Application of small molecule compound in preparation of anti-aging preparation

Abstract

The invention relates to the field of biological medicine, in particular to application of a small molecule compound in preparation of an anti-aging preparation. The invention provides an application of a small molecule compound in preparation of a preparation for treating and / or preventing senescence of a subject, and is characterized in that the small molecule compound comprises quinacrine and / or rimonaban. According to the present invention, both the quinacrine and the rimonabant can provide the anti-aging effect by adjusting the insulin / insulin-like growth factor (ILS) signal channel, such that the quinacrine and the rimonabant can be adopted as the novel insulin / insulin-like growth factor signal channel regulator so as to provide the life prolonging effect on the subject.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a use of a small molecule compound in preparation of an anti-aging preparation. BACKGROUND

[0002] Aging is an inevitable complex biological process, which is generally defined as the progressive loss of physiological integrity of the body with age, eventually leading to deterioration of body function and death, and is an irreversible phenomenon. With the intensification of global population aging, the susceptibility of aging-related diseases increases and becomes a major public health problem, which brings a heavy burden to the global medical system. With the increasing proportion of the elderly population in the population, how to enter the old age healthily becomes a crucial problem. Although aging is inevitable, it is possible to delay aging. Therefore, it is increasingly important to develop strategies to delay aging and extend healthy life span.

[0003] Currently, models applied in aging research include mice, fruit flies, nematodes, zebrafish and non-human primates, etc. In nematodes, Caenorhabditis elegans is widely used in aging research due to its clear genetic background, simple individual structure, transparent body, easy observation, short lifespan and easy control of growth conditions. The insulin / insulin-like growth factor (ILS) signaling pathway is a widely verified aging regulatory signaling pathway, and reducing the activity of the insulin signaling pathway can significantly prolong the lifespan of nematodes, fruit flies and mice, and the polymorphism of ILS gene in human population is closely related to the longevity phenotype. SUMMARY

[0004] In a first aspect, the present application provides a use of a small molecule compound in preparation of a preparation for treating and / or preventing aging of a subject, characterized in that the small molecule compound comprises quinacrine and / or rimonabant.

[0005] In some embodiments, the concentration of the small molecule compound in the preparation comprises 5 - 50 µM.

[0006] In some embodiments, the concentration of the small molecule compound in the preparation comprises 10 - 20 µM.

[0007] In some embodiments, the preparation takes quinacrine and / or rimonabant as the main active ingredient.

[0008] In some embodiments, the preparation takes quinacrine and / or rimonabant as the only active ingredient.

[0009] As used herein, "subject" refers to a subject in need of treatment and / or prevention of aging, more particularly, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow. In some embodiments, the subject can also be a nematode, for example, Caenorhabditis elegans.

[0010] In some embodiments, the preparation includes an injection, a spray, or an oral agent.

[0011] In some embodiments, the preparation is an anti-aging drug.

[0012] In some embodiments, the use includes inhibiting an insulin receptor of the subject.

[0013] In some embodiments, the insulin receptor includes DAF-2.

[0014] In some embodiments, the use includes inhibiting an insulin receptor of the subject by modulating an insulin / insulin-like growth factor signaling pathway of the subject to activate a downstream transcription factor of the subject.

[0015] In some embodiments, the downstream transcription factor includes at least one of DAF-16, CLK-1, and HSF-1.

[0016] In some embodiments, the use includes prolonging a lifespan of the subject.

[0017] In some embodiments, the aging includes aging caused by a metabolic abnormality.

[0018] In some embodiments, the metabolic abnormality is a lipid metabolic abnormality.

[0019] In a second aspect, the present application provides a use of a small molecule compound in the preparation of an insulin / insulin-like growth factor signaling pathway modulator, characterized in that the small molecule compound includes quinacrine and / or rimoprofen.

[0020] In some embodiments, the concentration of the small molecule compound includes 5 - 50 µM.

[0021] In some embodiments, the concentration of the small molecule compound includes 10 - 20 µM.

[0022] In some embodiments, the use includes inhibiting an insulin receptor of the subject by modulating an insulin / insulin-like growth factor signaling pathway of the subject to activate a downstream transcription factor of the subject.

[0023] In some embodiments, the insulin receptor includes DAF-2.

[0024] In some embodiments, the downstream transcription factor comprises at least one of DAF-16, CLK-1, and HSF-1.

[0025] In some embodiments, the insulin / insulin-like growth factor signaling pathway modulator is used for anti-aging.

