A method, system and device for screening ischemic stroke drugs based on GGCX

Abstract

The application discloses an ischemic stroke drug screening method, system and equipment based on GGCX. First, through a six-layer genetic evidence system such as Mendelian randomization, it is clear that the up-regulation of gamma-glutamyl carboxylase (GGCX) is the protective direction of ischemic stroke, and the genetic basis of GGCX as a drug reutilization target is established. Based on the three-dimensional structure of GGCX protein, the key residues such as active pocket ARG-108 and ASP-105 are used as docking anchor points to construct a molecular docking virtual screening model. Further, a three-dimensional comprehensive evaluation framework of'safety-mechanism consistency-clinical indication overlap' is constructed, and multi-index quantitative scoring and hierarchical sorting are carried out on candidate compounds. Through network pharmacology screening, molecular docking and comprehensive evaluation, it is confirmed that ifenprodil is the preferred conversion candidate drug from 41 candidate compounds. The application overcomes the limitation that the traditional virtual screening only depends on the docking score, and provides a systematic and reproducible method for the targeted drug screening and precise treatment of ischemic stroke.

Description

Technical Field

[0001] This invention relates to the field of computer-aided drug screening technology, specifically to a method, system, and device for screening drugs for ischemic stroke based on GGCX. Background Technology

[0002] Ischemic stroke is a leading cause of death and disability worldwide, characterized by high morbidity, high disability rate, and high mortality rate, and its treatment faces multiple challenges. The key to clinical treatment lies in initiating reperfusion therapy (such as intravenous thrombolysis or mechanical thrombectomy) as early as possible, as the improvement in neurological function is more significant. However, current reperfusion therapies have significant limitations. Intravenous thrombolysis (such as using recombinant tissue plasminogen activator rt-PA) is limited by a strict treatment time window (usually within 4.5 hours) and a high risk of hemorrhagic transformation. While mechanical thrombectomy extends the time window, it is limited by the uneven distribution of medical resources and the complexity of the surgical procedure.

[0003] Traditional drug discovery processes are lengthy, costly, and have low success rates, making it difficult to quickly respond to the demand for drug development targeting new targets. The rapid development of Computer-Aided Drug Design (CADD) technology has provided an efficient and economical solution for innovative drug development based on new targets. However, CADD does not consider the functional regulation of target proteins by candidate compounds and lacks validation of causal relationships with diseases, resulting in low clinical translation success rates. Therefore, how to integrate genetic causal evidence into the drug screening process and build more predictive screening models is a critical issue that urgently needs to be addressed.

[0004] Gamma-glutamyl carboxylase (GGCX) is the only key enzyme in the human body that catalyzes the vitamin K-dependent γ-carboxylation of glutamate. However, the role of GGCX in ischemic stroke has not been systematically elucidated, and its potential as a drug target remains to be explored. Therefore, it is necessary to develop a systematic drug screening method that integrates genetic causal validation of GGCX with computer-aided drug design techniques. Summary of the Invention

[0005] This invention provides a method, system, and device for screening drugs for ischemic stroke based on GGCX. This invention demonstrates for the first time that gamma-glutamyl carboxylase (GGCX) is significantly underexpressed in patients with ischemic stroke and is negatively correlated with stroke risk, indicating that GGCX is a protective target. Specifically, genetic causal inference (such as Mendelian randomization analysis, MR) confirms that promoting GGCX expression or increasing its activity can significantly reduce the risk of ischemic stroke. Addressing the shortcomings of existing technologies, this invention provides a computer-aided drug screening method based on GGCX for screening drugs to treat ischemic stroke.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of the present invention provides a computer-aided method for screening therapeutic drugs for ischemic stroke based on GGCX, wherein GGCX is negatively correlated with ischemic stroke, the method comprising: acquiring GGCX protein data, and screening compounds and analogues that regulate the expression level or biological activity of GGCX as candidate compounds for therapeutic drugs.

[0007] GGCX (γ-glutamyl carboxylase) includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed GGCX, as well as any form of GGCX derived from cell-processed sources. The term also encompasses naturally occurring variants of GGCX (e.g., splice variants or allelic variants). As a preferred embodiment, in this invention, GGCX is a human gene with gene ID 2677.

[0008] In this invention, the regulation includes, but is not limited to, binding to the active pocket of GGCX or affecting the key enzymatic catalytic function of GGCX, thereby intervening in GGCX-mediated biological processes. Since GGCX is negatively correlated with ischemic stroke, the above-mentioned regulatory effects can achieve a protective intervention against ischemic stroke by enhancing GGCX expression or activity, thus playing a protective role in the treatment of ischemic stroke. Therefore, compounds derived or optimized from compounds capable of regulating GGCX expression or activity can also serve as candidate compounds for the preparation of drugs for treating ischemic stroke.

[0009] In some embodiments, the compounds that regulate GGCX expression or activity include compounds that target the active pocket binding capacity of GGCX and its key enzyme activity and catalytic function.

[0010] Furthermore, the screening includes the following steps: (1) Based on the protein-compound interaction database, molecular docking technology was used to screen candidate compounds that have the ability to bind to the active pocket of GGCX protein; (2) The candidate compounds obtained in step (1) are evaluated in multiple dimensions; (3) Based on the comprehensive evaluation results of step (2), the candidate compounds are ranked in a hierarchical manner.

