Use of inhibiting hexokinase isozyme 2 in treatment of herpes simplex viral keratitis

Abstract

Disclosed is use of inhibiting hexokinase isozyme 2 in the treatment of herpes simplex viral keratitis. The present application provides use of an inhibitor of hexokinase isozyme 2 in the preparation of a pharmaceutical composition for treating or preventing herpes simplex viral keratitis in an individual. The present application further provides a pharmaceutical composition for treating or preventing herpes simplex viral keratitis. The present application further provides a method for screening for a drug for treating or preventing herpes simplex viral keratitis. The present application further provides a screening system and a screening device for a drug for treating or preventing herpes simplex viral keratitis. The present application further provides a related computer-readable storage medium.

Description

Application of Inhibition of Hexokinase Isozyme 2 in the Treatment of Herpes Simplex Keratitis Technical Field

[0001] The present application belongs to the field of biotechnology and relates to the application of inhibiting hexokinase isoenzyme 2 in the treatment of herpes simplex virus keratitis. Background Art

[0002] Herpes simplex keratitis (HSK) is a serious, blinding eye disease primarily caused by infection of the cornea with herpes simplex virus type 1 (HSV-1). Global epidemiological statistics indicate an annual global incidence of 1.5 million cases of HSK, accounting for approximately 40,000 new cases of severe monocular vision impairment or blindness worldwide. The incidence of HSK is significantly higher in developing countries than in developed countries, and it has become one of the primary indications for penetrating keratoplasty in China, necessitating significant attention. It is generally believed that HSV-1 initially infects mucosal epithelial cells, replicates for 6-7 days, and then remains latent in the trigeminal ganglion and corneal stroma. Under stress, ultraviolet light, corneal trauma, or immunosuppression, the virus can be reactivated by retrograde transport to the eye. In these recurrent infections, inflammation and angiogenesis trigger each other, leading to corneal scarring and, in severe cases, blindness. Summary of the Invention

[0003] Since the main receptor of HSV-1 is widely distributed in corneal tissues and cells, it can regulate cell death of different cell types, activate various cell signal transductions, and trigger the release of inflammatory mediators, ultimately leading to corneal damage. Herpes simplex keratitis is one of the main indications for penetrating keratoplasty. However, the shortage of corneal donors limits treatment options. Therefore, in order to more effectively treat herpes simplex keratitis (HSK), it is urgently needed to inhibit viral replication in the human cornea and reduce the damage suffered by host cells during HSV-1 infection. In order to solve the technical problems existing in the prior art, the present application provides the following technical solutions:

[0004] In a first aspect, the present application provides the use of an inhibitor of hexokinase isoenzyme 2 in the preparation of a pharmaceutical composition for treating or preventing herpes simplex virus keratitis in an individual.

[0005] Furthermore, the individual includes human and / or non-human individuals.

[0006] In some embodiments, the individual includes humans and / or non-human individuals, including birds and non-human mammals, such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, cows), and non-domesticated animals such as large cats. Regardless of which stage in the life cycle of an organism, the term individual is applicable. Therefore, the term individual is applicable to organisms in the womb or in the egg, depending on the organism (that is, whether the organism is a mammal or a bird, such as domesticated or wild fowl). In some specific embodiments, individual is used interchangeably with object, patient, subject, and detector.

[0007] Furthermore, after preparation, the pharmaceutical composition can be used for perilesional administration, lesion surface administration, intravenous administration, local application, corneal stroma injection administration, subconjunctival injection administration, and oral administration.

[0008] Furthermore, the pharmaceutical composition is prepared for administration to the ocular surface, local injection, oral administration, or intravenous injection.

[0009] The term "administering" and similar terms used herein refer to a single administration of a pharmaceutical composition, and the term "administering" is also intended to include administering a pharmaceutical composition according to a complete treatment regimen or dosing regimen. The term "administering" is also intended to include treatment regimens in which the pharmaceutical compositions are not necessarily administered by the same route of administration or at the same time.

[0010] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0011] As used herein, the term "HSV-1" refers to herpes simplex virus type 1.

[0012] As used herein, the term "HSV-2" refers to herpes simplex virus type 2.

[0013] The term "hexokinase isozyme 2" used in this application is also called HK2 (hexokinase 2), which is the first enzyme in the glycolysis pathway and also the rate-limiting enzyme in the glycolysis pathway.

[0014] The term "herpes simplex keratitis" used in this application is also called herpes simplex keratitis, abbreviated as HSK, which is an inflammatory disease of the cornea caused by herpes simplex virus infection of the cornea.

[0015] Furthermore, the inhibitor of hexokinase isoenzyme 2 includes lonidamine.

[0016] Furthermore, the lonidamine includes lonidamine and its derivatives, and the derivatives include pharmaceutically acceptable salts.

[0017] The application uses term " pharmaceutically acceptable salt " to comprise the conventional salt formed by pharmaceutically acceptable inorganic acid or organic acid or inorganic base or organic base and the acid addition salt of quaternary ammonium.The more specific example of suitable acid salt comprises the salt of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, hydroxyacetic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, steroic, tannic acid etc.Other acid, as oxalic acid, although itself is not pharmaceutically acceptable, can be used for the preparation of the salt used as intermediate, to obtain the present application compound and pharmaceutically acceptable salt thereof. More specific examples of suitable base salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. When referring to a pharmaceutically acceptable salt of salinomycin, it generally means that it can be used in the pharmaceutical field, is not harmful to the product or to mammals, or has a reasonable or acceptable benefit / risk ratio.

[0018] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0019] Furthermore, the auxiliary materials include adjuvants, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators, buffers, and emulsifiers.

