blue indicator reaches the bottom of the gel. After electrophoresis, disassemble the apparatus, transfer the gel to a plastic plate, and carefully peel off the glass plate. Transfer the gel to a nylon membrane or silica gel plate, and use a chemiluminescence imaging system to detect the RNA restriction bands and assess enzyme activity levels.

[0076] (2) Experimental results This application's embodiments used in vitro enzyme activity assays to determine the inhibitory effects of small molecule compounds DEL1, DEL2, and DEL3 on NSP14 protease activity. According to the in vitro enzyme activity assay results, DEL1, DEL2, and DEL3 all exhibited inhibitory effects on enzyme cleavage, and the inhibitory effect improved with increasing concentration. Furthermore, the best effect was observed when all three drugs were used together (see...). Figure 7 This experiment confirmed that the small molecule compound has the effect of inhibiting the SARS-CoV-2 exonuclease NSP14.

[0077] Example 5 Validation of the interaction between small molecule inhibitors and NSP14 exonuclease This embodiment uses isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) to study the binding characteristics of small molecule DEL compounds with NSP14 protein, in order to verify its molecular mechanism of action as an exonuclease inhibitor.

[0078] 1. ITC detection of the binding energy between proteins and small molecules In this embodiment, a VP-ITC microcalorimeter was used to perform titration experiments on NSP14 protein and small molecule compounds. The purified NSP14 protein was dissolved in buffer (20 mmol / L HEPES, 150 mmol / L NaCl, pH 7.5) and placed in the sample cell. The experimental temperature was kept constant at 25°C. The test compounds DEL1, DEL2, and DEL3 were prepared in the same buffer solution, with concentrations controlled in the range of 100-200 μmol / L, and placed in a titration syringe.

[0079] The instrument injects the compound into the sample cell in increments of 5-10 μL. The system records the thermal changes after each injection and maintains thermal equilibrium between the reference and sample cells using a differential power method. The obtained heat flux signal is integrated to generate a binding isotherm. Data fitting is performed using analysis software such as Origin to obtain the binding constant (K0). d Thermodynamic parameters such as enthalpy change (ΔH) and entropy change (ΔS) were measured. The results showed that the DEL series compounds exhibit measurable thermal changes with the NSP14 protein and possess specific binding behavior.

[0080] 2. SPR detection of real-time interactions between proteins and small molecules The SPR system was used to further verify the binding kinetics between the small molecule compound and the NSP14 protein. The Ni²⁺-reinforced polymerase chain was then used. + The 6×His-tagged NSP14 protein captured by NTA affinity was immobilized on the surface of a CM5 chip. The immobilization process used 50 mmol / L HEPES buffer (pH 7.5) and 150 mmol / L NaCl as the running buffer.

[0081] The chip channel pretreatment buffer solution is prepared as follows: Flow buffer: 50 mM HEPES buffer (pH 7.5), 150 mM NaCl; Nickel ion fixative: 50 mM NiSO4·H2O; Chip regeneration buffer: 6 M urea, 350 mM EDTA, 50 mM NaOH; Washing buffer: 3 mM ethylenediaminetetraacetic acid (EDTA); After NSP14 protein immobilization, different concentrations (100, 50, 25, and 12.5 μmol / L) of compounds DEL-1, DEL-2, and DEL-3 were sequentially injected. The system recorded refractive index changes in real time, and response unit (RU) curves were plotted to assess the stability and reversibility of the binding process. If the bound protein could be effectively eluted by the run buffer, it indicated rapid and reversible binding; if the binding was stable, chip regeneration buffer was used to regenerate the chip surface.

[0082] All samples were filtered through a 0.22 μm filter membrane and degassed to ensure the accuracy of the experimental data. The experimental data were fitted and analyzed using BIAevaluation software to calculate the binding rate constant (k). a ), dissociation rate constant (k d ) and affinity constant (K D (This is used to quantify the binding strength between proteins and small molecules.)

[0083] The ITC and SPR analysis methods used in this embodiment corroborate each other, enabling a systematic evaluation of the binding ability between the DEL series of small molecule compounds and the NSP14 protein, clarifying their mechanism of action as NSP14 exonuclease inhibitors, and providing experimental basis for their subsequent efficacy evaluation and optimization.

[0084] (2) Experimental results Isothermal titration calorimetry (ITC) is a method for accurately measuring the heat released or absorbed throughout a titration experiment. From a single experiment, changes in enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) accompanying binding and binding stoichiometry can be determined. The difference in heat capacity (δcp) between the free and bound states can also be determined by conducting experiments at different temperatures. ITC is commonly used for simple 1:1 binding reactions but can also be used to characterize more complex systems where ligands bind synergistically at multiple sites and / or are involved in coupling with other equilibrium states, such as protonation / deprotonation, anisotropic allosteric effectors, changes in oligomeric states, or conformational changes. Surface plasmon resonance (SPR) techniques can be used to study protein-ligand pair interactions, including protein-protein interactions, protein-DNA interactions, enzyme-substrate (inhibitor or receptor drug) interactions, lipid bilayer-protein interactions, protein-polysaccharide interactions, and cell-virus protein interactions.

