Anti-cataract compound based on cryab structure and screening method thereof

Abstract

The application discloses an anti-cataract compound based on a CRYAB structure and a screening method thereof, and relates to the technical field of drug screening. The method comprises the following steps: obtaining a crystal structure of CRYAB, and establishing a molecular docking screening model; setting an active pocket; obtaining a plurality of compounds with docking scores below-24 and different structures; and screening out the anti-cataract compound based on CRYAB. Through the structure-based virtual screening technology, the lens protein CRYAB is taken as a target protein, and the compounds with docking scores below-24 and different structures are screened out from the ZINC Database through molecular docking. The problems of complicated work and high cost in the cataract drug screening work are solved.

Description

Technical Field

[0001] This invention relates to the field of drug screening technology, specifically to an anti-cataract compound based on the CRYAB structure and its screening method. Background Technology

[0002] Currently, there are a variety of marketed anti-cataract drugs or compounds with reported anti-cataract activity, which can be mainly divided into the following categories: anti-oxidative stress drugs, anti-quinone accumulation drugs, aldose reductase inhibitors, and anti-lens protein aggregation drugs, as shown in Table 1.

[0003] Table 1. Reported anticataract drugs or active compounds

[0004]

[0005]

[0006]

[0007] Antioxidant stress-reducing drugs, such as carotenoids like lutein and zeaxanthin, vitamins E / C, glutathione, and L-cysteine, have attracted considerable attention for their role in regulating cataract development, partly due to their relatively low cost and availability in daily diets. Additionally, a Ganoderma lucidum extract—ganoderic acid A—has been reported to inhibit apoptosis and oxidative stress in lens epithelial cells, thus delaying lens opacity. Numerous studies have confirmed the positive effects of these antioxidants in slowing cataract progression; however, their effectiveness as human anti-cataract drugs remains controversial due to a lack of conclusive clinical trials.

[0008] Antiquinone accumulation drugs, such as cataractin (pirenoxine sodium) eye drops, have been used since 1958 to prevent early cataracts. They competitively inhibit the binding of quinones to the sulfhydryl groups of lens proteins, thus preventing lens protein denaturation. Furthermore, cataractin (pirenoxine sodium) eye drops effectively prevent ultraviolet radiation, selenite, and calcium-induced lens opacities, especially in the early stages. Additionally, phacolysin eye drops, marketed as Phacolisin (Japan), Quinax (USA), or Lutrax, have been used clinically since the 1980s. As a proteolytic enzyme activator, phacolysin activates β-protease to break down denatured proteins and competitively inhibits the binding of quinones to soluble proteins in the lens, thereby treating cataracts. However, production has been discontinued due to safety concerns. Some clinical trial evidence suggests that these drugs can prevent the early development of cataracts, but are ineffective in patients with advanced cataracts.

[0009] Aldose reductase inhibitors, such as Shapusi (benzyl lysine) eye drops, have been marketed as a cataract medication for many years to prevent early age-related cataracts and slow the progression of cataracts. However, they may be accompanied by adverse reactions such as transient burning sensation and tearing in the early stages of use, and their true therapeutic effect remains controversial. Recent studies have shown that the novel aldose reductase inhibitor diosgenin can significantly reduce the permeation and expansion of lens epithelial cells and delay the progression of galactosemia-induced cataracts in rats. Fereshteh et al. established a rat model of diabetic cataracts using streptozotocin (STZ) and treated it with L-lysine, finding that L-lysine could inhibit α-crystal protein glycosylation and aggregation, thereby suppressing catalase-induced aggregation and cataract formation in diabetic rats in vitro. Soni et al. discovered that a novel isoflavone isolated from Alstonia scholaris can prevent cataract formation by reducing lens oxidative stress in systemic hypertension and diabetes.

[0010] Anti-lens protein aggregation drugs, such as 25-HC and lanosterol, reported in Science and Nature in 2015 respectively, can prevent and reverse lens protein aggregation, significantly improving lens transparency and are considered a new hope for cataract treatment. Subsequently, a series of lanosterol derivatives were synthesized based on the structure of lanosterol. It was found that compound 6' (hereinafter referred to as compound 6' to distinguish it from compound 6 obtained by virtual screening in this invention) with lanosterol backbone and 25-HC side chain has good efficacy in depolymerizing aggregates formed by overexpression of CRYAB (R120G) in human lens epithelial cells. The 2-fluorination of this compound (compound 35) further enhances the depolymerization effect. Additionally, there are reports that rosmarinic acid can restore lens transparency; gold nanorods or gold micro-roses coated with levodopa and aspirin-formed nanorods can act as regulatory compounds, inhibiting and depolymerizing both α-lens protein model peptides and natural α-lens protein aggregation; fusidic acid (FA), when directly treated with human cataract lens samples, can depolymerize amyloid fibrils to some extent. While levodopa and aspirin have good solubility, current reports only indicate depolymerization effects at the protein level. Compared to the previous three classes of drugs or compounds, anti-lens protein aggregation drugs, although more direct and obvious in their effects, have limited structural options for 25-HC, lanosterol and its derivatives (compounds 6' and 35), and fusidic acid, as they are all sterol structures with low solubility and limited structural modification. Summary of the Invention

[0011] To address the shortcomings of the aforementioned background technologies, this invention focuses on the scarcity of anti-cataract drugs, the limited efficacy of existing marketed anti-cataract drugs, and the fact that reported anti-cataract compounds have simple structures, mostly sterol structures (such as 25-HC, lanosterol, compound 6', and compound 35), limiting further structural modification and exhibiting deficiencies in solubility, stability, and ocular penetration. Using 25-HC and lanosterol as positive controls, this invention employs virtual screening based on the CRYAB structure, thermal stability analysis, and solubility analysis to screen for active compounds with strong apparent activity, high affinity, and high solubility. In vitro aggregation and depolymerization experiments of CRYAB (R120G) were conducted, and the compounds were used to treat a sodium selenite-induced SD rat cataract model in vivo. The activity of these compounds was evaluated, and an active anti-cataract compound with strong affinity, good solubility, and a structure different from sterols was screened.

