Study On The Mechanism Of Inhibitors With Target Methionine Aminnopeptidase 1(MetAP1)

Methionine aminopeptidase (MetAP) carries out the important function of removing the initiator methionine residue from nascent peptides and proteins. MetAPs are potential targets for developing antibacterial, antifungal and anticancer candidates.Photoaffinity labeling is a newly developed technique, which is the combination of molecular biology, cell biology and organic chemistry. The method of photoaffinity labeling enables the direct labeling of target protein through a covalent bond which is photochemically introduced between a ligand and its specific receptor. Thus, photoaffinity labeling could be applied in two levels. If the binding site of a target protein with the particular pharmacophore is not defined, the photoaffinity labeling will give the structural information of receptor binding domain at micro-level. At macro-level, the method is useful for discovery of target protein from the evaluation of affinity by cross-linking. By photoaffinity labeling many proteins involved in diseases were found, and the ways interaction with lead compound were determined. These informations are very important for rational drug design.The work was. to detect the antifungal activity of some inhibitors of Saccharomyces cerevisiae methionine aminnopeptidase 1 (ScMetAP1) in vivo which were got by screening in vitro. And we investigated the interaction of PCAT series compounds with EcMetAP1 using photoaffinity labeling.The first part of this work was to detect the antifungal activity of inhibitors of ScMetAP1. These compounds prevented the growth of WT and Amap2 yeast, but has no influence on â–³mapl yeast. It suggested those compounds act on the target ScMetAPl in vivo.But no perfect relationship between the antifungal activity in vivo and the inhibitory effect on Co(II)-ScMetAP1 in vitro. It is possibly due to the metal ion of MetAP1 in yeast is not Co(II), but other divalent metal ions as Mn(II), Zn(II) or Fe(II). Next we investigated the inhibition on the different divalent metal activated ScMetAP1. The results indicated that these compounds have selective activity for ScMetAP1 activited by different divalent metal. It pave the way for finding anddesigning the potent inhibitior of ScMetAP1 in vivo.The second part of this work was to investigate the interaction of PCAT series compounds with EcMetAPl using photoaffinity labeling at micro-level. According to the structure -activity relationship of the compounds of PCAT, we designed and synthesized inhibitor of EcMetAP1 with Diazirine photoreactive group. This inhibitor was used as a probe and to label EcMetAPl. It was detected by Mass Spectrum that EcMetAP1 covalently labelled the probe successfully. After EcMetAP1/probe complex was digested by trypsin, the peptide covalent labelled with probe was separated and collected by HPLC. Molecular weight of the peptide is 1142 Da by mass spectrum (MS) detection and its sequence is deduced as "DGWTVK". The conclusion is PCAT pyridinium 3-substitute group binds into S1 pocket of EcMetAP1.