[0011] In some embodiments, the molecular libraries used in virtual screening mainly include the following: ZINC-22: Contains over 37 billion molecular compounds for virtual screening of small molecules. PubChem: Contains a broad range of bioactive substances for virtual screening of small molecules. DrugBank: Contains drugs and small molecules for drug design and discovery. ChEMBL: Contains small molecules for drug discovery and chemical genomics research. ChemDB: Contains a large number of known small molecules for studying the molecular interactions of compounds corresponding to biological genes and for drug discovery. HMDB: Contains a large number of known small molecules for identifying endogenous target interactions or screening metabolite mimics. BindingDB: Contains a large amount of experimentally determined binding affinity data (such as K+) between known small molecules and proteins. i， IC 50 (e.g., protein-ligand molecule binding data). SMPDB: Contains a large number of known small molecules and molecules that interact with related biological pathways, which can be used for drug discovery. In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M, and Interbioscreen to ensure the availability of candidate compounds.

[0012] Furthermore, the candidate compounds are sourced from: newly synthesized or existing databases; where existing databases include, but are not limited to, general natural product databases (COCONUT, Super Natural II, NPASS); source-specific natural product databases, including plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), traditional Chinese medicine natural product databases (CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), and marine natural product databases (MNPD, SWMD); regionally specific databases, including natural product databases from different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB); and function and activity-guided databases, including food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet). Databases; toxicology and environmental databases, including toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiCon Discovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol); structure verification databases, including MS data-based databases (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), and NMR data-based databases (NMRShiftDB, NAPROC-13); commercial supplier databases (ChemDiv, Enamine, TargetMol, InterBioScreen), etc.

[0013] Furthermore, the key structural features of the active pocket of the GGCX protein deduced by the molecular docking analysis in step (1) include: a conserved polar binding hotspot composed of the key GGCX amino acid residues ARG-108 and ASP-105, an auxiliary binding region containing the key GGCX amino acid residue ARG-453, and a hydrophobic sub-pocket enclosed by the key GGCX amino acid residues VAL-98 and PRO-102.

[0014] In some embodiments, the conservative polar binding hotspot is a component of the catalytic active center.

[0015] In some embodiments, the auxiliary binding region forms an additional anchorage through π-cation interactions.

[0016] In some embodiments, in molecular docking, affinity refers to the tightness of the binding between a molecule and its receptor. High affinity means a more stable binding, while low affinity indicates a less stable binding. Affinity is typically calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd). During molecular docking, affinity depends on the interactions between the molecule and the receptor, including hydrogen bonds, van der Waals forces, electrostatic interactions, etc. These interactions collectively determine the way the molecule and receptor bind, thus affecting affinity. To assess affinity, scoring systems or methods are commonly used to quantify the interactions between the molecule and the receptor. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type, and affinity between the molecule and the receptor.

[0017] Furthermore, the computer-aided screening process is as follows: 1) Perform molecular docking between the small molecules in the small molecule library and the active pocket of the GGCX protein and score the docked small molecules to obtain their scores; sort the scores and select the compounds with the lowest energy scores (Affinity) from the top 10 to the top 100 as candidates.

[0018] 2) Obtain the molecular structure of the GGCX protein and output the molecular structure to the pharmacophore module library for matching. Cluster all action sites based on the interaction pattern with GGCX to obtain a pharmacophore model. Input the pharmacophore model to the molecular compound database for high-throughput screening to obtain candidate drugs and generate a pharmacophore model based on receptor structure.

[0019] 3) First, obtain the structure of a known small molecule agonist that promotes GGCX expression or activity; then, based on the molecular structure of the small molecule agonist, screen to obtain a library of small molecules with similar structures; then, perform molecular docking between the library of small molecules with similar structures and the GGCX protein activity pocket to obtain the score of the docking molecules; finally, sort them to obtain candidate drugs.

[0020] 4) Small molecule compounds and similar compounds based on the GGCX protein active pocket are screened to obtain candidate drugs. The specific screening logic is as follows: screening is performed using the GGCX protein active pocket and molecular docking technology.

[0021] In some embodiments, the multidimensional assessment includes: (i) Safety assessment: Evaluate the clinical safety data and bleeding risk of the candidate compound; (ii) Mechanism consistency assessment: assess whether the regulatory direction of candidate compounds on GGCX is consistent with the direction of genetic causal inference; (iii) Clinical indication overlap assessment: assess the known or potential indications of the candidate compound.

[0022] In some embodiments, the known or potential indications include, but are not limited to, indications for cerebrovascular diseases (stroke, ischemic stroke, hemorrhagic stroke, cerebral hemorrhage), general treatments for cerebrovascular diseases (such as anticoagulation, antiplatelet therapy), indications for other diseases (such as post-traumatic stress disorder, PTSD), and general treatments for other diseases (such as drug therapy).

[0023] In some embodiments, the hierarchical sorting includes: Tier 1: Priority translation candidates, namely, candidate drugs that combine safety, consistency in mechanism direction, and overlap in clinical indications; Second level: Mechanism complement candidates, i.e., candidates that can serve as cofactors or indirect regulators of GGCX; The third level: Excluded or unverified candidates, namely candidate compounds whose regulatory direction is inconsistent with genetic inferences or whose direction is unclear.

[0024] Furthermore, activity tests were conducted on the candidate drugs: 1) For small molecule candidate drugs, surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) methods were used to determine their binding affinity to GGCX protein, and cell models with GGCX overexpression or knockdown were used to detect the promoting effect of candidate drugs on GGCX transcription or protein expression levels.

