Study On The Design, Synthesis And Bioactivity Of Small Molecular Inhibitors

Sphingomyelin (SM) synthase (SMS) as an integral membrane protein is the last enzyme involved in the SM biosynthesis which transfers the phosphorylcholine moiety from phosphatidylcholine onto the primary hydroxyl of ceramide-producing SM and diglyceride. SMS plays diverse roles in physiology and pathology, thus, may be a potential disease marker and/or drug target. Especially, recent studies suggest that SMS overexpression may play a promoting role in the development of atherosclerosis, as well as SMS deficiency significantly decreased atherosclerosis. Such an effect might contribute to the increased or decreased plasma SM levels observed in the animal models of atherosclerosis as well as in human coronary artery disease. These findings raise the possibility that SMS may be a useful target for coronary artery disease therapy. Therefore, seeking to develop clinical therapies for this largely unmet medical need, the discovery of inhibitors of SMS seems to be a strategic imperative.In chapter1, SM is one of the major lipid components in plasma and cell membranes. Plasma SM level is an independent risk factor for coronary artery disease. The ability to regulate SM biosynthesis could become a promising treatment for atherosclerosis. In chapter1, we reviewed the effect of SM biosynthesis on lipid protein metabolism and atherosclerosis. The potential of anti-atherosclerosis from reducing SM level by regulation of the pathways of SM biosynthesis were described. The advances on potential of SMS as an anti-atherosclerosis target, as well as researches about SMS inhibitors were reviewed.In chapter2, No specialized SMS inhibitors screening assay method was reported in literature. On the basis of quantitative analysis in HPLC, we reported a novel and sensitive method for SMS activity measurement, using ICR mice liver homogenate as the enzyme source. This method is suitable for quantifying SMS enzyme activity in SMS inhibitor screening studies. In addition, SMS is a membrane protein, containing two isoforms:SMS1and2. To obtain a pure SMS enzyme system was extremely difficult. Our cooperator tried hard to express SMS1and SMS2in non-SMS insect cells, and obtained only a high expression cell of SMS2finally. The SMS2high expression of cell homogenate can be considered equivalent to pure SMS2enzyme system. On this basis, we further establish SMS2inhibitor screening system by using the SMS2high expression cells homogenate as enzyme source. SMS1and2were located in different subcellular unit and there was no different in term of biosynthesis function. So the inhibitor screened by using the SMS2high expressing cell homogenate as the enzyme source also has inhibitory effect SMS1theoretically.In chapter3, In previous studies of our group, homology model of human sphingomyelin synthase1(hSMS1) had been simulated and used for virtual screening of the SPECS libraries, and we eventually found an SMS inhibition hit (Dy105). Then the inhibitory of Dy105was verified by the screening method reported in chapter2. In this chapter, structure modifications of Dy105were made by using methods of bioisosteric principle, skeleton hopping and similarity search. A series of small molecules SMS inhibitors were discovered, which included a number of new skeleton compounds such as3-4e,3-7b and3-10L, which could be used as new leads. The structure-activity relationship studies revealed the binding mode of Dy105with SMS. Also, the structure-activity relationships were used for further optimizing of hSMS1three-dimensional model. These results provided an important clue for the discovery of new SMS inhibitors. In addition, in this chapter, Dy105and3-4e were employed for preliminary pharmacology studies in vivo. And the results showed both Dy105and3-4e were able to play SMS inhibition in vivo, these findings strong support for using these compounds as tools for studies of relationship between SMS and atherosclerosis. In chapter4, In order to further develop the structural diversity of small molecule inhibitors of SMS, and better verify the potential of SMS as a drug target, the dynamics optimized hSMS1three-dimensional structure model was used for virtual screening of the SPECS compound libraries. A new skeleton SMS inhibitor Dx121(IC50=12μM) was found, and its preliminary modifications were made.15title compounds were synthesized and evaluated by screening method reported in chapter2. The results showed even a small modification of Dx121can decrease the activity extremely. The binding model of Dx121with ASMS1showed Dx121as a long flexible chain compound, sit in the narrow pocket of ASMS1from the outside of hydrophobicity pocket to the bottom of the active pocket. It explained that even a small modification could lead to the loss of the active conformation and the SMS activity disappeared. Based on this analysis, we have developed a plan to further optimize Dx121. These results paved a way for the discovery of new SMS inhibitors.