[0020] In a second aspect, the present application provides a pharmaceutical composition for treating or preventing herpes simplex virus keratitis, wherein the pharmaceutical composition comprises a therapeutically effective amount or a preventively effective amount of a hexokinase isoenzyme 2 inhibitor.

[0021] The term "therapeutically effective amount" as used herein refers to an amount of a compound sufficient to treat a disease when administered to a subject. A "therapeutically effective amount" may vary with the compound, the disease and its severity, the age and weight of the subject to be treated, and the like.

[0022] As used herein, the term "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in the prevention of a condition. The term "prophylactically effective amount" can encompass an amount that improves overall prevention or increases the prophylactic efficacy of another prophylactic agent.

[0023] As used herein, the terms "prevent," "preventing," or "suppressing" refer to preventing the onset of a preclinically apparent disorder in a subject or preventing the onset of a preclinically apparent stage of a disorder in a subject. Prevention includes, but is not limited to, prophylactic treatment of a subject at risk of developing a disorder.

[0024] As used herein, the term "treatment" includes palliative, restorative, and prophylactic treatment of a subject. The term "palliative treatment" refers to treatment that lessens or reduces the effects or intensity of a condition in a subject without curing the condition. The term "prophylactic treatment" refers to treatment that prevents the onset of a condition in a subject. The term "restorative treatment" refers to treatment that halts the progression of a condition in a subject, reduces the pathological manifestations, or completely eliminates the condition.

[0025] Furthermore, the hexokinase isoenzyme 2 inhibitor includes lonidamine.

[0026] Furthermore, the lonidamine includes lonidamine and its derivatives, and the derivatives include pharmaceutically acceptable salts.

[0027] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0028] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0029] Furthermore, the auxiliary materials include adjuvants, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, and emulsifiers.

[0030] The application uses the term "adjuvant" to mean a pharmacologically inert substance, which is used for the design of the carrier and / or pharmaceutical product preparation of active substances. The term "adjuvant" should include pharmaceutically acceptable, pharmacologically inert ingredients, such as adhesives, fillers, coatings, compound formations, plasticizers for coatings, and compounds that mask odor. Some examples of selectable adjuvants include pigments, disintegrants, antioxidants, flavorings, sweeteners, coloring agents, opacifiers, anti-adhesives, preservatives, glidants, lubricants, adsorbents, and barrier layer forming agents. Suitable materials known in the art. The term "adjuvant" used for the pharmaceutical preparation of the application also refers to a diluent or a medium administered together with an active substance. Such pharmaceutical adjuvants can derive from animals, plants, or synthetic raw materials.

[0031] The present application also provides a method for treating or preventing herpes simplex virus keratitis, which comprises the step of administering the aforementioned pharmaceutical composition to a patient.

[0032] The third aspect of the present application provides a method for screening drugs for treating or preventing herpes simplex virus keratitis for non-therapeutic purposes, the method comprising the steps of applying a candidate drug to herpes simplex virus keratitis cells or tissues in vitro, and detecting the expression of hexokinase isoenzyme 2 after the candidate drug is administered.

[0033] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0034] Furthermore, the drug that can cause the method to detect a decrease in the expression level of hexokinase isoenzyme 2 is the screened drug.

[0035] Furthermore, the method further comprises detecting the degree of glycolysis or aerobic oxidation after administration of the candidate drug.

[0036] Furthermore, the degree of aerobic oxidation includes the rate of ATP production.

[0037] Furthermore, the detection of the ATP generation rate includes detecting the expression levels of ATPB, UQCRC2, SDHB, and NDUFV2.

[0038] Furthermore, the detection of the degree of glycolysis includes detecting the level of lactate or lactate dehydrogenase.

[0039] Furthermore, the screened drugs can also reduce the degree of glycolysis or increase the degree of aerobic oxidation.

[0040] Furthermore, the decreased degree of glycolysis includes a decreased lactate level or an increased lactate dehydrogenase level.

[0041] Furthermore, the increase in the degree of aerobic oxidation includes an increase in the rate of ATP production.

[0042] Furthermore, the increased ATP production rate includes increased expression levels of ATPB, UQCRC2, SDHB, and NDUFV2.

[0043] In some embodiments, the method for screening drugs for treating or preventing herpes simplex keratitis for non-therapeutic purposes comprises the following steps: applying a candidate drug to in vitro herpes simplex keratitis cells or tissues, and then detecting the expression level of hexokinase isoenzyme 2, the degree of glycolysis, and the degree of aerobic oxidation in the in vitro herpes simplex keratitis cells or tissues; when it is found that the expression level of hexokinase isoenzyme 2 decreases, the candidate drug is a drug for treating or preventing herpes simplex keratitis; if the degree of glycolysis decreases and the degree of aerobic oxidation increases while the expression level of hexokinase isoenzyme 2 decreases, the candidate drug has a stronger therapeutic effect as a drug for treating or preventing herpes simplex keratitis.

[0044] The term "candidate drug" used in this application generally refers to substances that may be used as drugs. These substances can be evaluated by appropriate methods to obtain information on their biological activity, pharmacological effects and efficacy. "Candidate drug" also means a compound with potential therapeutic properties and / or commercial interests. These compounds can be any molecules that need to be tested for biological activity, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc. The compound can directly or indirectly change the Warburg effect. The Warburg effect refers to the fact that glycolytic metabolism in cancer cells takes precedence over cellular respiration metabolism. In this article, the Warburg effect refers to the fact that the physiological process of herpes simplex virus keratitis is similar to the physiological process in cancer cells, that is, glycolytic metabolism rather than cellular respiration metabolism takes precedence in herpes simplex virus keratitis.

[0045] In a fourth aspect, the present application provides a screening system for drugs for treating or preventing herpes simplex keratitis. The screening system comprises: a processing unit for applying candidate drugs to herpes simplex keratitis cells or tissues.