[0012] To achieve the above objectives, the first objective of this invention is to provide a method for screening anti-cataract compounds based on the CRYAB structure, comprising the following steps:

[0013] Obtain the crystal structure of CRYAB and establish a molecular docking screening model;

[0014] After the screening model is locally minimized in its internal coordinate space using the conjugate gradient algorithm and derived analytical tools, hydrogen atoms and heavy metal atoms are added to the receptor structure to set up the active pocket.

[0015] Virtual screening was performed by docking the active pocket with compounds in the ZINC Database, and docking scores were assigned to obtain a variety of compounds with docking scores below -24 and different structures.

[0016] Multiple compounds selected through virtual screening were analyzed for affinity, thermal stability, and solubility with CRYAB and / or CRYAB mutants to identify CRYAB-based anti-cataract compounds.

[0017] The second objective of this invention is to provide an anti-cataract compound based on the CRYAB structure.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention provides an anti-cataract compound based on the CRYAB structure and a screening method thereof. This method utilizes structure-based virtual screening technology, targeting the lens protein CRYAB, and employs molecular docking to virtually screen compounds with docking scores below -24 and diverse structures from the ZINC Database. First, the affinity between the compound and CRYAB / CRYAB(R120G) is measured to verify the reliability of the screening results. Further, the Tm values ​​of CRYAB / CRYAB(R120G) in systems with different compounds are measured. Finally, combined with the CLogP values ​​of the compounds, novel cataract drug candidates with strong affinity and high solubility are screened. This method solves the problems of tedious workload and high cost in cataract drug screening. Attached Figure Description

[0020] Figure 1 Changes in ThT fluorescence intensity during aggregation (A) or depolymerization (B) experiments of CRYAB (R120G). *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 2After treatment, the degree of lens opacity and protein content were observed in SD rats. (A) Images of the lens of SD rats recorded under a stereomicroscope. (B) Area of ​​the opaque region of the lens of SD rats estimated using ImageJ. (C) Detection of soluble protein content. nsp>0.05, ***p<0.001.

[0022] Figure 3 Antioxidant capacity of the lens of SD rats after treatment with different compounds. (A) SOD activity; (B) CAT activity; (C) GSH & GSSG activity; (D) MDA content. nsp>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<1×10 -4 .

[0023] Figure 4 The virtual screening model was validated using positive compounds (lower docking scores indicate better performance). (A) Binding of 25-HC to CRYAB, docking score -5.46. (B) Binding of lanosterol to CRYAB, docking score -4.20.

[0024] Figure 5 (A) Tm values ​​of CRYAB in different compound systems (3σ = 0.78℃); (B) Tm values ​​of CRYAB (R120G) in different compound systems (3σ = 2.25℃).

[0025] Figure 6 This represents the CLogP value of the compound. Detailed Implementation

[0026] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0027] This invention addresses the scarcity of anti-cataract drugs, the limited efficacy of existing marketed anti-cataract drugs, and the fact that reported anti-cataract compounds have simple structures, mostly sterol structures (such as 25-HC, lanosterol, compound 6', and compound 35), limiting further structural modification and exhibiting deficiencies in solubility, stability, and ocular penetration. Using 25-HC and lanosterol as positive controls, this invention employs virtual screening based on the CRYAB structure, thermal stability analysis, and solubility analysis to screen for active compounds with strong apparent activity, high affinity, and high solubility. In vitro aggregation and depolymerization experiments of CRYAB(R120G) were conducted, and the compound was used to treat a sodium selenite-induced SD rat cataract model in vivo to evaluate its activity. This resulted in the screening of an active anti-cataract compound with strong affinity, good solubility, and a structure different from sterols. It should be noted that CRYAB(R120G) represents the CR YAB mutant.

[0028] The first aspect of this invention provides a method for screening anti-cataract compounds based on the CRYAB structure, comprising the following steps:

[0029] Obtain the crystal structure of CRYAB and establish a molecular docking screening model;

[0030] After the screening model is locally minimized in its internal coordinate space using the conjugate gradient algorithm and derived analytical tools, hydrogen atoms and heavy metal atoms are added to the receptor structure to set up the active pocket.

[0031] Virtual screening was performed by docking the active pocket with compounds in the ZINC Database, and docking scores were assigned to obtain a variety of compounds with docking scores below -24 and different structures.

[0032] Multiple compounds selected through virtual screening were analyzed for affinity, thermal stability, and solubility with CRYAB and / or CRYAB mutants to identify CRYAB-based anti-cataract compounds.

[0033] During the docking process, the ECEPP3 potential, mmff partial charge, and energy of each molecule are calculated and minimized to represent the fraction of the binding strength between the ligand and the acceptor. Finally, the fraction of the ligand with the optimal conformation is selected as the ligand fraction.

[0034] When obtaining multiple compounds from the ZINC Database through virtual screening, the selected compounds are eliminated based on the modified Lipinski five-rule system, with the following conditions set: molecular weight < 650; number of hydrogen bond donors < 5; number of hydrogen bond acceptors < 10; lipid-water partition coefficient < 9; number of rotatable bonds ≤ 10.

[0035] A virtual screening was performed using the ZINC Database, selecting 11 compounds with docking scores below -24, diverse structures, and structures different from those of the positive compounds. Their structural formulas are as follows:

[0036]

[0037]

[0038] Among them, affinity K was screened by analyzing affinity, thermal stability, and solubility. D ≤5.358×10 -5 mol / L, apparent ΔT m ≥2℃, solubility characterization parameter CLogP (化合物) ≤CLog P (25-HC) It is a novel anti-cataract compound with a skeleton that differs from that of sterols.