[0026] In some embodiments, the insulin / insulin-like growth factor signaling pathway modulator is used for prolonging the lifespan of the subject.

[0027] In some embodiments, the aging comprises aging caused by metabolic abnormality.

[0028] In some embodiments, the metabolic abnormality is lipid metabolic abnormality.

[0029] In a third aspect, the present application provides a method for rapid screening of anti-aging drugs, which comprises: S101 crossing a nematode of strain KH2235 with a nematode of sqt-3 (e2117) mutant to obtain a KH2235;sqt-3 nematode; S102 adding a candidate small molecule drug to the KH2235;sqt-3 nematode and culturing at a first temperature for a first time; In some embodiments, the first temperature comprises 25℃.

[0030] In some embodiments, the KH2235;sqt-3 nematode is a synchronized L1 stage nematode.

[0031] In some embodiments, the first time is ≥5 days.

[0032] In some embodiments, the first time is 9 days.

[0033] In some embodiments, the concentration of the candidate small molecule drug is ≥10 μM.

[0034] S103 performing a first screening of the candidate small molecule drug according to the ratio of GFP to RFP in the KH2235;sqt-3 nematode obtained in S102, and when the ratio of GFP to RFP is ≥0.4, the candidate small molecule drug is evaluated as a small molecule drug with anti-aging effect.

[0035] In some embodiments, the method further comprises adding the small molecule drug with anti-aging effect obtained in S103 to a nematode of KH2235 and / or wild type for a second screening.

[0036] In a fourth aspect, the present application provides use of a KH2235;sqt-3 nematode in the preparation of a kit for rapid high-throughput screening of anti-aging drugs.

[0037] In some embodiments, the KH2235; sqt-3 nematode is obtained by crossing a nematode of strain KH2235 with a nematode of sqt-3 (e2117) mutant.

[0038] In some embodiments, the KH2235; sqt-3 nematode is a synchronized L1 stage nematode.

[0039] In some embodiments, the use comprises adding a candidate small molecule drug to the KH2235; sqt-3 nematode and culturing for a first time at a first temperature, and rapidly screening the candidate small molecule drug according to the ratio of GFP to RFP in the KH2235; sqt-3 nematode.

[0040] In some embodiments, when the ratio of GFP to RFP in the KH2235; sqt-3 nematode is ≥ 0.4, the candidate small molecule drug is evaluated as an anti-aging drug.

[0041] In some embodiments, the first temperature comprises 25 °C.

[0042] In some embodiments, the KH2235; sqt-3 nematode is a synchronized L1 stage nematode.

[0043] In some embodiments, the first time is ≥ 5 days.

[0044] In some embodiments, the first time is 9 days.

[0045] In some embodiments, the concentration of the candidate small molecule drug is ≥ 10 μΜ.

[0046] Caenorhabditis elegans has become an important model for aging research due to its short lifespan, clear genetic background, and highly conserved key aging pathways, such as the highly conserved insulin / insulin-like growth factor signaling pathway. Current methods for screening anti-aging drugs using Caenorhabditis elegans mainly involve inoculating wild-type (or other strains) nematode individuals into culture plates containing candidate drugs and culturing at 20 °C for a long period of time to screen for anti-aging drugs. Although the existing method can screen for anti-aging drugs, nematodes are prone to continuous reproduction and produce new offspring during long-term culture. That is, the existing method not only takes a long time, but in order to ensure the accuracy of the screening results, the nematodes and their offspring (such as eggs and / or larvae) must be separated every day, otherwise the newly born offspring (i.e., the life cycle of nematode offspring is longer than that of the parent) will seriously affect the accuracy of anti-aging drug screening.

[0047] The rapid high-throughput screening method (and kit) of anti-aging drugs provided by the present application not only does not have a negative impact on the aging process of the KH2235;sqt-3 nematodes themselves, but also can accurately and efficiently screen out potential small-molecule drugs with anti-aging effects in a rapid high-throughput manner by combining the ratio of GFP to RFP (for example, thousands of candidate small-molecule drugs can be screened to dozens of drugs, thereby saving at least several hundred hours of work). The rapid high-throughput screening method (and kit) of anti-aging drugs provided by the present application can effectively avoid the problem that the generation of nematode offspring interferes with the screening of anti-aging drugs, and effectively avoid the repetitive and labor-intensive work of separating nematodes from their offspring (such as eggs and / or larvae), saving experimental resources and labor costs, and providing an effective tool for developing and intervening in new therapeutic strategies for age-related diseases and extending healthy lifespan. The small-molecule drugs (such as quinacrine and rimonabant) screened by the present application have been verified on nematodes of other strains (such as KH2235 and / or wild-type nematodes), that is, the rapid screening method (and kit) of anti-aging drugs provided by the present application can effectively and high-throughput screen out potential small-molecule drugs with anti-aging effects.