[0025] 2) For candidate drugs of protein analogs / antibodies / RNA, establish ischemic stroke cell models (such as oxygen-glucose deprivation / re-glucose re-oxygenation models) or animal models (such as middle cerebral artery occlusion models), treat with candidate drugs, and detect indicators such as neurological function scores, cerebral infarction volume, and neuronal apoptosis to screen out candidate drugs that can improve the prognosis of ischemic stroke.

[0026] A second aspect of the present invention provides a computer-aided drug screening system for ischemic stroke based on GGCX, the screening system comprising the following units: Data acquisition unit: used to acquire protein-compound interaction database data, compound database data for subsequent unit screening, and GGCX protein structure data; Molecular docking unit: used for screening candidate compounds that can bind to the active pocket of GGCX protein based on molecular docking technology; Evaluation unit: used to perform multi-dimensional evaluation of candidate compounds, including safety evaluation, mechanism consistency evaluation, and clinical indication overlap evaluation; Hierarchical ranking unit: used to rank candidate compounds based on comprehensive evaluation results.

[0027] Furthermore, the key structural features of the GGCX protein active pocket in the molecular docking unit include: a conserved polar binding hotspot composed of GGCX key amino acid residues ARG-108 and ASP-105, an auxiliary binding region containing GGCX key amino acid residue ARG-453, and a hydrophobic sub-pocket enclosed by GGCX key amino acid residues VAL-98 and PRO-102.

[0028] In some embodiments, the hierarchical sorting unit divides candidate compounds into a first level, a second level, and a third level, wherein the first level consists of priority conversion candidates, the second level consists of mechanism complementarity candidates, and the third level consists of excluded or unverified candidates.

[0029] A third aspect of the present invention provides a computer-aided drug screening device for ischemic stroke based on GGCX. The screening device includes a memory and a processor. The memory is used to store program instructions. The processor is used to call the program instructions, and when the program instructions are executed, the method described in the first aspect is implemented.

[0030] A fourth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program, when executed by a processor, implements the method described in the first aspect.

[0031] The fifth aspect of this invention provides a medicament for treating or preventing ischemic stroke, said medicament being screened by the method described in the first aspect. Specifically, this invention provides a model construction, screening process, and application of screening candidate compounds that can interact with the GGCX protein, drug screening based on the corresponding candidate compounds, and their application in drugs for ischemic stroke.

[0032] In some embodiments, the drug has a dosage form selected from: solution, suspension, emulsion, tablet, pill, powder, granule, capsule, syrup, sterile aqueous solution, non-aqueous solution, lyophilized preparation, suppository. Furthermore, it can be administered once or multiple times. In this case, the drug or its related active ingredient is administered in the form of a liquid preparation, powder, aerosol, capsule, or suppository. Routes of administration may include, but are not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, rectal, etc. When administered orally, it may be formulated with a coating to protect the active ingredient in the biological agent from degradation in the stomach. Furthermore, the active ingredient can be administered via any device capable of transfer to the target tissue. In specific embodiments, the drug or its related active ingredient provided by the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a clinician based on factors such as the subject's type, age, weight, general disease condition, and route of administration. The route of administration may include, for example, injection or any other suitable route of administration known to those skilled in the art. An injection is preferred for rapid administration during the acute phase.

[0033] In some embodiments, the drug is fenfenadil or a pharmaceutically acceptable salt thereof.

[0034] In this invention, the pharmaceutically acceptable salt or pharmaceutically usable salt refers to a salt of an active compound (such as levonidil) and is prepared by reacting the active compound with a suitable organic or inorganic acid or acid derivative. Those skilled in the art can confirm that the pharmaceutically acceptable salt has the same chemical or active properties as the active compound. Pharmaceutically usable salts include, but are not limited to, hydrochlorides, sulfates, phosphates, citrates, hydrobromides, acetates, benzoates, benzenesulfonates, tartrates, carbonates, citrates, gluconates, lactates, malates, methanesulfonates, stearates, valerates, nitrates, sodium salts, calcium salts, potassium salts, zinc salts, and meglumine salts, particularly preferably levonidil hydrochloride.

[0035] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier, specifically efendanil or a pharmaceutically acceptable salt thereof. In this invention, experiments using cell and animal models have demonstrated that efendanil has a significant affinity for GGCX protein, and in the corresponding cell models, it has been shown to upregulate GGCX protein levels, and in the corresponding animal models, it has been shown to improve manifestations such as ischemic stroke-related neurological damage. The pharmaceutically acceptable carriers are detailed in Remington's Pharmaceutical Sciences, and these substances are used as needed to aid in formulation stability or to enhance the activity or bioavailability of the active ingredient. In some embodiments, the medicament is administered to humans in a safe and effective amount. There are no particular limitations on the dosage and route of administration of the medicament; a skilled physician can usually readily determine the prescription and the dosage effective for the desired treatment and / or prevention, and the route of administration may be, for example, injection or other treatment methods.

[0036] As used herein, the term “treatment” means the complete or partial relief or reduction of a disease or symptom or disorder, or its associated symptoms, adverse effects or consequences, or phenotype. Desired therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or relief of the disease state, and reduction or improvement of prognosis. This term does not imply a complete cure of the disease or the complete elimination of any symptom or effect or all symptoms or consequences.