[0046] The first detection unit is used to detect the expression level of hexokinase isoenzyme 2 in the herpes simplex virus keratitis cells or tissues treated by the treatment unit.

[0047] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0048] Furthermore, the screening system further comprises a second detection unit, which is used to detect the aforementioned degree of glycolysis or aerobic oxidation.

[0049] Furthermore, the screening system further includes a judgment unit for obtaining a result from the detection data as to whether the candidate drug can be used as a drug for treating or preventing herpes simplex virus keratitis.

[0050] As used herein, the term "screening system" refers to a system for processing and analyzing biological data to derive insights therefrom. A screening system may also refer to the control instrumentation, control circuitry, and / or data processing system used to operate the screening system and obtain results related to the biological data. Notably, the screening system significantly reduces random error and distortion when determining insights from the biological data and provides greater accuracy when inferring results from different parts of the biological data.

[0051] The screening system includes a control circuit. The control circuit refers to a computing element that is operable to respond to and process instructions that drive the screening system. Optionally, the control circuit includes but is not limited to a microprocessor, a microcontroller, a complex instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, or any other type of processing circuit. In addition, the term "control circuit" can refer to one or more separate processors, processing devices, a portion of an artificial intelligence (AI) system, and various elements associated with the screening system.

[0052] A fifth aspect of the present application provides a device for screening drugs for treating or preventing herpes simplex virus keratitis, the screening device comprising: a memory and a processor;

[0053] The memory is used to store program instructions;

[0054] The processor is used to call program instructions, and when the program instructions are executed, it is used to execute the method described in the third aspect of this application.

[0055] The term "memory" is used herein to refer to and may be used interchangeably with the terms "volatile memory," "non-volatile memory," "physical memory," "non-volatile memory device," "NVM," "NVM device," "non-volatile random access memory," "non-volatile memory array," "flash memory," "phase change memory," "resistance random access memory," "correlated electronic material memory," "CeRAM," "correlated electronic switch," "CES," or "correlated electronic switch (CES) element."

[0056] The terms "processor" and "processing device" as used herein may refer to a single processor or any number of processors in a collection of processors that together perform a set of operations, such as a central processing unit (CPU), a graphics processing unit (GPU), a remote server, or a combination of these. Read-only memory (ROM), random access memory (RAM), flash memory, hard drives, and other devices capable of storing electronic data constitute memory devices. A memory device may include a single device or a collection of devices that store data and / or instructions.

[0057] Furthermore, the program instructions control the processor to execute the step of applying the candidate drug to cells or tissues in vitro with herpes simplex keratitis.

[0058] Furthermore, the program instructions control the processor to execute the step of detecting the expression of hexokinase isoenzyme 2, which is performed after the candidate drug is used.

[0059] Furthermore, the program instructions control the processor to execute a judgment step, wherein the judgment step can judge the drug for which a decrease in the expression level of hexokinase isoenzyme 2 is detected as an effective drug.

[0060] Furthermore, the program instructions control the processor to execute the step of detecting the degree of glycolysis or aerobic oxidation, which is performed after the candidate drug is used.

[0061] Furthermore, the degree of aerobic oxidation includes the rate of ATP production.

[0062] Furthermore, the detection of the ATP generation rate includes detecting the expression levels of ATPB, UQCRC2, SDHB, and NDUFV2.

[0063] Furthermore, the detection of the degree of glycolysis includes detecting the level of lactate or lactate dehydrogenase.

[0064] Furthermore, the program instructions control the processor to execute a judgment step, wherein the judgment step can judge the drug that reduces the degree of glycolysis or increases the degree of aerobic oxidation as an effective drug.

[0065] Furthermore, the decreased degree of glycolysis includes a decreased lactate level or an increased lactate dehydrogenase level.

[0066] Furthermore, the increase in the degree of aerobic oxidation includes an increase in the rate of ATP production.

[0067] Furthermore, the increased ATP production rate includes increased expression levels of ATPB, UQCRC2, SDHB, and NDUFV2.

[0068] The sixth aspect of the present application provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the method for screening drugs for treating or preventing herpes simplex virus keratitis described in the third aspect of the present application.

[0069] The present application also provides a method for diagnosing herpes simplex virus keratitis, which comprises analyzing the level of hexokinase isoenzyme 2 in a subject, wherein when the level of hexokinase isoenzyme 2 is higher than a predetermined amount, it indicates that the subject suffers from herpes simplex virus keratitis.

[0070] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0071] The present application also provides a method for treating herpes simplex virus keratitis, comprising: 1) analyzing the level of hexokinase isoenzyme 2 in a subject, wherein when the level of hexokinase isoenzyme 2 is higher than a predetermined amount, it indicates that the subject has herpes simplex virus keratitis; 2) when the subject is diagnosed with herpes simplex virus keratitis, using an inhibitor of hexokinase isoenzyme 2 for treatment.

[0072] Furthermore, the inhibitor of hexokinase isoenzyme 2 includes lonidamine.

[0073] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0074] The present invention also provides the use of hexokinase isoenzyme 2 level in the preparation of products for diagnosing herpes simplex virus keratitis.

[0075] Furthermore, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

[0076] The terms "computer program" and "program" are used herein to refer to one or more application programs, software components, sets of instructions, procedures, functions, objects, classes, instances, related data, or portions thereof suitable for implementation in suitable computer-readable program code.