[0039] According to the present invention, during the virtual screening process, compounds are preliminarily screened by analyzing affinity, thermal stability and solubility; then, from the preliminarily screened compounds, compounds that reduce the apparent Tm of CRYAB and / or CRYAB mutants and have the strongest affinity or the highest solubility are selected for in vivo and in vitro activity evaluation to screen out CRYAB-based anti-cataract compounds.

[0040] The molecular docking screening model is a CRYAB screening model containing a molecular binding pocket.

[0041] The crystal structure of CRYAB is derived from the PDB protein structure database, PDB ID: 2WJ7.

[0042] A second aspect of the present invention provides an anti-cataract compound based on the CRYAB structure.

[0043] The compound includes

[0044] In one embodiment, a method for screening anti-cataract compounds based on the CRYAB structure includes the following steps:

[0045] (1) Establish a virtual screening model

[0046] The crystal structure of human CRYAB (PDB: 2WJ7) was selected, and a molecular docking screening model was established using the molecular docking software Molsoft ICM (version 3.9-1d) for verification of the reliability of step (2) and virtual screening of step (3). After local minimization using the conjugate gradient algorithm and analytical derivatives in the internal coordinate space, hydrogen atoms and heavy metal atoms were added to the receptor structure. The binding pocket of the protein (receptor) was displayed using a grid, and each compound (ligand) in the database was docked with the receptor's binding pocket. Molsoft ICM performed molecular docking using flexible ligand-receptor, calculating the ECEPP3 potential, mmff partial charge, and energy minimization for each molecule. The fraction representing the "binding strength" between the ligand and receptor was calculated, and the optimal conformation of the ligand was ultimately selected as the ligand's score.

[0047] (2) Reliability verification of the virtual screening model

[0048] The reliability of the model established in step (1) was verified using lanosterol and 25-hydroxycholesterol, two compounds that have been reported in the literature to have therapeutic effects on cataracts.

[0049] (3) Perform virtual screening from the compound library

[0050] Using a compound database as the screening target, molecular docking software Molsoft ICM (version 3.9-1d) was used for screening based on the molecular docking screening model established in step (1). To ensure the discovery of active compounds, the screened compounds were eliminated based on the modified Lipinski five rules for drug class, with the following conditions set: ① molecular weight < 650; ② number of hydrogen bond donors < 5; ③ number of hydrogen bond acceptors < 10; ④ lipid-water partition coefficient < 9; ⑤ number of rotatable bonds ≤ 10. The compounds obtained from the virtual screening were used for affinity determination in step (4), thermal stability analysis in step (5), and solubility assessment in step (6).

[0051] (4) Determine the affinity of the compound for CRYAB or its mutants.

[0052] The affinity of the compounds obtained from the virtual screening in step (3) to CRYAB or its mutant CRYAB (R120G) was determined using a protein interaction workstation (Biacore X100).

[0053] (5) Determine the Tm of CRYAB or its mutants.

[0054] The thermal stability of CRYAB or its mutants was analyzed by differential scanning fluorescence in a real-time PCR instrument, and the Tm value of the system containing CRYAB or its mutants obtained from the virtual screening in step (3) was determined.

[0055] (6) Solubility assessment of compounds

[0056] The CLogP value of the compounds obtained from the virtual screening in step (3) was calculated using Chem BioDraw Ultra 14.0 software. The value reflects the solubility of the compounds.

[0057] In this embodiment, during the screening process, the effectiveness of the docking screening model provided by the present invention is verified using existing compounds 25-HC and lanosterol, as detailed below:

[0058] (1) Virtual screening model

[0059] Based on the crystal structure of human CRYAB (PDB:2WJ7), a molecular docking screening model was established using molecular docking software. The model was then validated using compounds 25-HC and lanosterol, both reported in the literature. The results are as follows: Figure 4 As shown, the docking score of 25-HC with CRYAB was -5.46, and the docking score of lanosterol with CRYAB was -4.20, demonstrating the reliability of the established screening model. This invention establishes a reliable structure-based virtual screening model for anti-cataract drugs.

[0060] (2) Virtual screening of compounds

[0061] Using the validated screening model described above, a virtual screening was conducted from the compound library, selecting 11 compounds with docking scores below -24, diverse structures, and structures different from the positive compounds. Their structural formulas, molecular formulas, CAS numbers, and docking scores are shown in Table 2. This invention has identified potentially effective compounds for cataract treatment.

[0062] Table 2. Compounds with docking scores below -24 from virtual screening.

[0063] (Note: The compounds used in this method for virtual screening include, but are not limited to, those listed in Table 2.)

[0064]

[0065]

[0066]

[0067] (3) Affinity test

[0068] The affinity of the above 11 compounds for CRYAB / CRYAB(R120G) was determined by surface plasmon resonance (SPR) using a protein interaction workstation (Biacore X100). The results are shown in Table 3. The measured affinity of CRYAB(R120G) for 25-HC was (1.22 ± 0.14) × 10⁻⁶. -5 This is basically consistent with the literature reports. D =1.01±0.44)×10 -5 These 11 compounds (mol / L) all showed binding affinity to CRYAB / CRYAB(R120G), experimentally verifying the reliability of the virtual screening results.

[0069] Table 3. Affinity of CRYAB / CRYAB(R120G) to the compound as determined by SPR.