[0037] Advantages and beneficial effects of this invention: This invention constructs a six-layer progressive genetic evidence system consisting of "forward Mendelian randomization (MR) + SMR and HEIDI tests + Steiger directionality test + reverse MR test + subtype specificity analysis + colocalization". It establishes GGCX upregulation as a protective direction for ischemic stroke treatment from a genetic perspective, providing precise biological evidence for drug screening. Specifically, this system is the cornerstone for determining drug screening evaluation criteria from candidate compounds, providing solid genetic directionality support for GGCX as a therapeutic target (GGCX upregulation as a protective direction). Simultaneously, subtype specificity analysis confirms that the protective effect of GGCX specifically targets ischemic stroke, providing a clear direction for subsequent target validation and drug screening.

[0038] This invention proposes a screening strategy that couples the direction of regulation of a candidate compound on a target with the direction of genetic causal inference, which significantly improves the success rate of drug transformation. Specifically, this invention uses "the direction of regulation of GGCX by the candidate compound is consistent with the direction of MR causal inference" as one of the core evaluation criteria for drug screening. This is different from the traditional single-dimensional screening mode of "docking score priority". It overcomes the limitation of traditional screening models that rely only on molecular docking scores and ignore the direction of functional regulation. This helps to promote the discovery of potential drugs and the transformation based on the druggability of candidate compounds. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating a method for screening drugs for ischemic stroke based on GGCX.

[0040] Figure 2 This is a schematic diagram of a computer-aided drug screening system for ischemic stroke based on GGCX.

[0041] Figure 3 This is a schematic diagram of a computer-aided drug screening device for ischemic stroke based on GGCX.

[0042] Figure 4 This is a schematic diagram of a GGCX-based target screening and candidate drug screening process for ischemic stroke, provided as an embodiment of the present invention.

[0043] Figure 5 This invention provides evidence for Mendelian randomization causal inference of GGCX and ischemic stroke in embodiments of the present invention. Figure A shows a forest plot of the PsychENCODE brain tissue discovery dataset in stroke MR analysis results; Figure B shows a forest plot of the eQTLGen blood discovery dataset in stroke MR analysis results; Figure C shows a forest plot of the GTEx brain tissue validation dataset in stroke MR analysis results; Figure D shows the GTEx blood validation dataset; and Figure E shows forest plots of the four datasets in ischemic stroke MR analysis results.

[0044] Figure 6 The SMR / HEIDI test and Steiger directionality test results provided in this embodiment of the invention are shown in Figure A, which shows the SMR and HEIDI test results of GGCX in ischemic stroke; Figure B shows the Steiger directionality test results of GGCX in ischemic stroke.

[0045] Figure 7 The following figures illustrate the results of reverse MR and Steiger directionality tests provided in embodiments of the present invention. Figure A shows the results of reverse MR analysis of ischemic stroke and GGCX; Figure B shows the Steiger directionality test results of reverse MR analysis of ischemic stroke and GGCX.

[0046] Figure 8 The identification results of MR subtypes excluding hemorrhagic stroke provided in the embodiments of the present invention are shown. Figure A shows the MR analysis results of GGCX and cerebral hemorrhage; Figure B shows the MR analysis results of GGCX and subarachnoid hemorrhage.

[0047] Figure 9 The colocalization analysis and fine localization results provided in this embodiment of the invention are shown in Figure A. Figure B shows the colocalization analysis results for GGCX; Figure C shows the result of the sentinel variant rs1972297 of brain tissue signals being located within the GGCX gene region; Figure D shows the result of the sentinel variant SNP rs6547623 of blood signals being located within the GGCX gene region; Figure D shows the linkage disequilibrium results of the sentinel variant rs1972297 associated with brain tissue; and Figure E shows the linkage disequilibrium results of the sentinel variant rs6547623 associated with blood.

[0048] Figure 10 The molecular docking affinity ranking and binding mode / GGCX active pocket structure feature inference provided for embodiments of the present invention. Figure A shows the molecular docking results and binding parameters of the first six candidate compounds with GGCX; Figure B shows the molecular docking analysis results of the interactions between 1,5-isoquinolinediol, primidone, 2,3-dimethoxy-1,4-naphthoquinone, nilumid, fenfenidil, and apigenin with GGCX, respectively.

[0049] Figure 11 This provides a multi-dimensional comprehensive evaluation framework for embodiments of the present invention. Detailed Implementation

[0050] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0051] Figure 1 This is a flowchart illustrating the method for screening drugs for ischemic stroke based on GGCX provided by the present invention. Specifically, the method includes the following steps: 101: Obtain GGCX protein data.

[0052] In this invention, brain and blood eQTL and GWAS data are first obtained from a database, and targets for ischemic stroke are screened using the following genetic evidence system. In a specific embodiment of this invention, the following is included: 1. Forward MR causal inference and target orientation confirmation Table 1 presents the overall genome-wide association study (GWAS) data for stroke obtained from the GWAS Catalog (ebi-a-GCST90038613), which includes 6,925 clinically confirmed stroke cases and 477,673 control samples; it also presents the GWAS Catalog (ebi-a-GCST90018864) data for ischemic stroke, which includes 11,929 ischemic stroke cases and 472,192 control samples.