[0048] Compared with the prior art, the present application has at least the following beneficial effects: The present application uses the splicing reporter system to screen a chemical library and determines that quinacrine and rimonabant are potential anti-aging drugs. Quinacrine has been reported to be able to treat malaria, autoimmune diseases, cancer, Alzheimer's disease and protein aggregation diseases, but its therapeutic function does not depend on the aging regulatory signaling pathway. Specifically, quinacrine can interfere with the DNA replication and transcription of Plasmodium to treat malaria; can regulate the function of T cells and B cells, inhibit antibody production (especially the response to self-antigens) to treat autoimmune diseases; can inhibit tumor cell proliferation, induce tumor cell apoptosis, and inhibit tumor angiogenesis to play an anti-tumor role. In addition, quinacrine has been found to be able to directly bind to beta-amyloid protein, inhibit its aggregation process, and thus reduce the formation of amyloid plaques or other protein aggregates to treat Alzheimer's disease and protein aggregation diseases. Rimonabant (SR141716) as a CB1 receptor antagonist, mainly reduces the activity of central nervous system receptors, reduces the appetite and food intake of patients, and reduces the hepatic fat production to treat diseases such as atherosclerosis, obesity, non-alcoholic fatty liver disease and kidney disease. In other words, quinacrine and rimonabant have not been reported to be able to play an anti-aging (such as extending lifespan) role by regulating the insulin / insulin-like growth factor signaling pathway.

[0049] Drug intervention has been proven to effectively prolong healthy lifespan, and the experimental results of the present application show that the inhibition constant (Ki) value of another selective cannabinoid 1 type (CB1) receptor (CB1 receptor mainly located in the central nervous system, which regulates mood, cognition, pain and appetite, and its activation will produce psychoactive effects) antagonist, Otenabant (CP-945598) is 0.7 nanomolar, but it has not been proven to effectively prolong lifespan, and two selective cannabinoid 2 type (CB2) receptor (CB2 receptor located in the immune system and peripheral tissues, which regulates inflammation and immune response, can provide neuroprotection and has no obvious psychoactive effect) antagonists AM1241 and GW842166X also failed to prolong lifespan. The above results show that the inhibition of aging (such as prolonging lifespan) is actually very complex, and not any small molecule compound that can resist inflammation and provide neuroprotection has anti-aging effect. Based on the drug screening results, the present application verifies the effects of quinacrine and rimonabant on the lifespan extension of C. elegans and its molecular mechanism. The results show that quinacrine and rimonabant can regulate the insulin / insulin-like growth factor (ILS) signaling pathway by affecting the insulin receptor (such as DAF2) to play an anti-aging role, and therefore, quinacrine and rimonabant can be used as a new type of insulin / insulin-like growth factor signaling pathway regulator, which can prolong the lifespan of the subject by inhibiting the insulin receptor and activating the downstream transcription factor. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without paying creative labor.

[0051] Figure 1A Schematic diagram of ret-1 splicing reporter gene; Figure 1B Flow chart of anti-aging small molecule drug screening; Figure 1C Experimental result graph of the 9th day of feeding KH2235 nematodes with quinacrine (10 μM) and rimonabant (10 μM) (scale: 100 μm); Figure 1D Experimental result graph of the working concentration of quinacrine and rimonabant; Figure 1E Result graph of the effect of quinacrine and rimonabant on the survival rate of wild type nematodes N2; Figure 1F Graph showing the effect of quinalphos and lirimconab on the pharyngeal pump frequency of nematodes; Figure 2 Graph showing the aging process of KH2235;sqt-3 double mutant nematodes; Figure 3 Graph showing that quinalphos extends the lifespan of nematodes through the insulin / insulin-like growth factor signaling pathway (ILS); Figure 4A Graph showing that differentially expressed genes are enriched in oxidoreductase activity; Figure 4B Graph showing that metabolic pathways are changed due to quinalphos treatment; Figure 4C Graph showing the effect of quinalphos on metabolic pathways (pyroglutamyl-glutamyl- lysine peptides); Figure 4D Graph showing the effect of quinalphos on metabolic pathways (phosphatidylserine); Figure 5 Graph showing that lirimconab extends the lifespan of nematodes through the insulin / insulin-like growth factor signaling pathway (ILS); Figure 6 Graph showing that the effect of lirimconab on extending lifespan is regulated by hsf-1; Figure 7 Graph showing that AM1241 cannot effectively extend the lifespan of nematodes; Figure 8 Graph showing the results of screening quinalphos and lirimconab using KH2235;sqt-3 nematodes. DETAILED DESCRIPTION