[0070]

[0071]

[0072] (4) Thermal stability analysis

[0073] The thermal stability of CRYAB / CRYAB(R120G) was analyzed by differential scanning fluorometry in a real-time quantitative PCR instrument, and its Tm value was determined. This was performed according to Protein Thermal Shift. TM The Dye Kit instructions first add 5 μL of Protein Thermal Shift to the eight-tube pack. TM Add 12.5 μL of the compound and a CRYAB / CRYAB (R120G) mixture to the buffer, and finally add 2.5 μL of Protein Thermal Shift. TM Dye(40×) was applied to achieve a final compound concentration of 40 μM, a final CRYAB / CRYAB(R120G) concentration of 25 μM, and a Protein Thermal Shift. TMThe final concentration of dye was 5×. After centrifuging the eight-tube sample pack, the samples were placed in a real-time quantitative PCR instrument (Quant Studio5). The Experiment Type was set to MeltCurve, the Chemistry to Other, and a 20 μL system was used. The temperature was increased from 25℃ to 99℃ in increments of approximately 1℃ / min. The Melt Curve Filter was set to x1m3, and the Plate Attributes to None. After setting up the program, the process was started. The data was imported into Protein Thermal Shift Software 1.4 for analysis to obtain the Tm value of CRYAB / CRYAB(R120G) in the presence of each compound.

[0074] like Figure 5 As shown, compounds 2 and 5 reduced the Tm of CRYAB to below three standard deviations (3σ = 0.78℃) of the control group (62.30℃), specifically 61.26℃ and 60.28℃, respectively. Meanwhile, the Tm of CRYAB (R120G) was reduced to below three standard deviations (3σ = 2.25℃) of the control group (62.38℃) by compounds 1, 3, 4, 5, and 10, specifically 59.38℃, 59.74℃, 59.57℃, 59.01℃, and 59.52℃, respectively.

[0075] (5) Comparison of solubility

[0076] The CLogP values ​​of reported and screened compounds were calculated using Chem BioDraw Ultra 14.0 software, such as... Figure 6 As shown. Among these compounds, lanosterol had the highest CLogP value (10.593), while compound 35 had a CLogP value 2.399 lower than lanosterol, indicating that the calculation method is reliable. Of the 11 screened compounds, compound 3 had the lowest CLogP value (-2.163), and compound 5 had the highest (5.212), but both were lower than the CLogP values ​​of 25-HC (7.313) and lanosterol (10.593). These results indicate that the solubility of the 11 screened compounds is higher than that of 25-HC and lanosterol.

[0077] In summary, this invention has screened out CRYAB-based anti-cataract compounds with strong affinity, high activity, high solubility, and novel skeletons.

[0078] It should be noted that the screening of active compounds in this invention mainly includes:

[0079] (1) Virtual screening of active compounds: A screening model was established based on the crystal structure of CRYAB (PDB:2WJ7), and active compounds were virtually screened from the compound library for the following steps (2) verification of the reliability of the virtual screening results, (3) thermal stability analysis and (4) solubility assessment.

[0080] (2) Verify the reliability of the virtual screening results: The affinity of the compounds obtained from the virtual screening in step (1) to CRYAB or its mutant CRYAB (R120G) was determined using a protein interaction workstation (Biacore X100).

[0081] (3) Thermal stability analysis: The thermal stability of CRYA B or its mutants was analyzed by differential scanning fluorescence in a real-time PCR instrument. The Tm value of the system containing the compound obtained from the virtual screening in step (1) was determined.

[0082] (4) Solubility analysis: The CLogP value of the compounds obtained by virtual screening in step (1) was calculated using Chem BioDraw Ultra 14.0 software. The value reflects the solubility of the compounds.

[0083] (5) Select compounds that reduce the apparent Tm of CRYAB or CRYAB(R120G) and have the strongest affinity or highest solubility for in vitro and in vivo activity evaluation. 25-HC and lanosterol were used as positive controls in the screening process.

[0084] To further illustrate the anti-cataract activity of the compounds screened in this invention, the compounds were evaluated in vitro and in vivo. Specifically, compounds that reduced the apparent Tm of CRYAB or CRYAB(R120G) and had the strongest affinity or highest solubility for CRYAB were selected for in vitro and in vivo activity evaluation. 25-HC and lanosterol were used as positive controls in the screening process.

[0085] In vitro evaluation: Aggregation and depolymerization of CRYAB (R120G)

[0086] (1) Solution preparation: Dilute CRYAB (R120G) with 1×PBS to a solution of 0.2-1 μg / μL and use it in steps (2) and (3) below; prepare a 1-10 mM stock solution with DMSO for positive compounds or screening compounds and use it in steps (2) and (3) below; dissolve ThT in DMSO to prepare a 50 mM stock solution and use it in steps (2) and (3) below.

[0087] (2) Aggregation experiment: Add the stock solution of the positive compound or the selected compound from step (1) to the CRYAB (R120G) dilution solution from step (1) to make the final concentration 50-500 μM. Use DM SO as a control and incubate at 20-40°C with shaking at 150-300 rpm for 20-60 min. Then use it in step (4).

[0088] (3) Depolymerization experiment: Place the CRYAB (R120G) dilution solution from step (1) at 20-40℃ and shake at 150-300 rpm for 20-60 min. Then add the stock solution of the positive compound or the selected compound from step (1), with DMSO as the control, and continue to shake and incubate at 20-40℃ and 150-300 rpm for 36-60 h. Then use it in step (4).

[0089] (4) Fluorescence measurement: Dilute the 50mM ThT solution in step (1) to 2mM, add it in equal volume to the mixed solution after incubation in step (2) or (3) in a dark environment, vortex mix, and dispense 200μL into a 96-well plate. Use 1×PBS as a blank control, and measure the fluorescence intensity at excitation and emission wavelengths of 440nm and 480nm, respectively, using a microplate multifunction detector.

[0090] In vivo evaluation: Evaluation of the therapeutic effect and efficacy of sodium selenite-induced cataract in SD rats;

[0091] Establishment and treatment of sodium selenite-induced cataract model in SD rats:

[0092] (1) Solution preparation: Sodium selenite was prepared into a 20mM stock solution with physiological saline and diluted to 4mM with physiological saline before use in step (2); the positive compound and the active compound were prepared into a 5mM solution with cyclodextrin solution and used in step (3).