[0077] The term "computer-readable storage medium" as used herein shall be deemed to include a single medium or multiple media (e.g., a centralized or distributed database, and / or associated cache memories and servers) that stores one or more sets of computer-executable instructions or data. The term "computer-readable storage medium" shall also be deemed to include any medium that can store or encode a set of instructions for execution by a processor and cause the processor to perform any one or more methods of the present disclosure. Therefore, the term "computer-readable storage medium" shall be deemed to include, but not be limited to, solid-state memories, optical media, and magnetic media. For example, a computer-readable storage medium can be one or more volatile, non-transitory, or non-volatile tangible computer-readable media.

[0078] The terms "including," "having," "comprising," and "containing" used herein should be interpreted as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the numerical ranges recited herein are intended merely as a convenient method of referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the present application and does not limit the scope of the present application, unless otherwise requested. No language in the specification should be construed as indicating that any unclaimed element is necessary for implementing the present application.

[0079] Advantages and beneficial effects of this application:

[0080] The present application discovered that inhibitors of hexokinase isoenzyme 2 can effectively treat herpes simplex virus keratitis, wherein the inhibitor includes lonidamine. Experiments have shown that lonidamine can activate the aerobic oxidation pathway of the HSK cornea, inhibit the glycolysis pathway, treat herpes simplex virus keratitis, protect the cornea, and reduce viral replication. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 shows the gene expression dynamics and interaction network results for a multigene module. (A) This heat map illustrates the correlation between modules (rows) and traits (columns), with red indicating positive correlation and blue indicating negative correlation. The color block on the left represents the multigene module. (B) GO biological process term enrichment for genes in the turquoise module. (C) KEGG pathway enrichment in the turquoise module. (D) HSK volcano plot of energy metabolism-related genes, with blue indicating downregulated genes and red indicating upregulated genes. The right side shows the protein-protein interaction network (PPI) of upregulated genes, while the left side shows the PPI of downregulated genes. (E) GO pathway enrichment in the PPI network.

[0082] Figure 2 shows the expression analysis of respiratory chain complex proteins and HK in HSK patients. (A) Western blot analysis showed decreased expression of respiratory chain complex-related proteins in HSK patients compared with normal controls. (C) Increased expression of HK type II isoenzymes. (B & D) Relative expression levels of each protein (ns, P > 0.05; *p < 0.05; ****p < 0.0001). (E) Mitochondrial respiratory chain complexes I, II, and III, and HK activities (*P < 0.05, **P < 0.01, ***P < 0.001). (F) Lactate expression levels.

[0083] Figure 3 shows the temporal changes in HSV1-gD and ICP0 expression after in vitro drug treatment. (A) Western blotting shows that HSV1-gD and ICP0 protein expression peaks 2 to 4 hours after infection. (B) Drug administration 2 hours after infection. (C) Representative Western blotting results show decreased HSV1-gD and ICP0 protein expression after lonidamine treatment. (D) Statistical graph of the relative expression of the indicated proteins (*P < 0.05, **P < 0.01).

[0084] Figure 4 shows the effects of lonidamine on viral protein expression and metabolic enzymes in HSV-1-infected corneas. (A) Administration 3 days after infection. (B) Photographs of fluorescein-stained and unstained corneas after lonidamine treatment. (C) HK activity after lonidamine treatment. (D) Lactate expression levels. (E) Representative Western blotting (WBs) showing decreased HSV1-gD and ICP0 protein expression after lonidamine treatment. (F) Statistical graph of HSV1-gD and ICP0 protein expression (**P<0.01, ****P<0.0001). (G) Representative Western blotting showing UQCR2, NDUFV2, and SDHB protein expression after lonidamine treatment. (H) Statistical graph of UQCR2, NDUFV2, and SDHB protein expression (ns, P>0.05; *P<0.05). (I) Mitochondrial respiratory chain complexes I, II, and III (*P<0.05). (J) Shows the expression of HSV-1-gD and ICP0 in different treatment groups (GCV: ganciclovir; CM: combined drug). (K) Statistical graph of HSV1-gD and ICP0 protein expression (*P<0.05, **P<0.01). DETAILED DESCRIPTION

[0085] Example 1

[0086] 1. Experimental Materials

[0087] This trial was registered with the Chinese Clinical Trial Registry (ChiCTR220060105) and fully complied with the principles of the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. Patients were enrolled at the Wenzhou Medical University Eye Hospital. Clinical data collected included general information (age and sex), age at HSK diagnosis, and ocular history. Herpes simplex keratitis examinations were performed by a corneal fellowship-trained ophthalmologist using slit-lamp biomicroscopy. The ocular surface, visual acuity, degree of corneal vascularization, and degree of anterior chamber inflammation were observed and photographed. Patients with a history of prior ocular surgery or any other ocular disease (such as lens-induced uveitis or glaucoma) or a history of ocular trauma were excluded.

[0088] 2. Experimental methods

[0089] (1) Sample collection and processing

[0090] Epithelial tissue was collected from 10 corneal transplant recipients at the Wenzhou Medical University Eye Hospital for the experimental group. The control group consisted of 10 corneal limbuses obtained from donors undergoing corneal transplantation. After corneal harvest during surgery, the tissue was placed in pre-chilled cryopreservation tubes and stored at -80°C until RNA extraction.

[0091] (2) RNA purification and library construction

[0092] RNA from 10 HSK groups and 10 control groups was used for library preparation for RNA sequencing. Total RNA was extracted from frozen tissue using the RNA extraction reagent TRIZOL (Invitrogen Life Technologies) according to the standard acid guanidine phenol chloroform method. RNA degradation and contamination were monitored on a 1% agarose gel. RNA purity was checked by spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNANano 6000 assay kit (Agilent Technologies, CA, USA) for the Bioanalyzer 2100 system. A total of 1 μg of RNA was used as input material for RNA sample preparation for each sample. RNA was purified using the PCR amplification kit (Implen, CA, USA) according to the manufacturer's recommendations. (Northeastern University, USA) Sequencing libraries were generated using the Ultra™ RNA Library Preparation Kit, and index codes were added to the attribute sequences of each sample. Finally, PCR products were purified (AMPure XP System), and library quality was assessed on an Agilent Bioanalyzer 2100. The TruSeq PE Cluster Kit v3 cBot HS (Illumia) was used for power generation according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on the Illumina Novaseq platform.