[0052] In order to make the objects, technical solutions, and advantages of the embodiments of the present application clearer, the following will be combined with the accompanying drawings for the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0053] In this document, “and / or” includes any and all combinations of one or more of the associated items.

[0054] In this document, “a plurality of” means two or more, that is, it includes two, three, four, five, and the like.

[0055] It is to be appreciated that the terms "comprises", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a" does not, without more constraints, exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0056] As used herein, the term "about", in the context of a given value, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0057] In this specification, certain embodiments can be disclosed in a format that is a range. It is to be understood that such a "range" format is used only for convenience and brevity and should be interpreted in the context of the description as a whole. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a description of a range 1-6 should be considered to have specifically disclosed the sub-ranges like 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers 1, 2, 3, 4, 5, and 6. This same logic should be applied to ranges of any magnitude.

[0058] Example One Materials and Methods Worm Strains and Culture The following Caenorhabditis elegans strains were originally obtained from the Caenorhabditis elegans Genetic Center: N2 wild type, CB4121 [sqt - 3(e2117)], CB1370 [daf - 2(e1370)], CF1038 [daf - 16(mu86)], PS3551 [hsf - 1(sy441)], MQ130 [clk - 1(qm30)], KN259 [sod - 3::GFP + rol - 6(su1006)], VC222 [raga - 1(ok386)], and CL4176 [myo3p::A - Beta (1 - 42)::let - 8513'UTR) + rol - 6(su1006)]. KH2235 (lin15(n765)ybIs2167[eft - 3p::ret - 1E4E5(+1)E6 - GGS6 - mCherry + eft3p::ret - 1E4E5(+1) E6(+2)GGS6 - GFP + lin - 15(+)+ pRG5271Neo]) was derived from the laboratory of Dr. H. Kuroyanagi. Nematodes were cultured and maintained on growth medium (NGM) supplemented with E. coli OP50. All strains were cultured at 20°C unless otherwise stated.

[0059] Drug screening using KH2235 nematodes 1) Prepare NGM medium on the first day, add 1.5 mL medium per well of a 24-well plate, invert and dry at room temperature for about 24 h.

[0060] 2) On the second day, pick single colonies of E. coli OP50 and initially shake in 4 mL of LB medium for 8 h, inoculate 300 μL of the bacterial solution into 20 mL of LB liquid medium and continue to culture for 12-14 h.

[0061] 3) On the third day, take 15 μL of the OP50 bacterial solution and spread it on the 24-well plate, so that the OP50 grows on the NGM overnight.

[0062] 4) On the fourth day, irradiate with ultraviolet light at a wavelength of 254 nm for 30 min-1 h, completely kill the OP50 on the surface of the nematode medium. Dilute the small molecule compounds using DMSO and add them to the plate after ultraviolet irradiation, so that the final concentration of the small molecule compounds is 10 μM. Add the synchronized L1 nematodes of KH2235 and place them in a 20°C incubator, which is recorded as the first day of feeding.

[0063] 5) Observe nematodes without starvation and contamination, if no starvation and contamination, when nematodes grow to the second day of adult (the fifth day of feeding), use picker to pick about 20 P0 adults to new 24-well plates. On the ninth day of feeding, pick adult P0 for fluorescence observation, calculate G / R value, and calculate P value of molecular drug group and control group.

[0064] Total RNA extraction After collecting nematodes, wash them with M9 buffer. Put the washed nematodes into TRIzol reagent and vortex vigorously. Then, freeze-thaw the sample in liquid nitrogen for 3 times to fully lyse the nematode cells. Add TRIzol reagent to the sample again and vortex vigorously to ensure thorough mixing. Then, add chloroform to the mixture, invert the mixture vigorously for 15 seconds, and then stand for 3 minutes to allow the mixture to layer. Centrifuge the sample at 14000 g for 15 minutes at 4°C, transfer the supernatant to a new tube, and add an equal volume of isopropanol, mix the sample and stand at room temperature for 10 minutes to promote RNA precipitation. Centrifuge the sample at 14000 g for 20 minutes at 4°C, remove the supernatant. Rinse the precipitate with 1 mL of pre-cooled 75% ethanol for 3 times, finally dissolve the precipitate with RNAse-free water to obtain nematode total RNA. Store the extracted RNA at -80°C to maintain its stability and integrity.