[0093] (2) Establishment of cataract animal model: 11-day-old SD rats were selected and injected subcutaneously with 4mM Na2SeO3 saline solution in the neck and back. The injection volume was 20nmol / g (body weight) to establish the cataract animal model, which was used in step (2). The normal group was injected with the corresponding volume of saline.

[0094] (3) Treatment of cataract animal models: Starting from the 12th day of age (after opening the eyes), 5 μL of 5 mM saturated β-cyclodextrin solution of positive compound or active compound was instilled into the eyes of each juvenile mouse in step (1) twice a day for 2 weeks.

[0095] Lens opacity and soluble protein content:

[0096] (1) Compound tropicamide eye drops were used to dilate the pupils of SD rats in each group, twice, with an interval of 10 min.

[0097] (2) Anesthetize the patient by injecting 500 μL of 20% maltodextrin solution and take a picture under a stereomicroscope;

[0098] (3) Use ImageJ software to delineate the opaque areas of the lens and calculate their area;

[0099] (4) After lens removal, the protein content in the lens was measured using a protein content assay kit. Lens antioxidant system parameter detection:

[0100] (1) SOD activity detection: SOD activity in rat lens was detected using an SOD activity detection kit.

[0101] (2) CAT activity detection: CAT activity in rat lenses was detected using a CAT activity detection kit.

[0102] (3) GSH & GSSG activity detection: The GSH and GSSG activity in rat lenses was detected using a GSH & GSSG activity detection kit.

[0103] (4) MDA content detection: The MDA content in the lens of rats was detected using an MDA content detection kit.

[0104] In in vitro and in vivo evaluations, the effects of selecting compounds that reduce the apparent Tm of CRYAB or CRYAB(R120G) and have the strongest affinity or highest solubility for in vitro and in vivo activity evaluations are explained as follows:

[0105] (1) Screening of active compounds

[0106] Active compounds with docking scores below -24 were virtually screened using the molecular docking software Molsoft ICM (version 3.9-1d). The affinity of the compounds for CRYAB or CRYAB(R120G) was determined by surface plasmon resonance (SPR), validating the reliability of the virtual screening results. The results showed that all compounds could bind to CRYAB or CRYAB(R120G). Thermostability analysis of CRYAB or CRYAB(R120G) was performed using differential scanning fluorometry (DSF) to screen compounds with apparent activity. The CLogP values ​​of the reported and screened active compounds were calculated using Chem BioDraw Ultra 14.0 software to compare their solubility. Compounds that lowered the apparent Tm of CRYAB or CRYAB(R120G) and exhibited the strongest affinity or highest solubility were selected for in vitro and in vivo activity evaluation. The screened active compounds are shown in Table 4.

[0107] Table 4. Positive compounds and active compounds obtained from screening.

[0108]

[0109]

[0110] (2) Evaluation of aggregation and deaggregation of CRYAB (R120G)

[0111] After co-incubating the compound with CRYAB(R120G) for 30 min or the CRYAB(R120G) aggregate with the compound for 48 h, ThT was added to measure the fluorescence intensity of the system. The results are as follows: Figure 1 As shown in the figure. In the aggregation experiment, compared with the control group, the fluorescence intensity of the 25-HC, lanosterol, and compound 3 and 4 treatment groups decreased by 5.86%, 3.84%, 5.89%, and 5.58%, respectively, while the fluorescence intensity of the compound 5 treatment group increased by 17.27%, which was statistically significant. In the depolymerization experiment, compared with the control group, the fluorescence intensity of the 25-HC, lanosterol, and compound 3 and 4 treatment groups decreased by 4.69%, 8.57%, 29.18%, and 29.98%, respectively, which was statistically significant, while the fluorescence intensity of the compound 5 treatment group increased by 0.12%, which was not statistically significant. The positive compound showed a significant effect in preventing or reversing CRYAB(R120G) aggregation. Compounds 3 and 4 also prevented or reversed CRYAB(R120G) aggregation, and their effect in reversing CRYAB(R120G) aggregates was more significant than that of the positive compound. However, compound 5 promoted the formation of aggregates, indicating that compounds 3 and 4 may be potentially active compounds with therapeutic effects.

[0112] (3) Assessment of lens opacity and determination of soluble protein content

[0113] After treatment, the lens opacity of the SD rats was observed under mydriasis. The observation and photography were performed using a stereomicroscope, and the results are as follows: Figure 2 As shown in (A), the area of ​​the turbid region is calculated using ImageJ, as follows. Figure 2As shown in (B), compared to the model group, the area of ​​the opaque region of the lens in the 25-HC, lanosterol, and compound 4 treatment groups decreased by 24.84%, 27.27%, and 26.34%, respectively, showing statistically significant differences. The area of ​​the opaque region of the lens in the compound 3 and compound 5 treatment groups decreased by 1.12% and increased by 8.64%, respectively, with no statistically significant difference. Compounds 3 and 5 did not significantly change the degree of lens opacity, while compound 4 and the positive control compound significantly delayed Na2SeO3-induced lens opacity in SD rats, with similar effects. Furthermore, the content of soluble and insoluble proteins in the lens of SD rats after treatment with different compounds was determined using a BCA protein content assay kit, and the percentage of soluble protein content was calculated. The results are shown below. Figure 2 As shown in (C), compared with the model group, the percentage of soluble protein content in the 25-HC, lanosterol, and compound 3, 4, and 5 treatment groups increased by 25.53%, 20.50%, 20.37%, 22.65%, and 14.18%, respectively, showing statistical differences. However, there was no significant difference in the percentage of soluble protein content between the compound 4 treatment group and the 25-HC treatment group. The increase in soluble protein content indicates that the compound at least bound to CRYAB and stabilized its soluble state, partially preventing or reversing the aggregation of lens proteins, thereby preventing or delaying the development of cataracts.