[0093] (3) Bioinformatics analysis

[0094] The raw data obtained by high-throughput sequencing were converted into raw sequencing reads by CASAVA base calling and stored in FASTQ format. Afterwards, the raw data were filtered to obtain high-quality clean data for subsequent analysis by removing joint sequences and low-quality reads. The reference genome and gene model annotation files were downloaded directly from the genome website. The index of the reference genome was constructed using Hisat2 v2.0.5, and paired end clean reads were aligned to the reference genome using Hisat 2v2.0.5. FeatureCounts v1.5.0-p3 was used to calculate the number of reads mapped to each gene. Differential expression analysis was performed between the two groups using DESeq2 (version 4.1.0). The threshold for significant differential expression was set as FDR < 0.01 & |log2(foldchange)| ≥ 1. Co-expression analysis was performed using Pearson correlation based on the relevant lncRNA and mRNA expression data.

[0095] Gene Ontology (GO) and KEGG enrichment analysis (Kyoto Encyclopedia of Genes and Genomes (KEGG)) were performed using the R package clusterProfiler, which corrected for gene length bias. GO terms and KEGG terms with adjusted P values ​​less than 0.05 were considered significantly enriched for differentially expressed genes.

[0096] Unsigned weighted gene co-expression network analysis (WGCNA) was performed on the normalized (log2-counts per million) gene expression data using the R package WGCNA to determine whether co-expressed gene modules were significantly associated with herpes simplex keratitis.

[0097] We analyzed the top 5,000 most variably expressed mRNAs in HSK samples. A hybrid tree-cutting algorithm was used to form unmerged modules. Module eigengenes were calculated and associated with herpes simplex keratitis by double-mean correlation. Modules with the highest correlation were considered to be of potential interest. To determine the functions of the selected modules, GO and KEGG enrichment analysis was performed on all modules using the R package clusterProfiler (https: / / guangchuangyu.github.io / clusterProfiler). After module identification, genes with gene significance (GS) > 0.9 and module membership (MM) > 0.9 were considered hub genes.

[0098] The Molecular Signature Database (MSigDB, http: / / software.broadinstitute.org / gsea / msigdb / ) was queried to identify 29 gene sets related to energy metabolism. From these gene sets, 592 cross-talking genes were identified for differential expression analysis. Protein-protein interaction (PPI) networks of up-regulated and down-regulated DEGs were constructed using the STRING database (http: / / string-db.org). GO enrichment analysis was performed on all proteins within the PPI network.

[0099] Pearson correlation coefficients were calculated between lncRNA expression and mRNA DEGs. Intersections of lncRNAs with |r| > 0.8 and co-expressed with ≥ 10 mRNAs and hub genes were selected. lncRNAs with |r| > 0.8 interacting with more than 10 mRNAs were identified and then intersected with the hub lncRNAs in the selected modules. Finally, a lncRNA-mRNA correlation network was constructed using Cytoscape.

[0100] 3. Experimental results

[0101] To elucidate the transcriptomic alterations in HSK, we performed RNA sequencing on corneal samples from 10 patients and 10 healthy controls. Weighted correlation network analysis (WGCNA) further classified HSK samples into 14 distinct gene co-expression modules, of which the turquoise module showed the strongest correlation with HSK (Figure 1A). To gain deeper insights into the biological functions of the turquoise module, we performed GO and KEGG pathway enrichment analyses. GO analysis revealed altered mitochondrial function in HSK, with significant enrichment of processes related to cytochrome c release and apoptosis regulation (Figure 1B). KEGG analysis confirmed these findings by highlighting the enrichment of the oxidative phosphorylation pathway (Figure 1C), suggesting that HSK may be involved in energy metabolism disorders.

[0102] To assess metabolic changes, we analyzed the expression patterns of genes related to energy metabolism in HSK. Further analysis of energy metabolism revealed 173 differentially expressed genes in the HSK group, with a threshold of |log2(fold change)| ≥ 1 and p_adjusted < 0.05, of which 82 were upregulated and 91 were downregulated (Figure 1D). A protein-protein interaction (PPI) network was constructed using the Searching for Interacting Genes (STRING) database (Figure 1D). GO enrichment analysis of proteins in the PPI network revealed that pathways related to mitochondrial aerobic respiration were significantly downregulated, including the mitochondrial electron transport chain, mitochondrial respiratory chain complex I, respiratory chain complex, mitochondrial respiratory chain complex III, and mitochondrial respiratory chain complex IV. Furthermore, hexokinase (HK), the rate-limiting enzyme in the glycolytic pathway, was significantly downregulated (Figure 1E). These results suggest that HSV-1 infection may induce metabolic reprogramming in corneal cells, shifting from mitochondrial respiration to aerobic glycolysis. This change resembles the Warburg effect often observed in cancer cells, indicating alterations in energy production pathways. This metabolic reprogramming may represent a cellular adaptation to viral infection, providing new insights into the pathophysiology of HSK and potentially new targets for therapeutic intervention.

[0103] Example 2

[0104] 1. Experimental Materials

[0105] The experimental reagents are shown in Table 1.

[0106] Table 1

[0107] 2. Experimental methods

[0108] (1) Western blot

[0109] A. Sample Preparation

[0110] (a) Material preparation: 1.5 mL EP tube, RIPA lysis buffer containing PIC placed on ice, ice box.