[0053] Table 2 presents the quantitative trait locus (eQTL) data for GGCX gene expression obtained from PsychENCODE (brain tissue), eQTLGen (blood), and GTEx v8 (brain tissue and blood). PsychENCODE included 1387 brain tissue samples covering multiple brain regions; eQTLGen included 31684 samples; and GTEx v8 came from 838 donors, encompassing 670 brain tissue samples and 175 blood samples. The study employed multiple Mendelian randomization (MR) methods, including inverse variance weighted (IVW), Wald, MR-Egger, and MR-PRESSO, to assess the causal relationship between γ-glutamyl carboxylase (GGCX) expression and stroke and its subtypes (ischemic stroke and hemorrhagic stroke).

[0054] Table 1. Sources of GWAS data and sample characteristics used in MR analysis

[0055] Table 2. Sources of eQTL data and sample characteristics used in MR analysis

[0056] GGCX and overall stroke: Results are shown Figure 5 In the four datasets of PsychENCODE brain tissue, GTEx brain tissue, eQTLGen blood, and GTEx blood, the odds ratios (ORs) for the three targets GGCX, SLC33A1, and ALDH16A1 were all <1, with P < 0.05. However, the OR direction (OR > 1) of HTR6 in the eQTLGen blood and GTEx blood datasets was inconsistent with the OR direction (OR < 1) in the PsychENCODE brain tissue and GTEx brain tissue datasets.

[0057] GGCX and ischemic stroke (the major subtype of stroke, accounting for 80% of all strokes): Results are shown in [link to results]. Figure 5E, brain tissue OR=0.916, P=0.012; blood OR=0.902, P=0.027. Notably, GGCX is the only gene that maintains a significant protective association in both brain and blood tissues, and the effect direction is completely consistent in the discovery and validation datasets of both tissues (OR<1, P<0.05). See results below. Figure 5 E, the forest plot shows a significant causal association between GGCX and ischemic stroke.

[0058] Further sensitivity analysis showed that the Cochran's QP of the GGCX blood signal was 0.197, indicating no heterogeneity; MR-Egger P was 0.821; and MR-PRESSO P was 0.652, indicating no level of pleiotropic effects.

[0059] 2. SMR and HEIDI tests and Steiger directionality test Summary-based Mendelian randomization (SMR) combined with the HEIDI test was used to rule out linkage disequilibrium (LD) artifacts. The p-values ​​(SMR) for all four independent datasets (PsychENCODE, eQTLGen, GTEx brain, and GTEx blood) were <0.05, and the p-values ​​(HEID) were >0.05, confirming that the association between GGCX expression and ischemic stroke was due to non-linkage disequilibrium. Results are shown below. Figure 6 A. The SMR and HEIDI tests exclude linkage disequilibrium spurious phenomena.

[0060] 3. Steiger Directionality Test Meanwhile, the Steiger orientation test further confirmed the causal direction as GGCX (exposure) → ischemic stroke (outcome). Results are shown below. Figure 6 B, Steiger directionality test reinforces causal orientation.

[0061] 4. Reverse MR exclusion Using significant GWAS variants in ischemic stroke as instrumental variables, we conducted a reverse causal test to examine whether ischemic stroke affects GGCX expression. Steiger directionality tests showed a FALSE causal direction for all datasets, ruling out reverse causality and further confirming the causal direction as GGCX (exposure) → ischemic stroke (outcome). Results are shown below. Figure 7 AB and reverse MR analysis ruled out the possibility that ischemic stroke affects GGCX expression.

[0062] 5. Subtype specificity analysis In one embodiment, as shown in Table 3, the subtype data of hemorrhagic stroke were obtained from the FinnGen database. This included two main subtypes: intracerebral hemorrhage (ICH, 1,224 cases / 163,533 controls) and subarachnoid hemorrhage (SAH, 1,019 cases / 163,508 controls).

[0063] Table 3. Sources and sample characteristics of GWAS data used in MR analysis of hemorrhagic stroke.

[0064] No significant association was observed between GGCX and intracerebral hemorrhage or subarachnoid hemorrhage (P>0.05), suggesting that the protective effect of GGCX is specifically targeted at ischemic stroke. See results below. Figure 8 A and B, MR subtype analysis excluded the association with hemorrhagic stroke, confirming that the protective effect of GGCX is subtype specific, that is, only for "ischemic stroke".

[0065] 6. Colocation Analysis The core purpose of colocalization analysis is to assess whether gene expression (eQTL signaling) and disease risk (GWAS signaling) within the same genomic region are driven by the same causal variant, which is crucial for the validity of MR analysis. To further elucidate the probability of genetic variants that may contribute to the potential causal relationship between GGCX and ischemic stroke, we performed Bayesian colocalization analysis, genetic sentinel variant analysis, and fine colocalization analysis.

[0066] PPH3 represents stroke risk and gene expression, but driven by different causal variants; PPH4 represents stroke risk and gene expression, driven by the same shared causal variant. This patent uses the PPH3+PPH4 ≥ 0.8 method to assess whether GGCX expression and ischemic stroke share the same causal variant. Results are shown below. Figure 9 A. In brain tissue, PPH3 + PPH4 = 0.846. In blood, PPH3 + PPH4 = 0.808.

[0067] Genetic sentinel variants provide a clear central anchor for subsequent fine mapping analysis to identify substitution variants and potential functional variants in high linkage disequilibrium with GGCX. Results are shown in [link to results]. Figure 9 B. The sentinel variant rs1972297, representing brain tissue signaling, is located near chromosome 2 at 85.8 Mb (chr2:85808573), with an allele of T / C and a minor allele frequency (MAF) of 0.4314. In the regional mapping, rs1972297 is the strongest associated site for both eQTL and GWAS signals: its eQTL... log 10A p-value exceeding 60 indicates that this variant has a highly significant effect on GGCX expression in brain tissue; simultaneously, its GWAS... log 10 The (P) value is approximately 4, corresponding to a P value of approximately 10. -4 This indicates that the variant is also statistically significantly associated with the risk of stroke.