[0114] (4) Determination of the antioxidant capacity of the lens

[0115] SOD activity in lens samples from SD rats treated with different compounds was determined using a SOD activity assay kit. The results are as follows: Figure 3 As shown in (A), compared with the model group, the SOD activities of the 25-HC lanosterol and compound 3, 4, and 5 treatment groups increased by 35.92%, 20.86%, 28.42%, 40.63%, and 31.02%, respectively, showing statistically significant differences. The SOD activity of the compound 4 treatment group was not significantly different from that of the 25-HC treatment group, but was significantly higher than that of the lanosterol treatment group. The CAT activity in the lens of SD rats after treatment with different compounds was measured using a CAT activity assay kit, and the results are as follows: Figure 3 As shown in (B), compared to the model group, the CAT activities of the lanosterol, compound 3, 4, and 5 treatment groups increased by 67.96%, 78.20%, 73.63%, and 59.91%, respectively. The CAT activities of the compound 3 and 4 treatment groups were 31.96% and 17.69% higher than those of the lanosterol treatment group, respectively, showing statistically significant differences. The GSH and GSSG activities in the lens of SD rats treated with different compounds were measured using a GSH and GSSG activity assay kit. The results are as follows: Figure 3As shown in (C), compared with the model group, the total glutathione (GSH+GSSG) activities in the 25-HC, lanosterol, and compound 3, 4, and 5 treatment groups increased by 90.19%, 89.34%, 87.89%, 89.56%, and 84.11%, respectively, showing statistically significant differences. Compound 4 showed no significant difference compared with the lanosterol treatment group. The MDA content in the lens of SD rats after treatment with different compounds was determined using an MDA content assay kit, and the results are as follows: Figure 3 As shown in (D), compared to the model group, the MDA content in the 25-HC, lanosterol, and compound 3 and 4 treatment groups decreased by 30.54%, 18.47%, 32.14%, and 22.66%, respectively, showing statistically significant differences; the MDA content in the compound 5 treatment group decreased by only 1.38%, with no statistically significant difference. The positive compound and compounds 3, 4, and 5 can all enhance the antioxidant capacity of the lens.

[0116] In general, positive compound and compounds 3, 4 and 5 can improve the antioxidant capacity of the lens, but compound 3 did not significantly improve the opacity of the lens, compound 5 significantly aggravated the opacity of the lens, and only compound 4 alleviated or inhibited the development of cataracts, with effects similar to those of positive compound.

[0117] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0118] Example 1

[0119] The therapeutic effects of positive compounds were studied using ThT fluorescence assays and animal models of cataracts.

[0120] First, the positive compounds (25-HC and lanosterol) were dissolved in DMSO to prepare a 1 mM stock solution. CRYAB (R120G) was diluted with 1×PBS to a 0.5 μg / μL solution. ThT was dissolved in DMSO to prepare a 50 mM stock solution. In the aggregation experiment, the positive compound solution was added to the CRYAB (R120G) dilution to a final concentration of 200 μM and incubated at 37°C and 220 rpm for 30 min. In the depolymerization experiment, the CRYAB (R120G) dilution was incubated at 37°C and 220 rpm for 30 min, then the positive compound solution was added to a final concentration of 200 μM and incubated at 37°C and 220 rpm for 48 h. The 50 mM ThT solution was diluted to 2 mM and added to the incubated mixture in an equal volume in the dark. After vortexing and mixing, the mixture was dispensed into 200 μL portions in a 96-well plate. 1×PBS was used as a blank control. The fluorescence intensity at excitation and emission wavelengths of 440 nm and 480 nm was detected.

[0121] A cataract model was established in 11-day-old SD rats by subcutaneous injection of 4 mM Na2SeO3 saline solution at a concentration of 20 nmol / g (body weight) into the neck and back. After eye opening, 5 μL of 5 mM β-cyclodextrin solution (a positive compound) was instilled into each eye of the SD rats twice daily for two weeks. At the end of treatment, compound tropicamide eye drops were instilled twice at 10-minute intervals to dilate the pupils. After anesthesia with 500 μL of 20% maltose solution injected intraperitoneally, the degree of lens opacity was observed under a stereomicroscope. Following the removal of vitreous lens tissue, the content of soluble proteins and antioxidant indicators such as SOD, CAT, GSH & GSSG, and MDA in the lens were measured.

[0122] In this embodiment, compared with the control group, the fluorescence intensity of the 25-HC and lanosterol treatment groups decreased by 5.86% and 3.84%, respectively, in the aggregation experiment. Figure 1 A); In the depolymerization experiment, the fluorescence intensity of the 25-HC and lanosterol-treated groups decreased by 4.69% and 8.57%, respectively. Figure 1 B); In animal model cataract treatment experiments, the area of ​​the opaque region of the lens in SD rats decreased by 24.84% and 27.27% respectively after treatment with 25-HC and lanosterol. Figure 2 The soluble protein content of A and B increased by 25.53% and 20.50%, respectively. Figure 2 C), SOD activity increased by 35.92% and 20.86%, respectively. Figure 3 A), the CAT activity in the lanosterol-treated group increased by 67.96% ( Figure 3 B), the total glutathione (GSH+GSSG) activity increased by 90.19% and 89.34%, respectively. Figure 3 C), the MDA content decreased by 30.54% and 18.47%, respectively. Figure 3 D), all showed statistically significant differences. The results indicated that positive compounds 25-HC and lanosterol could prevent or reverse the aggregation of CRYAB (R120G), increase the content of soluble proteins in the lens, and enhance the antioxidant capacity of the lens, thereby delaying or inhibiting the development of cataracts.

[0123] Example 2

[0124] The therapeutic effects of active compound 3 were studied using ThT fluorescence assays and an animal model of cataracts.