[0111] (b) Sample collection: Samples were collected 24 and 48 hours after administration. First, the cap of the EP tube was placed upside down on a temporary ice box. RIPA lysis buffer was added at 25 μL / eye. After anesthesia, the mouse was killed by cervical dislocation. The corneal epithelial tissue of the mouse cornea was removed using ophthalmic scissors and placed in RIPA lysis buffer. Four ball mill beads were added to each EP tube and labeled.

[0112] (c) Protein extraction: The collected tissue was ball milled for 15 min at 65 times / s. The tissue was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was transferred to a new EP tube and the protein concentration was determined by the BCA assay. The supernatant was stored at -20°C until use.

[0113] (d) Based on the protein concentration determined by BCA, 20 μg of protein was taken and added to 5× SDS loading buffer. All samples were adjusted to the same volume using pure water (ddH2O).

[0114] (e) Denature the prepared protein in a 100°C incubator for 10 min.

[0115] (f) Remove the protein and immediately place it on ice to stop denaturation. Centrifuge and place on ice until use.

[0116] B. PAGE gel preparation:

[0117] (a) First, clean the glass plate, align the glass plate, clamp it with an electrophoresis stand, and add water to check for leaks.

[0118] (b) Prepare separating gel: Prepare separating gel at the desired concentration, mix thoroughly, and immediately pipette it into the glass sandwich along the edge of the glass plate, avoiding bubbles as much as possible. Add a layer of water on top of the separating gel. Incubate at 37°C for half an hour and remove.

[0119] (c) Remove the separating gel, pour out the water in the glass plate, and absorb it dry with paper.

[0120] (d) Prepare 4% stacking gel: Prepare stacking gel at the desired concentration, shake well, and immediately add it to the remaining space on the glass plate. Insert a comb, taking care to avoid creating bubbles. Incubate at 37°C until the gel solidifies.

[0121] C. Sample loading:

[0122] Remove the PAGE gel, fill the inner tank with 1× electrode solution, and pull out the comb; slowly add the denatured samples to the sample wells in sequence, and add 5μL protein marker on both sides of the sample wells. Change the pipette tip for different samples.

[0123] D. Electrophoresis:

[0124] Fill with 1× electrode solution; maintain a constant voltage of 80V for protein concentration. When the protein is at the junction of the stacking gel and the separation gel, adjust the voltage to 120V. When bromophenol blue is about 1 cm from the bottom of the gel, stop electrophoresis. The total duration is about 3 hours.

[0125] E. Transfer:

[0126] (a) Prepare enough 1× electroporation buffer in advance and pre-cool it at 4°C.

[0127] (b) Gel cutting: Remove the glass plate and gently pry it open with a ruler. Refer to the marker position to retain the target protein and cut away the excess gel. Measure the length and width with a ruler and place in pre-cooled electroporation solution.

[0128] (c) Cutting the membrane: Cut the NC membrane of the same size according to the length and width of the gel and soak it in the electrotransfer solution.

[0129] (d) Preparation of sandwich electroporation structure: In the electroporation solution, prepare a sandwich structure in the following order: black plate (negative electrode) → sponge → filter paper → PAGE gel containing target protein → NC membrane → filter paper → sponge → white plate (positive electrode).

[0130] (e) Electroporation: Place the sandwich in an electroporation tank. Add enough 1x electroporation buffer and place the tank in a basin covered with ice to prevent overheating. Maintain a current of 300 mA for 1.5 hours.

[0131] (f) Blocking: After transfer, remove the NC membrane and block it with BSA rapid blocking solution for 10 min.

[0132] (g) Membrane cutting: After sealing, place the NC membrane in a disposable film glove and cut it according to the molecular weight using a blade and ruler.

[0133] (h) Primary antibody incubation: Prepare an appropriate amount of primary antibody using Western blotting primary antibody diluent and incubate overnight at 4°C in a humidified chamber.

[0134] (i) Washing the membrane: Wash the membrane three times with PBST on a shaker, 5 min each time.

[0135] (j) Secondary antibody incubation: dilute the secondary antibody at a ratio of 1:5000 with blocking buffer and incubate at room temperature for 1 hour.

[0136] (k) Washing the membrane: Wash the membrane three times with PBST on a shaker, 5 min each time.

[0137] (l) Scanning: Chemical exposure: Prepare an appropriate amount of ultrasensitive ECL chemiluminescence detection reagent in a 1:1 ratio, add it to the membrane, detect it using an automatic chemiluminescence analyzer, and save the image.

[0138] (2) Measurement of enzyme activity

[0139] A. Homogenize the tissue at a ratio of 1:5-10 (tissue weight (g): extract volume (mL)). Centrifuge at 8000 g, 4°C for 10 min, remove the supernatant, and place on ice for testing.

[0140] B. Preheat the UV spectrophotometer or microplate reader for more than 30 minutes, adjust the wavelength to 340 nm, and zero the UV spectrophotometer with distilled water.

[0141] C. Add reagents according to the volumes in Table 2.

[0142] Table 2

[0143] Add the reagents in Table 2 in order to a micro-quartz cuvette or 96-well UV plate. Mix thoroughly immediately and measure the absorbance at 340 nm after 20 seconds (A1). Immediately incubate at 37°C (for mammals) or 25°C (for other species) for 5 minutes (microplate reader with temperature control can adjust the temperature to 37°C). Remove, quickly dry, and measure the absorbance at 5 minutes and 20 seconds (A2). Record the absorbance at 340 nm at 20 seconds (A1) and the absorbance after 5 minutes and 20 seconds (A2). Calculate ▲A = A2 - A1. HK activity (U / mg prot) = [▲A × V total ÷ (Σ × d) × 10 9 ]÷(V model × Cpr)÷T=643×▲A÷Cpr.