[0065] Survival curve experiment The experiment was carried out at a temperature of 20°C. Synchronized nematodes were fed with ultraviolet-inactivated E. coli OP50. Survival curves were plotted using GraphPad Prism 6. P values were calculated using the log-rank (Mantel Cox) analysis method.

[0066] Pharyngeal pump frequency analysis On the ninth day of feeding small molecule drugs in C. elegans, count the pharyngeal pumping frequency of N2. Specifically, observe pharyngeal pumping under a 5x microscope, record the number of pharyngeal pumping of a single nematode within 20 seconds, and count 10 nematodes in each group.

[0067] RNA sequencing Synchronized to L1 stage KH2235 nematodes were transferred to nematode growth medium (NGM) containing dimethyl sulfoxide (DMSO), quinacrine hydrochloride or rimobrotan. After 15 days, C. elegans were collected and washed with M9 buffer. Total RNA was extracted and sent to Shanghai Pinengnobio Technology Co., Ltd. for sequencing. Quality control analysis was performed using Agilent 2100 Bioanalyzer System. Samples were sequenced on the NovaSeq 6000 platform (Illumina). In terms of data analysis, read counts for each gene were quantified using HTSeq version (0.9.1), which were considered as raw gene expression levels. These values were then normalized using the FPKM (fragments per kilobase of transcript per million mapped reads) method to correct for differences in gene length and sequencing depth. Differential gene expression analysis was performed using DESeq (1.30.0) with a significance threshold set at |log2FoldChange| > 1 and P-value < 0.05.

[0068] Mass spectrometry analysis Nematodes were collected and washed with M9 buffer. Samples were sent to Novozymes for metabolite mass spectrometry analysis. After transferring nematodes in 100 μL M9 buffer to a glass tube, 900 μL water, 2 mL methanol and 0.9 mL dichloromethane were added, followed by thorough mixing. After adding 1.0 mL water and 0.9 mL dichloromethane, the sample was vortexed to an emulsified state and centrifuged at 3000 rpm for 5 minutes at 4°C. Next, the lower dichloromethane layer was transferred to a clean glass tube with a glass syringe. The upper water phase (original sample) was mixed with 2 mL dichloromethane and centrifuged at 3000 rpm for 5 minutes at 4°C. The lower dichloromethane layer was transferred to a glass tube with a glass syringe. The two lower liquid layers obtained were combined and freeze-dried under vacuum conditions. The dried metabolite precipitate was re-dissolved in 500 μL methanol / dichloromethane and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The raw data file was imported into Progenesis QI software (Waters Corporation). Peak alignment was performed for each compound based on parameters such as retention time, mass-to-charge ratio (m / z) and mass deviation (within 5 ppm). The processed mass spectrometry data were then compared with databases including Lipidmaps (http: / / www.lipidmaps.org), Human Metabolome Database (HMDB, http: / / www.hmdb.ca), National Institute of Standards and Technology database (NIST, https: / / chemdata.nist.gov) and a self-built lipid database. Qualitative and quantitative analysis was performed on the lipid data results.

[0069] Statistical analysis All experiments were repeated at least three times. Statistical analysis was performed using GraphPad Prism 7.0 software. Quantitative results were reported as mean ± standard deviation. All tests were two-tailed tests, and P < 0.05 was considered statistically significant.

[0070] Example Two High-throughput screening of anti-aging molecules Particular RNA splicing factors have been shown to be associated with lifespan in multiple species, including C. elegans, mice, and humans. As organisms age, their RNA splicing patterns change significantly. The C. elegans KH2235 strain expresses a reporter gene for ret-1 exon 5 with different frameshifts (ret-1 splicing reporter). When exon 5 is included, the reporter gene produces green fluorescent protein (GFP), while when exon 5 is skipped, it produces red fluorescent protein (RFP) (Figure 1). Figure 1A In 5-day-old C. elegans, the splicing pattern of exon 5 remains heterogeneous, reflecting a young state. However, by day 7, aging disrupts alternative splicing, and all worms lose GFP expression.