[0125] First, compound 3 was dissolved in DMSO to prepare a 1 mM stock solution. CRYAB (R120G) was diluted with 1×PBS to a 0.5 μg / μL solution. ThT was dissolved in DMSO to prepare a 50 mM stock solution. In the aggregation experiment, the compound 3 solution was added to the CRYAB (R120G) dilution to a final concentration of 200 μM and incubated at 37°C and 220 rpm for 30 min. In the depolymerization experiment, the CRYAB (R120G) dilution was incubated at 37°C and 220 rpm for 30 min, then the compound 3 solution was added to a final concentration of 200 μM, and incubated at 37°C and 220 rpm for 48 h. Dilute 50 mM ThT solution to 2 mM, add an equal volume of the incubated mixture to the solution in the dark, vortex mix, and dispense 200 μL into a 96-well plate. Use 1×PBS as a blank control and detect the fluorescence intensity at excitation and emission wavelengths of 440 nm and 480 nm, respectively.

[0126] A cataract model was established in 11-day-old SD rats by subcutaneous injection of 4 mM Na2SeO3 saline solution at a concentration of 20 nmol / g (body weight) into the neck and back. After eye opening, 5 μL of 5 mM cyclodextrin solution of compound 3 was instilled into each eye of the SD rats twice daily for two weeks. At the end of treatment, compound tropicamide eye drops were instilled twice at 10-minute intervals to dilate the pupils. After anesthesia with 500 μL of 20% maltose solution injected intraperitoneally, the degree of lens opacity was observed under a stereomicroscope. Following the removal of vitreous lens tissue, the content of soluble proteins and antioxidant indicators such as SOD, CAT, GSH & GSSG, and MDA in the lens were measured.

[0127] In this embodiment, compared with the control group, the fluorescence intensity of compound 3 treatment decreased by 5.89% in the aggregation experiment. Figure 1 A); In the depolymerization experiment, the fluorescence intensity of compound 3 decreased by 29.18% ( Figure 1 B); In the cataract animal model treatment experiment, the content of soluble protein in the lens of SD rats increased by 20.37% after treatment with compound 3. Figure 2 C), SOD activity increased by 28.41% ( Figure 3 A), CAT activity increased by 78.20% ( Figure 3 B), the total glutathione (GSH+GSSG) activity increased by 87.89% ( Figure 3 C), the MDA content decreased by 32.14% ( Figure 3 D), all showed statistical differences, but the area of ​​the opaque region of the lens decreased by only 1.12% ( Figure 2(A & B) No statistically significant difference. The results indicate that compound 3 can prevent or reverse the aggregation of CRYAB (R120G), increase the content of soluble proteins in the lens, and improve the antioxidant capacity of the lens, but it does not significantly improve the degree of lens opacity.

[0128] Example 3

[0129] The therapeutic effects of active compound 4 were studied using ThT fluorescence assays and an animal model of cataracts.

[0130] First, compound 4 was dissolved in DMSO to prepare a 1 mM stock solution. CRYAB(R120G) was diluted with 1×PBS to a 0.5 μg / μL solution. ThT was dissolved in DMSO to prepare a 50 mM stock solution. In the aggregation experiment, the compound 4 solution was added to the CRYAB(R120G) dilution to a final concentration of 200 μM and incubated at 37°C and 220 rpm for 30 min. In the depolymerization experiment, the CRYAB(R120G) dilution was incubated at 37°C and 220 rpm for 30 min, then the compound 4 solution was added to a final concentration of 200 μM, and incubated at 37°C and 220 rpm for 48 h. Dilute 50 mM ThT solution to 2 mM, add an equal volume of the incubated mixture to the solution in the dark, vortex mix, and dispense 200 μL into a 96-well plate. Use 1×PBS as a blank control and detect the fluorescence intensity at excitation and emission wavelengths of 440 nm and 480 nm, respectively.

[0131] A cataract model was established in 11-day-old SD rats by subcutaneous injection of 4 mM Na2SeO3 saline solution at 20 nmol / g (body weight) into the neck and back. After eye opening, 5 μL of 5 mM cyclodextrin solution of compound 4 was instilled into each eye of the SD rats twice daily for two weeks. After treatment, compound tropicamide eye drops were instilled twice at 10-minute intervals to dilate the pupils. Following anesthesia with 500 μL of 20% maltose solution injected intraperitoneally, the degree of lens opacity was observed under a stereomicroscope. After vitreous lens examination, the content of soluble proteins and antioxidant indicators such as SOD, CAT, GSH & GSSG, and MDA in the lens were measured.

[0132] In this embodiment, compared with the control group, the fluorescence intensity of compound 4 treatment decreased by 5.58% in the aggregation experiment. Figure 1 A); In the depolymerization experiment, the fluorescence intensity of compound 4 decreased by 29.98% ( Figure 1 B); In animal model cataract treatment experiments, compound 4 reduced the area of ​​the opaque region of the lens in SD rats by 26.34% after treatment. Figure 2 (A&B) The soluble protein content increased by 22.65% respectively. Figure 2 C), SOD activity increased by 40.63% ( Figure 3 A), CAT activity increased by 73.63% ( Figure 3 B), the total glutathione (GSH+GSSG) activity increased by 89.56% ( Figure 3 C), the MDA content decreased by 22.66% ( Figure 3 D), all showed statistically significant differences. The results indicated that compound 4 could prevent or reverse the aggregation of CRYAB (R120G), increase the content of soluble proteins in the lens, and enhance the antioxidant capacity of the lens, thereby delaying or inhibiting the development of cataracts, with effects similar to those of the positive compound.

[0133] Example 4

[0134] The therapeutic effects of active compound 5 were studied using ThT fluorescence assays and an animal model of cataracts.