[0144] V: total volume of the reaction system, Σ: NADPH molar extinction coefficient, 6.22×10 3 L / mol / cm; d: cuvette optical path, Vsample: added sample volume; T: reaction time, 5 min; Cpr: sample protein concentration, mg / mL.

[0145] 3. Experimental results

[0146] To confirm the hypothesis of metabolic reprogramming toward aerobic glycolysis in HSK, we examined the expression of mitochondrial respiratory chain complexes and HK in HSK and control corneas. Our data revealed that the respiratory chain complex proteins NDUFV2, SDHB, UQCR2, and ATPB were significantly downregulated in HSK corneas, while HK2 expression was upregulated (Figure 2A-D). Functional analysis confirmed decreased activity of respiratory chain complexes I, II, and III, increased HK activity, and elevated extracellular lactate levels in HSK corneas (Figure 2E-F), indicating a shift from oxidative phosphorylation to glycolysis. HK2, a key rate-limiting enzyme in the first step of glycolysis, showed significant increases in both expression and enzymatic activity, potentially contributing to the metabolic reprogramming. Our study extends this understanding by comprehensively analyzing the metabolic pathway changes that occur during HSV-1 infection. Specifically, we identified HK2-induced metabolic reprogramming, which had not previously been associated with HSV infection in ocular tissues. This detailed metabolic profile highlights the complexity of virus-host interactions and the potential role of metabolic alterations in disease pathogenesis. Our study uniquely links this metabolic reprogramming to HSV-1 infection in HSK.

[0147] Example 3

[0148] 1. Experimental Materials

[0149] (1) Animal origin

[0150] C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd.

[0151] (2) Drug source

[0152] Lonidamine was purchased from MedChemExpress.

[0153] (3) Virus source

[0154] The HSK strain was kindly donated by Shanghai Jiao Tong University.

[0155] 2. Experimental methods

[0156] (1) Steps of animal experiment

[0157] 1) Mouse anesthesia: The mice were anesthetized by intraperitoneal injection of avertin anesthetic at a dose of 30 μL / g body weight, and then the model was established.

[0158] 2) Infection Model: The left eye served as a normal control (control) and received no treatment; the right eye served as the experimental eye and was infected with herpes simplex virus keratitis. First, under a stereomicroscope, a drop of normal saline was placed on the mouse ocular surface and blotted dry with a cotton swab to clean the ocular surface. Then, topical anesthesia was achieved by applying proparacaine hydrochloride eye drops to the mouse ocular surface. Under a microscope, the back tip of a #11 scalpel blade was used to create a "#" incision in the center of the cornea, ideally penetrating the corneal epithelium to the Descemet's membrane. Then, using a pipette tip, 5 μL of HSV-1 virus-containing DMEM culture medium was dropped onto the corneal surface. After closing the eyelid, the eye was gently massaged for 30 seconds to ensure that the virus solution fully contacted the cornea.

[0159] 3) Drug application: 72 hours after infection, the corneal epithelial infection status was observed, and 5 μL of 500 μM / mL lonidamine solution was dripped into the affected eye of the mouse three times a day.

[0160] 4) Sampling steps: At 24 h and 48 h after administration, the mice were anesthetized and killed by cervical dislocation. The corneal epithelial tissue of the mice was removed using ophthalmic scissors to extract protein or RNA to provide samples for subsequent experiments.

[0161] 3. Experimental results

[0162] To investigate the role of HK2 in HSV-1 infection and explore potential therapeutic strategies targeting this enzyme, we utilized a corneal epithelial cell model infected with HSV-1 in vitro. Expression of the viral replication markers HSV-1 glycoprotein D (HSV1-gD) and infected cell protein 0 (ICP0) peaked between 2 and 4 hours postinfection. This increase in viral protein expression correlated with a corresponding decrease in host cell viability (Figure 3A). Given the critical role of HK2 in cellular energy metabolism and its potential as an antiviral therapeutic target, we next sought to assess the impact of HK2 inhibition on viral replication. To this end, we first needed to determine the optimal concentration of lonidamine for treatment. Cell viability assays allowed us to identify 100 μmol / mL as the optimal concentration for lonidamine treatment. We treated corneal epithelial cells with lonidamine 2 hours postinfection (Figure 3B), a time point when viral replication is expected to be active. Two hours after treatment, we detected the expression of viral replication markers HSV1-gD and ICP0 in treated cells, which were significantly reduced compared with those in the control group ( Figure 3C-D ), indicating that targeting HK2 can inhibit viral replication.

[0163] After establishing the antiviral effects of HK2 inhibition in vitro, we extended our findings to an in vivo model by topically applying the drug to HSV-1-infected mice starting on day 3 post-infection (Figure 4A). This treatment significantly reduced corneal epithelial defects on days 4 and 5 post-infection (Figure 4B). Furthermore, the levels of viral replication markers HSV1-gD and ICP0 in the cornea were significantly reduced compared with the control group (Figure 4C-D). HK enzyme activity (Figure 4E) and lactate production (Figure 4F) were significantly decreased in the lonidamine-treated group. Although key proteins involved in the respiratory chain (such as NDUFV2, SDHB, UQCR2, and ATPB) showed only minor changes, the activity of respiratory chain complexes was significantly increased (Figure 4G-I). This result indicates that lonidamine inhibition of HK successfully enhances aerobic respiration in HSK and reverses the shift to anaerobic glycolysis in HSK.