[0085] This application utilizes both ITC and SPR methods to verify the interaction between the synthesized small molecule compound and the NSP14 protein. According to the ITC experimental results, an interaction exists between DEL1 and the NSP14 protein, with a binding constant (K) of approximately 5 × 10⁻⁶. 6 M-1 corresponds to a KD value of approximately 2 × 10⁻⁶. -5M (see) Figure 8 (A). Further SPR experiments confirmed the protein-protein interaction between DEL1 and NSP14, with a measured KD value of approximately 1.65 × 10⁻⁶. -4 M (see) Figure 8 (B). This experiment demonstrated that the synthesized compound interacts with the purified protein NSP14, and further investigated its binding constant.

[0086] In summary, this application provides a class of small molecule compounds for treating novel coronavirus. The gene of the novel coronavirus NSP14 was cloned into prokaryotic and eukaryotic expression vectors via molecular cloning, enabling research under laboratory conditions. In the eukaryotic system, cell experiments verified that the NSP14 protein enhances the host's translation process. In the prokaryotic expression system, the NSP14 protein was purified via prokaryotic expression, and the C-terminal and N-terminal domains of NSP14 were purified separately based on their characteristics. This application utilizes the WuXi AppTec DELopen technology platform to perform high-throughput screening of a library of 4.4 billion DNA-encoded small molecules. Through ligand-receptor binding energy calculation and structural stability assessment, multi-level selection of lead compounds was completed. The final candidate molecules were systematically characterized using nuclear magnetic resonance spectroscopy (1H / 13C NMR), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and supercritical fluid chromatography (SFC) to confirm the accuracy of their chemical structures and verify the uniqueness of the synthesized products. This study further confirmed the significant inhibitory effect of the target compound on the NSP14 protein of the novel coronavirus using in vitro enzyme activity inhibition experiments. Simultaneously, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) techniques were used to quantitatively characterize the specific interaction mode between the compound and the target protein, successfully determining its binding affinity (K0). D The study used the values ​​and thermodynamic parameters to systematically reveal the energy change characteristics of the ligand-receptor binding process.

[0087] This application demonstrates that small molecule compounds DEL1, DEL2, DEL3, DEL4, and DEL5 can effectively inhibit the exonuclease activity of the novel coronavirus NSP14 protein, blocking the viral RNA processing mechanism at the molecular level and exhibiting significant antiviral effects. In summary, this application systematically revealed the structure-activity relationship of small molecule inhibitors targeting the novel coronavirus NSP14 protease using high-throughput screening technology and established a methodology for developing inhibitors targeting the N-terminal exonuclease functional domain of the NSP14 protein. The study not only elucidates the crucial role of exonucleases in viral genome replication and host immune response regulation but also constructs a theoretical model of synergistic inhibition across two functional domains through multi-dimensional mechanism analysis. This achievement will provide innovative insights for the field of antiviral drug development.

[0088] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of an NSP14 small molecule inhibitor in the preparation of drugs for the prevention and treatment of novel coronavirus infection, characterized in that, The NSP14 small molecule inhibitors include at least one of DEL1 as shown in structural formula I, DEL2 as shown in structural formula II, and DEL3 as shown in structural formula III. Wherein, structural formula I is Structural formula II is Structural Formula III is .

2. The application according to claim 1, characterized in that, The NSP14 small molecule inhibitor uses the NSP14 protein as a target site, wherein the target site includes the exonuclease domain or the N7-MTase domain of the NSP14 protein.

3. The application according to claim 2, characterized in that, The NSP14 small molecule inhibitors block the proofreading replication of viral RNA and 5'm7G cap modification by inhibiting the exonuclease activity or N7-MTase activity of the NSP14 protein.

4. The application according to claim 2, characterized in that, The binding constant between the NSP14 small molecule inhibitor and the NSP14 protein is 1.65 × 10⁻⁶. -4 M to 2×10 -5 M.

5. The application according to claim 1, characterized in that, The application of the NSP14 small molecule inhibitor in the preparation of drugs for preventing and treating the replication of the novel coronavirus.

6. The application according to claim 1, characterized in that, The application of the NSP14 small molecule inhibitor in the preparation of drugs for the prevention and treatment of novel coronavirus.

7. The application according to claim 1, characterized in that, The application of the NSP14 small molecule inhibitor in the preparation of drugs that inhibit the activity of NSP14 protein.

8. The application according to any one of claims 1 to 7, characterized in that, The drug is a formulation prepared using the NSP14 small molecule inhibitor as the active ingredient.

9. The application according to claim 8, characterized in that, The dosage form of the drug is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, syrups, and aerosols.

10. A pharmaceutical composition, characterized in that, It includes the NSP14 small molecule inhibitor as described in any one of claims 1 to 9, and a pharmaceutically acceptable carrier or excipient.

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