[0068] Fine-mapping analysis further narrows down the range of candidate functional variants within the high-probability regions identified in co-mapping analysis, and assesses the potential biological functions of GGCX. Results are shown in [link to results]. Figure 9 C. The sentinel variant rs6547623 of the blood signal is also located within the GGCX gene region (chr2:85753553), with an allele of T / A and a MAF of 0.326. In the regional localization map, rs6547623 is the strongest associated site of GWAS signal in this region (GWAS). log 10 (P) is approximately 4.1), while also exhibiting a moderate level of eQTL signal (eQTL log 10 (P) is approximately 5). Results are shown below. Figure 9 D. The sentinel variant rs1972297 associated with brain tissue is in complete linkage disequilibrium with the synonymous exon variant rs1009 (164 bp away) in the coding region of the GGCX gene. See results below. Figure 9 E. The blood-associated sentinel variant rs6547623 is located in the intron region of the GGCX gene.

[0069] The results of the Bayesian colocalization, genetic sentinel variant, and fine colocalization analyses above collectively confirm that GGCX expression shares causal variants with ischemic stroke.

[0070] In summary, the six-layer genetic evidence system confirms that GGCX upregulation is a protective effect, and that the protective effect is specific to stroke subtypes, providing a clear causal direction for subsequent target validation and drug screening.

[0071] Based on the aforementioned genetic evidence confirming that GGCX upregulation is a protective mechanism against ischemic stroke, it is clear that drugs capable of upregulating GGCX gene expression levels or enhancing GGCX protein activity can serve as candidate therapeutics for ischemic stroke. Therefore, this invention further utilizes computer-aided drug design technology, based on the three-dimensional structure of the GGCX protein, to screen candidate drugs.

[0072] 102: Screening compounds and their analogues that regulate GGCX expression levels or biological activity as candidate compounds for the treatment of ischemic stroke.

[0073] In this invention, candidate compounds are screened through steps such as initial screening using network pharmacology, fine analysis using molecular docking, and hierarchical ranking. Specific steps include: 102-1: Screening candidate compounds with binding ability to the active pocket of the GGCX protein using molecular docking technology based on a protein-compound interaction database. In a specific embodiment of this invention, the following steps are included: 1. Initial screening using online pharmacology Network pharmacology screening is the initial step in drug discovery, with the core objective of narrowing down the candidate pool from a vast space of compounds. This patent, based on protein-compound interaction databases (such as DGIdb v4.2.0 and DrugBank), uses GGCX as the target protein to identify candidate compounds that may interact with GGCX, initially identifying 41 candidate compounds. The results are shown in Table 4. These compounds exhibit a broad gradient distribution in docking affinity: from the highest affinity of 1,5-isoquinolinediol (-6.571 kcal / mol) to the lowest affinity of paclitaxel (-0.213 kcal / mol), spanning an energy range of approximately 6.4 kcal / mol.

[0074] Table 4. Molecular docking results and binding parameters of 41 candidate compounds

[0075] 2. Fine analysis of molecular docking Molecular docking analysis further quantified the binding strength of candidate compounds to the active site of the GGCX protein. Based on affinity, the top six compounds were subjected to AutoDock Vina molecular docking analysis based on their docking scores.

[0076] The top six candidate compounds were: 1,5-isoquinolinediol (-6.571), primidone (-5.796), DMNQ (2,3-dimethoxy-1,4-naphthoquinone (-5.565), nilutamide (-5.497), ifenprodil (-5.367), and apigenin (-5.361). See results below. Figure 10 A shows the molecular docking results and binding parameters of the first six candidate compounds with GGCX.

[0077] From the overall structure of the docking results, the key structural features of the active pocket of the GGCX protein include: a conserved polar binding hotspot composed of the key GGCX amino acid residues ARG-108 and ASP-105, which is part of the catalytic active center; an auxiliary binding region containing the key GGCX amino acid residue ARG-453, which forms an additional anchor through π-cation interactions; and a hydrophobic sub-pocket enclosed by the key GGCX amino acid residues VAL-98 and PRO-102. (See results below) Figure 10 B shows the ranking of molecular docking affinity, binding mode, and inference of GGCX active pocket structure characteristics of the top six candidate compounds.

[0078] 102-2: Multi-dimensional evaluation of the screened candidate compounds. In a specific embodiment of the present invention, this includes: 3. Multi-dimensional comprehensive evaluation and hierarchical ranking In one embodiment, candidate compounds are evaluated in three dimensions: safety assessment (marketing status, clinical safety data, and bleeding risk assessment); mechanistic consistency assessment (whether the regulatory direction of the candidate compound on GGCX is consistent with the causal inference of MR); and clinical indication overlap assessment (whether the known or potential indications of the candidate compound overlap with ischemic stroke). Results are shown in […]. Figure 11 This demonstrates a three-dimensional comprehensive evaluation framework for seven candidate compounds.