[0071] To identify small-molecule compounds that affect aging, this example high-throughput screens the splicing pattern as a marker of aging. Specifically, this example crosses the KH2235 strain with the sqt-3 (e2117) mutant, which grows and reproduces normally at 15°C but exhibits embryonic lethality at 25°C (Figure 2). Figure 1B

[0072] This double mutant enables efficient screening without time-consuming animal transfer procedures. The results show that aging of KH2235; sqt-3 nematodes results in increased RFP expression and decreased GFP expression (Figure 3), confirming that the double mutant is a reliable model for rapid, high-throughput screening of anti-aging compounds. Figure 2

[0073] This example uses KH2235; sqt-3 nematodes to screen a library containing 2189 compounds (Figure 4). Figure 1B ​​). Specifically, the present example crosses the KH2235 strain with the sqt-3 (e2117) mutant to obtain KH2235; sqt-3 nematodes. Synchronized L1 KH2335; sqt-3 (e2117) nematodes are cultured at 25 °C in 24-well NGM plates coated with UV-inactivated OP50 and small molecule compounds in a custom-made small molecule library (Cherry Pick Library, Selleck, L2000Z287029-30Ul). KH2335; sqt-3 (e2117) nematodes do not reproduce under the culture conditions at 25 °C, thus do not interfere with the screening of anti-aging drugs, and the anti-aging drugs can be quickly screened by calculating the green / red fluorescence ratio (GFP to RFP ratio, i.e., G / R value). When the G / R value is <0.4, the small molecule compound is screened as a non-anti-aging drug, and when the G / R value is ≥0.4, the small molecule compound is screened as an anti-aging drug.

[0074] After two biological repeats, the present example identifies 76 compounds that improve GFP splicing (i.e., potential anti-aging drugs). Further testing in KH2235 nematodes confirms that 16 compounds significantly enhance splicing. The above results show that the method of the present example for quickly screening anti-aging drugs can improve the efficiency of screening anti-aging drugs and quickly identify potential anti-aging drugs. After excluding compounds that have a negative impact on the growth of nematodes, the present example ultimately determines that quinacrine and rimonabant are the most promising candidate compounds, which show consistent and significant effects in multiple repeated experiments (p<0.05, two-tailed Student’s t-test). Figure 1C 、 Figure 8 ).

[0075] The optimal working concentration of quinacrine is ≥20 µM and that of rimonabant is ≥10 µM determined by concentration-response experiments. Figure 1D ).

[0076] To explore the molecular effects of these compounds, the present example also measures the mRNA levels of ret-1 and tos-1 genes, as well as the splicing pattern of the target tos-1 of the splicing factor SFA-1, which is known to affect the lifespan of nematodes. The results show that rimonabant reduces the mRNA levels of ret-1 and tos-1, while quinacrine has no significant effect on their expression. In addition, rimonabant increases the level of the tos-1 intronless splicing isoform. In summary, these results suggest that rimonabant may regulate transcription and splicing.

[0077] Example Three Quinacrine and rimonabant can extend the lifespan of Caenorhabditis elegans To investigate the effects of quercetin and rimobulin on lifespan, the present example investigated their effects on the lifespan of wild-type strain N2. The results showed that both rimobulin and quercetin significantly extended the lifespan of the nematodes ( Figure 1E ). These findings indicate that both quercetin and rimobulin promote longevity in C. elegans and maintain the health of C. elegans, i.e., do not negatively affect the health of C. elegans. Dietary restriction (DR) is known to extend the lifespan of C. elegans and slow down the rate of decline in splicing efficiency with age. To determine whether the lifespan-extending effects of quercetin and rimobulin are dependent on dietary restriction, the present example evaluated the feeding behavior of the nematodes by measuring the pharyngeal pump frequency. On the ninth day of the adult stage, the pharyngeal pump frequency of the nematodes treated with these two compounds was recorded. The data showed that there was no significant change in the pharyngeal pump frequency after treatment with quercetin or rimobulin ( Figure 1F ), indicating that the observed lifespan extension was not caused by a change in feeding ability.

