Discovery Of Novel Allosteric Activators For Cystathionine β-synthase And Donors For Hydrogen Sulfide And The Study Of Their Mechanisms

Endogenous hydrogen sulfide（H₂S）gas is recently reported as the third gaseous signal molecule.It has significantly physiological functions in human.Cystathionineβ-synthase（CBS）is one of the key enzymes in the production of H₂S in human bodies.It contains an allosteric CBS domain in the C-terminus,which can regulate the activity of the enzyme.The pathogenic missense mutations in the CBS domain are related to the homocystinuria disease.To date,no specific and allosteric activator of CBS has been identified,and the allosteric regulation mechanism of this enzyme is not completely revealed yet.In this study,I constructed a method for analyzing the level of H₂S in cells,which is based on the Laser Scanning Confocal Microscope.And I also developed a LC-MS/MS method to analyze the activity of CBS activators on CBS WT and its pathogenic mutants in vitro,in cells and in animal levels.Moreover,a preliminary study of the mechanism and the studies of the structure-activity of activators were carried out.Our results revealed that the Compound M was a novel,specific allosteric activator of CBS,it is bioactive in vitro with an EC₅₀of 20μmol/L,and in cells with 50μmol/L as well as in animals.Biochemical and biophycial experiments demonstrate that it specifically targeted at the CBS domain of the enzyme.In addition,the Compound M can also effectively activate the pathogenic mutants of homocystinuria in vitro and in the cellular level.Pro422 and Gln445 of hCBS were also discovered as the key residues for the binding of Compound M to CBS.We also found a new H₂S donor AZD1152,it had a novel structure and was effective in the cell level.It can release H₂S by reacting with Cysteine and Homocysteine to achieve the long-lasting release of H₂S at specific sites.In conclusion,I established the analysis methods to analyze the activities,specificities,and mechanisms of novel compounds targeting to the CBS and its pathogenic mutants in vitro,in cells and in animals.And I discovered some of these activators were novel,specific and bioactive CBS allosteric activators or the H₂S donors.These compounds can be used as the molecular probes to further promote our understanding of the allosteric activation mechanism of CBS and its pathological inactivation,and they can also provide us the new treatment strategy and candidates of drug leads for the related diseases.