[0079] 102-3: Based on the comprehensive evaluation results, candidate compounds are ranked in a hierarchical manner. In a specific embodiment of the present invention, this includes: Based on the comprehensive evaluation results, the candidate compounds were divided into three levels: Tier 1 (Level A, Priority Transformation Candidate): Candidate drugs with confirmed safety, consistent mechanism of action, and overlapping clinical indications. Ifenprodil is a marketed drug for the treatment of cerebrovascular disorders (a non-competitive antagonist of the NMDA receptor GluN2B subunit). It has been approved in Japan and other countries for the treatment of post-stroke sequelae, with no known bleeding risk, and a safety rating of 3 (strong). It can upregulate GGCX protein expression, which is highly consistent with the protective direction of Mendelian randomized causal inference, and its mechanism of action consistency rating is 3 (strong). Its approved indication is for sequelae of cerebrovascular disorders (such as hemorrhagic and vertigo sequelae of learning stroke). In addition, new formulations with ifenprodil as the active ingredient are undergoing clinical trials in indications such as adolescent PTSD, IPF, MS, and COVID-19, with a clinical indication rating of 2 (moderate). Its docking affinity is -5.367. The compound has a concentration of kcal / mol and binds to GGCX via a "hydrophobic framework anchoring + polar group hydrogen bonding / cationic-π assisted fixation" mechanism. The benzene ring forms a cation-π interaction with ARG-453, and the hydroxyl group forms hydrogen bonds with ASP-97 and PRO-109. The hydrophobic framework is embedded in a hydrophobic sub-pocket enclosed by VAL-98 and PRO-102. With a comprehensive score of 8, it is rated as a Grade A priority conversion candidate. Vitamin K1 / Phylloquinone is an approved vitamin drug and an essential cofactor for the GGCX catalytic reaction. It stabilizes the GGCX protein conformation, has manageable risks, and a safety score of 2 (moderate). As a natural cofactor for GGCX, its mechanism consistency score is 3 (strong). Its indications are vitamin K deficiency and anticoagulation-related bleeding, with a clinical indication score of 2 (moderate). With a comprehensive score of 7, it is also rated as a Grade A priority conversion candidate.

[0080] Tier 2 (Level B, Complementary Mechanism Candidates): Candidate drugs with a cofactor relationship or indirect regulatory association with GGCX. Apigenin / Chamomile is a natural product, still in the investigational stage, with no reported known risks (safety score 2, moderate); it shows a predictive association with GGCX but the evidence is insufficient (mechanistic consistency score 1, weak); it has only been used in healthy volunteer studies and has no disease indication (clinical indication score 0, none), with a comprehensive score of 3, it is rated Level B and requires further validation. Primidone is an approved antiepileptic drug with unknown risks (safety score 2, moderate); it shows a predictive association with GGCX and an indirect clinical association (mechanistic consistency score 1, weak); it is indicated for clopidogrel treatment of stroke or TIA (clinical indication score 1, weak), with a comprehensive score of 4, it is rated Level B. Phenobarbital is an approved antiepileptic drug with unknown risks (safety score 2, moderate); it has a predictive association with GGCX (mechanistic consistency score 1, weak); it is indicated for intracranial hemorrhage in premature infants (clinical indication score 1, weak), and its overall score is 4, thus it is rated as Grade B.

[0081] Tier 3 (Category C, Excluded or Candidates to be Verified): Candidate compounds whose regulatory direction is inconsistent with or unclear from genetic causal inference. Warfarin is an approved anticoagulant, but it has a known high risk of bleeding adverse reactions (safety score 1, weak); as a vitamin K antagonist, it indirectly inhibits GGCX catalytic activity, which is inconsistent with the protective direction of GGCX upregulation inferred by MR (mechanism consistency score 0, none); it is suitable for patients requiring anticoagulation therapy (clinical indication score 2, moderate), with a comprehensive score of 3, it is rated Category C and excluded. Anisindione is an approved synthetic anticoagulant with unknown risks (safety score 1, weak); as a vitamin K antagonist, it competitively inhibits GGCX catalytic activity, but the direction is also inconsistent (mechanism consistency score 0, none); its indication is narrow (clinical indication score 1, weak), with a comprehensive score of 2, it is rated Category C and excluded.

[0082] The above comprehensive evaluation results show that effendil performs excellently in three dimensions: safety, mechanism consistency, and clinical indications. It also has a clear molecular binding mode and target interaction mechanism, confirming it as a priority candidate drug for the treatment of ischemic stroke based on GGCX target.

[0083] Figure 2 This is a schematic diagram of the computer-aided drug screening system for ischemic stroke based on GGCX provided by the present invention.

[0084] The computer-aided drug screening system is programmed or otherwise configured to include a data acquisition unit 201, a molecular docking unit 202, an evaluation unit 203, and a hierarchical sorting unit 204.

[0085] Data acquisition unit 201: used to acquire protein-compound interaction database data and GGCX protein structure data.

[0086] Molecular docking unit 202: Used for screening candidate compounds that can bind to the active pocket of GGCX protein based on molecular docking technology.

[0087] In a specific embodiment of the present invention, the key structural features of the GGCX protein active pocket in the molecular docking unit include: a conserved polar binding hotspot composed of GGCX key amino acid residues ARG-108 and ASP-105, an auxiliary binding region containing GGCX key amino acid residue ARG-453, and a hydrophobic sub-pocket enclosed by GGCX key amino acid residues VAL-98 and PRO-102.

[0088] Evaluation unit 203: used to perform multi-dimensional evaluation of candidate compounds, including safety evaluation, mechanism consistency evaluation and clinical indication overlap evaluation.

