Studies of the biological activity of naamidine A

Malignant cells can occur due to deregulation of growth factor cascades. The binding of epidermal growth factor (EGF) to its receptor culminates in the activation of the MAP kinase pathway. This pathway has been identified as a vital link between membrane-bound Ras and nuclear events and therefore has great pharmacological potential as a target of chemotherapeutic drugs. Naamidine A is a dibenzylated 2-aminoimidazole alkaloid from the calcareous sponge Leucetta chagosensis which was reported to exhibit potent inhibition of EGF-stimulated DNA synthesis in high-throughput screens. Proliferation inhibition data collected in a cell line transfected with the EGF receptor (EGFR) gene showed modest cytotoxicity, and preliminary in vivo data demonstrated promising squamous cell carcinoma growth inhibition. Studies were undertaken to elucidate the molecular target(s) of naamidine A and to further investigate the mechanism of action. Naamidine A showed a significant effect on the mitogenic response by completely halting DNA synthesis in A-431 cells after 30 h. Also, cells examined in this time point were shown to be arrested in the G1 phase of the cell cycle. Treatment with naamidine A also produced charges in the phosphorylation states of the extracellular signal-regulated kinases (ERKs). These enzymes propagate the activation of several proteins, which can trigger proliferation, differentiation, or cell death. The hypothesis of this thesis in that ERK1 and ERK2 are the primary cellular targets of naamidine A in A-431 cells. In vitro and in-gel kinase experiments demonstrated that ERK1 is a primary molecular target for naamidine A. In-gel data, western blotting, and notable sequence similarity to ERK1 demonstrate that ERk2 is also a primary target of naamidine A. Treatment with naamidine A strongly induces the phosphotransferase activity of the ERK enzymes in A-431 cells. Naamidine A is the first known small molecular to cause this effect on the ERK kinases. This type of robust ERK signal, previously only achieved by the transfection of activated Raf of MEK, has been shown to induce and sustain increased levels of p21[CIP1] in various cell lines in the G1 phase of the cell cycle. The increase levels of p21[CIP1] bind to and inhibit cdk2, which cannot complex with cyclin E. causing cells to arrest in late G1.