[0135] First, compound 5 was dissolved in DMSO to prepare a 1 mM stock solution. CRYAB(R120G) was diluted with 1×PBS to a 0.5 μg / μL solution. ThT was dissolved in DMSO to prepare a 50 mM stock solution. In the aggregation experiment, the compound 5 solution was added to the CRYAB(R120G) dilution to a final concentration of 200 μM and incubated at 37°C and 220 rpm for 30 min. In the depolymerization experiment, the CRYAB(R120G) dilution was incubated at 37°C and 220 rpm for 30 min, then the compound 5 solution was added to a final concentration of 200 μM, and incubated at 37°C and 220 rpm for 48 h. Dilute 50 mM ThT solution to 2 mM, add an equal volume of the incubated mixture to the solution in the dark, vortex mix, and dispense 200 μL into a 96-well plate. Use 1×PBS as a blank control and detect the fluorescence intensity at excitation and emission wavelengths of 440 nm and 480 nm, respectively.

[0136] A cataract model was established in 11-day-old SD rats by subcutaneous injection of 4 mM Na2SeO3 saline solution at a concentration of 20 nmol / g (body weight) into the neck and back. After eye opening, 5 μL of 5 mM cyclodextrin solution of compound 5 was instilled into each eye of the SD rats twice daily for two weeks. At the end of treatment, compound tropicamide eye drops were instilled twice at 10-minute intervals to dilate the pupils. After anesthesia with 500 μL of 20% maltose solution injected intraperitoneally, the degree of lens opacity was observed under a stereomicroscope. Following the removal of vitreous lens tissue, the content of soluble proteins and antioxidant indicators such as SOD, CAT, GSH & GSSG, and MDA in the lens were measured.

[0137] In this embodiment, compared with the control group, the fluorescence intensity of compound 5 treatment increased by 17.27% in the aggregation experiment. Figure 1 A); In the depolymerization experiment, the fluorescence intensity decreased by 0.12% after treatment with compound 5. Figure 1 B); In an animal model treatment experiment for cataracts, the area of ​​the opaque region of the lens in SD rats increased by 8.64% after treatment with compound 5. Figure 2 (A&B) The soluble protein content increased by 14.18% respectively. Figure 2 C), SOD activity increased by 31.02% ( Figure 3 A), CAT activity increased by 59.91% ( Figure 3 B), the total glutathione (GSH+GSSG) activity increased by 84.11% ( Figure 3 C), the MDA content decreased by 1.38% ( Figure 3 D). The results showed that compound 5 promoted the aggregation of CRYAB(R120G). Although it increased the content of soluble proteins and antioxidant capacity in the lens to some extent, it significantly aggravated the opacity of the lens and could not be used as an anti-cataract drug.

[0138] This invention describes preferred embodiments and their effects. However, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to include both the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0139] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for screening anti-cataract compounds based on the CRYAB structure, characterized in that, Includes the following steps: Obtain the crystal structure of CRYAB and establish a molecular docking screening model; After the screening model is locally minimized in its internal coordinate space using the conjugate gradient algorithm and derived analytical tools, hydrogen atoms and heavy metal atoms are added to the receptor structure to set up the active pocket. Virtual screening was performed by docking active pockets with compounds in the ZINC Database, and docking scores were assigned to obtain a variety of compounds with docking scores below -24 and different structures. Multiple compounds selected through virtual screening were analyzed for affinity, thermal stability, and solubility with CRYAB and / or CRYAB mutants to identify CRYAB-based anti-cataract compounds.

2. The screening method for anti-cataract compounds based on the CRYAB structure according to claim 1, characterized in that, During the docking process, the ECEPP3 potential, mmff partial charge, and energy minimization of each molecule are calculated to represent the fraction of binding strength between the ligand and the acceptor. Finally, the optimal conformation ligand is selected as the docking fraction.

3. The screening method for anti-cataract compounds based on the CRYAB structure according to claim 1, characterized in that, When obtaining multiple compounds from the ZINC Database through virtual screening, the selected compounds are eliminated based on the modified Lipinski five-rule system, with the following conditions: molecular weight < 650; number of hydrogen bond donors < 5; number of hydrogen bond acceptors < 10; lipid-water partition coefficient < 9; number of rotatable bonds ≤ 10.

4. The screening method for anti-cataract compounds based on the CRYAB structure according to claim 1, characterized in that, A virtual screening was performed using the ZINC Database, selecting 11 compounds with docking scores below -24, diverse structures, and structures different from those of the positive compounds. Their structural formulas are as follows: The positive compounds are 25-hydroxycholesterol (25-HC) and lanosterol.

5. The screening method for anti-cataract compounds based on the CRYAB structure according to claim 4, characterized in that, Affinity K was selected by analyzing affinity, thermal stability, and solubility. D ≤5.358×10 -5 mol / L, apparent ΔT m ≥2℃, solubility characterization parameter CLogP (化合物) ≤CLogP (25-HC) It is a novel anti-cataract compound with a skeleton that differs from that of sterols.

6. The method for screening anti-cataract compounds based on the CRYAB structure according to claim 1, characterized in that, During the virtual screening process, compounds were initially screened by analyzing affinity, thermal stability, and solubility. Then, from the initially screened compounds, compounds that reduced the apparent Tm of CRYAB and / or CRYAB mutants and had the strongest affinity or the highest solubility were selected for in vivo and in vitro activity evaluation to screen out CRYAB-based anti-cataract compounds.

7. The method for screening anti-cataract compounds based on the CRYAB structure according to claim 1, characterized in that, The molecular docking screening model is a CRYAB screening model containing a molecular binding pocket.

8. The method for screening anti-cataract compounds based on the CRYAB structure according to claim 1, characterized in that, The crystal structure of CRYAB is derived from the PDB protein structure database, PDB ID: 2WJ7.

9. A CRYAB-based anticataract compound screened by the method described in any one of claims 1 to 8.

10. The anti-cataract compound based on the CRYAB structure according to claim 9, characterized in that, The compound includes

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