[0078] Example Four Quercetin extends lifespan through the insulin / insulin-like growth factor signaling pathway (ILS) The lifespan of C. elegans is regulated by multiple signaling pathways that respond to stress and nutritional availability, including the insulin / insulin-like growth factor (ILS) pathway, the target of rapamycin (TOR) pathway, and mitochondrial metabolism. To determine whether quercetin affects these pathways, the present example treated nematodes with quercetin and evaluated the effects on the lifespan of mutants lacking key genes in these pathways. The ILS pathway plays a key role in lifespan regulation, involving the FOXO transcription factor DAF-16 and the insulin receptor DAF-2. In addition, the heat shock factor HSF-1 is known to integrate signals related to cellular energy metabolism and various stressors, thereby affecting lifespan. The results showed that quercetin failed to extend the lifespan of daf-2(e1370), daf-16(mu86), or hsf-1(sy441) mutants ( Figure 3 A、 Figure 3 B、 Figure 3 C), indicating that the lifespan-extending effect of quercetin is dependent on the ILS pathway.

[0079] To further confirm this conclusion, the present example also detected the expression of sod-3, a downstream target gene of the ILS pathway. This gene encodes an extracellular superoxide dismutase that protects cells from oxidative damage by neutralizing superoxide anion radicals. After treatment with quercetin, the fluorescence of sod-3::gfp transgenic nematodes was enhanced, indicating an increase in the expression of this protective gene ( Figure 3 D). These results further confirm that quercetin extends lifespan by activating the ILS pathway.

[0080] Next, this example evaluated whether quinacrine affects mitochondrial metabolism, which also plays a role in lifespan regulation. CLK-1 is a homolog of coenzyme Q7 in *C. elegans* and is crucial for mitochondrial metabolism. In the clk-1(qm30) mutant, ubiquinone synthesis and mitochondrial electron transport are impaired. Quinacrine still extended the lifespan of the clk-1 mutant (…). Figure 3 E), indicating that the life-extending effect of quinacrine is not primarily mediated by mitochondrial metabolism.

[0081] Finally, this embodiment investigated the TOR signaling pathway, which regulates cell growth and metabolism based on nutrient availability. RAGA-1 is a Ras-associated GTP-binding protein that links amino acid sensing to the activity of rapamycin complex 1 (mTORC1). Quinacrine was able to prolong the lifespan of the raga-1 (ok386) mutant. Figure 3 (F), indicating that its life-extending effect is independent of the TOR pathway.

[0082] The above results indicate that quinacrine mainly prolongs the lifespan of *C. elegans* through the ILS signaling pathway.

[0083] Example 5 Mechanism of quinacrine's effect on aging in Caenorhabditis elegans To investigate the molecular mechanism by which quinacrine affects *C. elegans*, batch RNA sequencing was performed in this study. Quinacrine treatment led to significant changes in the transcriptome, with 260 genes upregulated (URGs) and 306 genes downregulated (DRGs) (|log2FC|>1, p<0.05). Gene ontology (GO) analysis of differentially expressed genes (DEGs) showed that these genes were significantly enriched in multiple biological processes, and significant changes were found in fatty acid metabolism (GO:0006082). Molecular functional analysis showed that the differentially expressed genes were significantly enriched in oxidoreductase activity (GO:0016491), which is conducive to the biochemical transformation of fatty acids and exogenous substances. Figure 4A These enzymes play a crucial role in the breakdown and detoxification processes, maintaining metabolic homeostasis and protecting organisms from harmful compounds. Further analysis using the Kyoto Encyclopedia of Genetics and Genomes (KEGG) revealed that metabolic pathways, particularly those related to fatty acid metabolism, were significantly altered by quinacrine treatment. Figure 4B ).

[0084] Mass spectrometry analysis further confirmed the effects of quinacrine on lipid metabolism pathways, identifying 2 downregulated and 8 upregulated metabolites (p<0.05, |log2FC|>1). Figure 4C , Figure 4D). Among the upregulated metabolites, phosphatidylserine (PS) (6:0 / 6:0) was identified, which is a key component of cell membranes and is essential for neuronal function. In addition, quinacrine induced the production of a pyroglutamyl-glutamine-lysine peptide (PyroGlu-Gln-Lys). Although its specific function is unclear, pyroglutamic acid is thought to play a protective role in neurons, participating in antioxidant defenses by neutralizing reactive oxygen species (ROS) associated with aging and neurodegenerative diseases.