[0164] Based on these findings, we next compared the efficacy of lonidamine with the classic antiviral drug, ganciclovir ophthalmic gel (GCV), for the treatment of ocular disease, evaluating both monotherapy and combination therapy (CM) approaches. Results demonstrated that lonidamine monotherapy allowed earlier intervention, with significant reductions in HSV-1 gD and ICP0 expression observed as early as 24 hours after treatment compared with the PBS control group (Figures 4J-K). Combination therapy, on the other hand, significantly reduced the suppression of ICP0 expression after 48 hours, indicating a significant reduction in viral replication. Both lonidamine monotherapy and combination therapy demonstrated superior efficacy compared to GCV monotherapy in the early stages of infection (Figures 4J-K). Our studies suggest that, compared with conventional antiviral therapies, lonidamine is a novel therapeutic agent that targets HSV-1-infected host cells, HK2, promoting a more rapid initial decline in viral load, thereby mitigating host cell damage and enhancing the overall therapeutic response. This strategy has the potential to avoid serious corneal complications such as ulceration and perforation. This breakthrough combination therapy significantly shortens disease duration and significantly reduces the risk of lasting complications, providing a more effective and comprehensive approach to treating epithelial herpetic keratitis.

[0165] The description of the above embodiments is only for understanding the method and core concept of the present application. It should be noted that, for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and such improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. Use of an inhibitor of hexokinase isoenzyme 2 in the preparation of a pharmaceutical composition for treating or preventing herpes simplex virus keratitis in an individual; Preferably, the individual includes a human and / or non-human individual; Preferably, the pharmaceutical composition is prepared for administration around the lesion, on the lesion surface, intravenous administration, topical application, corneal stroma injection, subconjunctival injection, or oral administration; Preferably, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

2. The use according to claim 1, wherein the inhibitor of hexokinase isoenzyme 2 comprises lonidamine; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary materials include adjuvants, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, and emulsifiers.

3. A pharmaceutical composition for treating or preventing herpes simplex virus keratitis, comprising a therapeutically effective amount or a prophylactically effective amount of a hexokinase isoenzyme 2 inhibitor; Preferably, the hexokinase isoenzyme 2 inhibitor comprises lonidamine.

4. The pharmaceutical composition according to claim 3, wherein the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary materials include adjuvants, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, and emulsifiers.

5. A method for treating or preventing herpes simplex virus keratitis, comprising the step of administering the pharmaceutical composition according to claim 3 or 4 to a patient suffering from the disease.

6. A method for screening a drug for treating or preventing herpes simplex keratitis for non-therapeutic purposes, the method comprising the step of applying a candidate drug to in vitro herpes simplex keratitis cells or tissues, and detecting the expression of hexokinase isozyme 2 after the candidate drug is administered; Preferably, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus; Preferably, the drug that can cause the method to detect a decrease in the expression level of hexokinase isoenzyme 2 is the screened drug.

7. The method of claim 6, further comprising detecting the extent of glycolysis or aerobic oxidation after administration of the candidate drug; Preferably, the degree of aerobic oxidation includes the rate of ATP production; Preferably, the detection of the ATP production rate includes detecting the expression levels of ATPB, UQCRC2, SDHB, and NDUFV2; Preferably, the detection of the degree of glycolysis comprises detecting the level of lactate or the level of lactate dehydrogenase; Preferably, the screened drug also reduces the degree of glycolysis or increases the degree of aerobic oxidation; Preferably, the decreased degree of glycolysis includes a decreased lactate level or an increased lactate dehydrogenase level; Preferably, the increased degree of aerobic oxidation includes an increased rate of ATP production; Preferably, the increased ATP production rate includes increased expression levels of ATPB, UQCRC2, SDHB, and NDUFV2.

8. A screening system for treating or preventing herpes simplex virus keratitis, comprising: a processing unit for applying a drug candidate to herpes simplex virus keratitis cells or tissues; a first detection unit, for detecting the expression level of hexokinase isoenzyme 2 in the herpes simplex virus keratitis cells or tissues treated by the treatment unit; Preferably, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus; Preferably, the screening system further comprises a second detection unit, the second detection unit being used to detect the degree of glycolysis or aerobic oxidation according to claim 7; Preferably, the screening system further comprises a judgment unit for obtaining a result from the detection data as to whether the candidate drug can be used as a drug for treating or preventing herpes simplex virus keratitis.

9. A screening device for treating or preventing herpes simplex virus keratitis, comprising: memory and processor; The memory is used to store program instructions; The processor is used to call program instructions, and when the program instructions are executed, is used to execute the method according to claim 6 or 7.

10. A computer-readable storage medium having a computer program stored thereon, wherein when the computer program is executed by a processor, the method for screening drugs for treating or preventing herpes simplex virus keratitis according to claim 6 or 7 is implemented.

11. A method for diagnosing herpes simplex virus keratitis, the method comprising analyzing the level of hexokinase isoenzyme 2 in a subject, wherein when the level of hexokinase isoenzyme 2 is higher than a predetermined amount, it indicates that the subject suffers from herpes simplex virus keratitis; Preferably, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

12. A method for treating herpes simplex keratitis, comprising: 1) analyzing the level of hexokinase isoenzyme 2 in a subject, wherein when the level of hexokinase isoenzyme 2 is higher than a predetermined amount, it indicates that the subject suffers from herpes simplex keratitis; 2) treating the subject with an inhibitor of hexokinase isoenzyme 2 when the subject is diagnosed with herpes simplex keratitis; Preferably, the inhibitor of hexokinase isoenzyme 2 comprises lonidamine; Preferably, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

13. Application of hexokinase isoenzyme 2 levels in the preparation of products for the diagnosis of herpes simplex virus keratitis; Preferably, the herpes simplex virus keratitis includes keratitis caused by HSV-1 virus and keratitis caused by HSV-2 virus.

Images