Screening Of A New Lipid Metabolism-Related Target ABHD5 Active Leads And Validation Of Its Inhibitory Effect On Colorectal Cancer

BackgroundColorectal cancer（CRC）is one of the most common gastrointestinal tumors in clinical practice.Due to the lack of popularity of gastrointestinal endoscopy,most patients are found to be in the advanced stage of colorectal cancer at the first examination,resulting in poor prognosis and low five-year survival rate.Systemic therapy,including chemotherapy,radiotherapy and targeted therapy,is the main treatment for m CRC patients,which has not significantly improved the prognosis of m CRC patients in the past 20 years.Metabolic reprogramming is one of the important characteristics of malignant tumors,and abnormal expression activity of a variety of metabolism-related genes is a common concomitant phenomenon during the occurrence and progression of tumors.In recent years,some inhibitors targeting glucose metabolism,lipid metabolism,and amino acid metabolism have achieved certain anti-tumor effects,which have become new strategies for targeting the occurrence and development of tumors.Therefore,it is of a great significance to screen and identify functional metabolism-related molecules that have important regulatory effects on the occurrence and development of colorectal cancer.To explore their mechanism of action,develop new structural and original small molecule drugs by taking colorectal cancer specific metabolic pathways,metabolites and metabolic enzymes as new targets for the effective prevention and treatment of colorectal cancer.ABHD5（a/b-hydrolase domain 5）,also known as CGI-58,acts as a cofactor of Adipose triglyceride lipase（ATGL）,increasing ATGL activity by nearly 20-fold and assisting in the catabolism of triglycerides.It is well known that ATGL catalyzes the first step of lipolysis,converting triglycerides（TGS）to diacylglycerols（DAGs）.ATGL requires ABHD5 for full TG hydrolase activity.Mutations in the human ABHD5 gene cause Chanargin-Dorfman syndrome,a rare autosomal recessive disease.At the same time,recent studies have shown that ABHD5plays an important role in the occurrence and development of colorectal cancer,and also plays a key role in the process of chemosensitivity and drug resistance of 5-FU-based colorectal cancer.Our group previously identified ABHD5 as a tumor suppressor gene with metabolic activity.In the occurrence spectrum of colorectal cancer,ABHD5 expression is reduced to varying degrees from the adenoma stage.The degree of reduction in its expression is closely related to triglyceride deposition,adenoma carcinogenesis,tumor stage,grade and prognosis of patients with colorectal cancer.Intestinal ABHD5 knockout can significantly promote the growth,malignant transformation,invasion and metastasis of intestinal adenomas in APC^min/+mice.Therefore,our team first proposed that ABHD5 is a metabolically active tumor suppressor gene,and its reduced expression activity is an important driver of colorectal carcinogenesis and progression.Subsequent studies have shown that Beclin1,a key molecule of autophagy in colorectal cancer cells,binds to ABHD5 with high affinity specifically,thereby promoting the degradation of ABHD5 through the lysosomal pathway,which is an important reason for the decrease of ABHD5 expression and its abnormal expression can promote the malignant progression of colorectal cancer.Based on our previous research on ABHD5,we used AI virtual screening combined with cytological drug entity screening to screen out 1-2 lead compounds with high activity to inhibit the occurrence and progression of colorectal cancer by up-regulating ABHD5 protein expression.It opens up a new direction for tumor therapy targeting metabolism-related targets.ObjectivesThrough high-throughput screening,we hope to find 1-2 original drug leads that can specifically target ABHD5-Beclin1（HSP90）and effectively up-regulate ABHD5 expression to inhibit the occurrence and development of colorectal cancer.This study provides a material basis for the development of drugs targeting ABHD5-Beclin1 to inhibit the carcinogenesis of intestinal adenoma and the malignant progression of intestinal cancer.Methods1.A virtual screening approach using artificial intelligence（AI）,along with a three-dimensional compound library comprised of self-built small molecules were used to screen inhibitors of the protein-protein interaction between ABHD5 and Beclin1.2.In vitro experiments（CCK8 detection,Western blotting,heat stability experiment,surface plasmon resonance（SPR）experiment,etc.）were used to verify the effect of small molecular compounds on the activity of SW480 cells,verify its binding to ABHD5 protein and its effect on the expression of ABHD5 protein.3.Animal experiment)was used to verify the in vivo effects of the target small molecule compounds.Results1.There were 99 high-scoring small molecule compounds that were screened virtually by artificial intelligence,and 65 of these compounds were purchased for preliminary screening.2.The results of CCK8 assay:most of the small molecular compounds（about 72%,47/65）（including 25,26,38,39,41,43,etc.）had a significant inhibitory effect on the viability of SW480 cells（cell survival rate<70%）.3.CCK8 test results:small molecular compounds（No.25,26,38,39,41,43）had a certain inhibitory effect on the viability of SW480,MC38 and CT26 cells,among which No.38 small molecule compound had the most obvious effect.Meanwhile,we found that small molecule compound 38 had low cytotoxicity on normal FHC cells.4.Western blotting experiment demonstrated that No.38 small molecule compound could significantly increase ABHD5 protein expression.5.Thermal stability experiment and SPR experiment showed a further confirmation of a specific binding between No.38 small molecule compound and ABHD5.6.The No.38 small molecule compound was confirmed to inhibit the tumorigenicity of colorectal cancer cells in mice.Conclusions1.Most of the small molecule compounds screened by AI showed significant inhibitory effects on the proliferation of CRC cells,among which No.38 small molecule compound showed significant inhibitory effects on SW480,MC38 and other colorectal cancer cells,and had little toxic effect on FHC.At the same time,the No.38 small molecule compound could significantly up-regulate the expression of ABHD5 protein in SW480.2.It was confirmed that No.38 small molecule compound could directly bind to ABHD5protein by CETSA and SPR experiment.3.The No.38 small molecule compound was validated to inhibit the proliferation of colorectal cancer xenografts in mice.