Computer Aided Design, Synthesis And Activity Study Of L-Lysine Derivates As Aminopeptidase N Inhibitors

Objective: Aminopeptidase N (APN), as known as CD13, is a member of zinc dependent metalloproteinase. It is widely expressed on the surface of renal and intestinal brush border cells, synaptic membranes in central systems and APN is over-expressed on the surface of some tumor cells. APN is demonstrated to play a key role in the process of tumor growth, invasion and metastasis. The functional aspects are briefly summarized: (1). Degrading extracellular matrix, promoting the growth and invasion of tumor cells; (2). As a regulator of novel vessels, promoting the angiogenesis of tumor tissues; (3). Degrading immunoactive substance and bioactive peptides, such as interleukin, thymopentin, enkephalin and so on.More and more researches focus on the field of anti-aminopeptidase N drug as the enzyme has close relationship with the: some cancers. Most of the research on the anti-aminopeptidase N drug is to find aminopeptidase N inhibitor possessed inhibitory activity. Bestatin, the first marketed drug as aminopeptidase N inhibitor, now is clinically used to prolong the survival of patients with acute adult nonlymphcytic leukemia. In recent years, many natural aminopeptidase N inhibitors have been reported, such as Probestatin, Amastatin, Curcumin. Moreover, numerous synthetic APN inhibitors have also been reported, such asα-aminophosphonates APN inhibitors,β-amino- thiols APN inhibitors. In our group, we also had reported numerous synthetic small molecular peptidomimetic compounds targeted to aminopeptidase N to find novel scaffolds which possess potential aminopeptidase N inhibitory activities as anticancer lead compounds in the past ten years.We used computer-aided drug design software to design and synthesize a series of compounds. Then we hope to find lead compounds with novel scaffold which possess potential aminopeptidase N inhibitory activity.Methods: According to the active site of APN, the APN inhibitors can be divided into three parts. Part A: heterocyclic rings or hydrophobic groups; Part B: linker with Zinc-Binding-Group (ZBG); Part C: heterocyclic rings or hydrophobic groups. Part A and C was linked by Part BFirst, four rational protein models of APN were built and tested by known active compounds. Secondly, the compounds which has zinc binding groups were picked out from NCI2000 database by SYBYL/Unity. Followly, these compunds were docked into the active site of APN models and the top ranked compounds were picked out. During this program, Bestatin (ID: 265489),Phebestin (ID: 702307),para-hydroxybestatin (ID: 327461) were taken as test compounds. At last, L-lysine derivates as our target compounds were built from top ranked compounds in docking and some known active compounds.As a result, the target compounds are designed rationally and synthesized easily. In addition, enzyme assay, cancer cell (HL-60, ES-2, K562, A549, H7402, 2PLC) proliferation assay and in vivo experiment were processed in this research.Results: We obtained 44 target compounds and all of them are novel without any report by now with the structures identified by IR, ~1H-NMR and ESI-MS.Preliminary activity evaluation against APN showed that compound C7 was better than positive control Bestatin and C20 was equivalent. For D series, compounds D9 and D15 were better than positive control Bestatin and compound D24 was equivalent.The results of in vitro growth inhibition against six tumor cells indicated that most potent APN inhibitors displayed good inhibitory effect against the growth of these tumor cells. Compounds C7,D9,D14,D15,D23 exhibited good potency against the proliferation of HL-60 cell line. Compounds C7,D14,D21,D23 showed better than Bestatin against ES-2 cell line and D23 was equivalent. Compound D21 showed better than Bestatin against H7402 cell line; and C7,D24 were equivalent. Compounds C7,C20,D9,D14,D15,D19,D21,D23,D24 showed better than Bestatin against PLC cell line.The structure activity relationship (SAR) was summarized based on the structure and the activity data of the target compounds with the scaffold L-lysine. Comparative Molecular Field Analysis (CoFAR) was utilized to establish the quantitative structure activity relationship (QSAR) of the target compounds. The steric contour map and the electrostatic contour map of the CoMFA model showed the model had good cross-validated coefficient q~2 and predictive potency.Finally, we obtain a complex of compound D24 and the mutational tricorn interacting factor F3 from thermoplasma acidophilum.Conclusions: In conclusion, based on the virtual screening of APN, L-lysine derivates as APN inhibitors were designed and synthesized. We reported a convenient and economical method of the synthesis of APN inhibitors. Preliminary activity assays showed that most compounds displayed good APN inhibitory effect. Compounds C7,D9,D15 showed better than positive control Bestatin and could be used as lead compounds in the future. We also established a QASR model of target compounds which is beneficial for the design APN inhibitor in the future.