[0085] Example Six Mechanism of action of rimonabant in extending the lifespan of C. elegans To investigate the mechanism of action of rimonabant in extending the lifespan of C. elegans, the effect of rimonabant on several genetic mutants was tested, including daf- 16(mu86), daf- 2(e1370), clk- 1(qm30) and hsf- 1(sy441). The survival curves of these mutants were compared with the survival curve of wild-type worms treated with rimonabant. The results showed that rimonabant failed to extend the lifespan of the insulin / insulin-like growth factor signaling pathway (ILS) upstream mutants daf- 2(e1370) or hsf- 1(sy441) mutants ( Figure 5 A, Figure 5 B). However, rimonabant did extend the lifespan of daf- 16(mu86) and clk- 1(qm30) mutants ( Figure 5 C, Figure 5 D). Rimonabant also extended the lifespan of raga- 1(ok386) mutant worms ( Figure 5 E). Further analysis using the ret- 1 splicing reporter in the hsf- 1(sy441) mutant confirmed that the lifespan-extending effect of rimonabant was regulated by HSF- 1 ( Figure 6 ). Overall, the above results indicate that rimonabant mainly affects the lifespan of worms by modulating the ILS signaling pathway, particularly through the DAF- 2 / HSF- 1 axis.

[0086] To further explore the molecular mechanism of rimonabant action, RNA sequencing was performed on C. elegans treated with rimonabant. In the C. elegans treated with rimonabant, 343 up-regulated genes (URG) and 309 down-regulated genes (|log2FC|>1, p<0.05) were identified. Gene ontology (GO) analysis of the differentially expressed genes showed that rimonabant significantly affected several biological processes, including exogenous substance metabolic process (GO:0006805), innate immune response (GO:0045087), and glutathione metabolic process (GO:0006749). Further molecular function analysis of the differentially expressed genes showed that genes related to oxidoreductase activity, steroid hydroxylase activity, monooxygenase activity, glutathione transferase activity, and heme binding were significantly enriched.

[0087] Rimonabant is a CB1 receptor antagonist that can be used for weight loss. In fact, the library of 2189 compounds mentioned above also contains Otenabant (CP-945598, which is another selective cannabinoid 1 type (CB1) receptor antagonist with an inhibition constant (Ki) value of 0.7 nanomolar), as well as the selective cannabinoid 2 type (CB2) receptor antagonists AM1241 and GW842166X. However, the drug screening of the present embodiment shows that neither Otenabant nor AM1241 and GW842166X can effectively prolong the lifespan of the nematodes, as shown in Figure 7 The above results show that rimonabant mainly prolongs the lifespan of C. elegans through the ILS signaling pathway, and the mechanism of lifespan extension is very complex, and not any selective cannabinoid 1 type (CB1) receptor antagonist (as well as selective cannabinoid 2 type (CB2) receptor antagonist) can effectively prolong the lifespan.

[0088] The embodiments of the present application are described above in conjunction with the drawings, but the present application is not limited to the specific embodiments described above, which are merely illustrative rather than limiting, and a person of ordinary skill in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, which are all within the protection of the present application.

Claims

1. Use of a small molecule compound for the manufacture of a preparation for the treatment and / or prevention of aging in a subject, characterized in that, The small molecule compound includes quinacrine and / or rimoprofen.

2. Use according to claim 1, characterized in that, The concentration of the small molecule compound in the preparation includes 5-50 µM.

3. Use according to claim 1, characterized in that, The preparation includes injection, spray or oral agent.

4. The use according to claim 1, characterized in that, The use includes inhibiting insulin receptor of the subject.

5. The use according to claim 1, characterized in that, The use includes prolonging the life of the subject.

6. Use according to claim 4, characterized in that, The use includes inhibiting insulin receptor of the subject by adjusting insulin / insulin-like growth factor signal pathway of the subject to activate downstream transcription factor of the subject.

7. Use of a small molecule compound for the manufacture of a modulator of the insulin / insulin-like growth factor signaling pathway, characterized in that, The small molecule compound includes quinacrine and / or rimoprofen.

8. Use according to claim 7, characterized in that, The concentration of the small molecule compound includes 5-50 µM.

9. Use of KH2235; sqt-3 nematode for the preparation of a kit for rapid high-throughput screening of anti-aging drugs, characterized in that, The use includes adding candidate small molecule drug into the KH2235;sqt-3 nematode and culturing for a first time at a first temperature, and rapidly screening the candidate small molecule drug according to the ratio of GFP to RFP in the KH2235;sqt-3 nematode; when the ratio of GFP to RFP of the KH2235;sqt-3 nematode is ≥0.4, the candidate small molecule drug is evaluated as an anti-aging drug.

10. The kit of claim 9, wherein The concentration of the candidate small molecule drug is ≥10 µM.

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