[0089] Hierarchical ranking unit 204: used to perform hierarchical ranking of candidate compounds based on comprehensive evaluation results.

[0090] In a specific embodiment of the present invention, the hierarchical sorting unit divides candidate compounds into a first level, a second level and a third level, wherein the first level is a priority conversion candidate, the second level is a mechanism complementation candidate, and the third level is an exclusion or candidate to be verified.

[0091] Figure 3 This is a schematic diagram of the structure of the computer-aided drug screening device for ischemic stroke based on GGCX provided by the present invention.

[0092] The computer device 300 includes a processor 301 and a memory 302 coupled to the processor 301. The memory 302 stores program instructions. When the program instructions are executed by the processor 301, the processor 301 performs the steps of the GGCX-based drug screening method for ischemic stroke described in any of the above embodiments.

[0093] It should be understood that the systems, devices, and methods described in this invention can be implemented in other ways. For example, the division of modules is merely a logical functional division, and in actual implementation, there may be other division methods. For instance, multiple modules or components may be combined or integrated into another system, or some features may be ignored or not executed. The modules described as separate components may or may not be physically separated, and the components shown as modules may or may not be physical units, i.e., they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of the technical solution of this embodiment according to actual needs.

[0094] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for computer-aided screening of drugs for ischemic stroke based on GGCX, characterized in that, The GGCX is negatively correlated with ischemic stroke, and the method includes: Acquire GGCX protein data and screen compounds and their analogues that regulate GGCX expression levels or biological activities as candidate compounds for therapeutic drugs; Preferably, the compounds that regulate GGCX expression or activity include compounds that target the active pocket binding ability of GGCX and its key enzyme catalytic function.

2. The method of claim 1, wherein, The screening process includes the following steps: (1) Based on the protein-compound interaction database, molecular docking technology was used to screen candidate compounds that have the ability to bind to the active pocket of GGCX protein; (2) The candidate compounds obtained in step (1) are evaluated in multiple dimensions; (3) Based on the comprehensive evaluation results of step (2), the candidate compounds are ranked in a hierarchical manner.

3. The method according to claim 1, characterized in that, The key structural features of the active pocket of the GGCX protein, as deduced by the molecular docking analysis in step (1), include: a conserved polar binding hotspot composed of the key GGCX amino acid residues ARG-108 and ASP-105, an auxiliary binding region containing the key GGCX amino acid residue ARG-453, and a hydrophobic sub-pocket enclosed by the key GGCX amino acid residues VAL-98 and PRO-102. Preferably, the conservative polar binding hotspot is a component of the GGCX catalytic active center; Preferably, the auxiliary binding region is an additional anchoring between the candidate compound and GGCX formed through π-cation interactions; Preferably, the hydrophobic subpocket is a contact between the auxiliary candidate compound formed in the GGCX protein structural conformation and GGCX, and an interaction is formed.

4. The method according to claim 1, characterized in that, The multi-dimensional assessment includes: (i) Safety assessment: Evaluate the clinical safety data and bleeding risk of the candidate compound; (ii) Mechanism consistency assessment: assess whether the regulatory direction of candidate compounds on GGCX is consistent with the direction of genetic causal inference; (iii) Clinical indication overlap assessment: assess the known or potential indications of the candidate compound.

5. The method according to claim 1, characterized in that, The hierarchical sorting includes: Tier 1: Priority conversion candidates, namely candidate compounds that combine safety, consistency in mechanism direction, and overlap in clinical indications as subsequent drug candidates; Second level: Mechanism complement candidates, i.e. candidate compounds or substances that can serve as cofactors or indirect regulators of GGCX. The third level: Excluded or unverified candidates, namely candidate compounds whose regulatory direction is inconsistent with genetic inferences or whose direction is unclear.

6. A computer-aided drug screening system for ischemic stroke based on GGCX, characterized in that, The screening system includes the following units: Data acquisition unit: used to acquire protein-compound interaction database data and GGCX protein structure data; Molecular docking unit: used for screening candidate compounds that can bind to the active pocket of GGCX protein based on molecular docking technology; Evaluation unit: used to perform multi-dimensional evaluation of candidate compounds, including safety evaluation, mechanism consistency evaluation, and clinical indication overlap evaluation; Hierarchical ranking unit: used to rank candidate compounds based on comprehensive evaluation results.

7. The screening system according to claim 6, characterized in that, The key structural features of the GGCX protein active pocket in the molecular docking unit include: a conserved polar binding hotspot composed of GGCX key amino acid residues ARG-108 and ASP-105, an auxiliary binding region containing GGCX key amino acid residue ARG-453, and a hydrophobic sub-pocket enclosed by GGCX key amino acid residues VAL-98 and PRO-102. Preferably, the hierarchical sorting unit divides candidate compounds into a first level, a second level, and a third level, wherein the first level consists of priority conversion candidates, the second level consists of mechanism complementarity candidates, and the third level consists of excluded or unverified candidates.

8. A computer-aided drug screening device for ischemic stroke based on GGCX, characterized in that, The screening device includes a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, and when the program instructions are executed, to implement the method described in any one of claims 1-5.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the method described in any one of claims 1-5.

10. A drug for treating or preventing ischemic stroke, characterized in that, The drug is obtained by screening using the method according to any one of claims 1-5; Preferably, the drug is fenfenadil or a pharmaceutically acceptable salt thereof.

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