The improved design of antineoplastic drugs is enabled by a knowledge of the drug pharmacophore and its binding surface on an intracellular target. Dequalinium, an antineoplastic agent that inhibits carcinoma growth in a number of animal models, is known to inhibit protein kinase C (PKC), a critical component in signal transduction pathways. Irradiation with 365 nm light photoactivates dequalinium in vitro causing it to modify PKC covalently and consequently to cause irreversible inhibition of PKC activity. Crosslinking of the drug in this manner provides a valuable tool for identifying drug recognition sites on the protein. The first objective of this proposal is to determine the binding surface(s) of deg=qualinium on PKC by use of genetic and chemical approaches (Specific Aim 1). Deletion mutants and site-directed mutagenesis will identify binding sites in the regulatory domain of PKC. In order to identify binding site(s) in the catalytic domain, crosslinking of PKC-beta1 with photoactivated 3H-dequalinium will be carried out, followed by proteolysis and microsequencing of tritiated peptide fragments. The functional significance of these sequences will be verified by site-directed mutagenesis. The molecular interaction of dequalinium with peptide sequences identified by the radiolabeling and genetic studies will be modeled by use of computer-assisted energy minimization analysis. The intention of these studies is to obtain a three-dimensional model of the interaction between dequalinium and pKC, in order to design and synthesize novel dequalinium analogues (Specific Aim 2). The second objective of these studies is to use photoactivated dequalinium or a more potent analogue to inhibit intracellular PKC (Specific Aim 3), and to intervene in PKC-mediated metastasis of B16 melanoma cells by comparing the effects of novel dequalinium analogues (Specific Aim 4). In order to determine the mechanistic significance of PKC activity to B16 cell metastasis, B16 cells will be either depleted of PKCalpha (by anti-sense methodology), or genetically engineered to overproduce PKCalpha or a mutant lacking the dequalinium binding site, and the metastatic activity of these cells will be assayed in syngeneic B57CL/6 mice; the anti-metastatic effect of dequalinium/UV in these modified cell systems will verify whether the drug is selecting PKC as a target (Specific Aim 5). Elucidation of the molecular pharmacology of dequalinium coupled with studies of photoactivated dequalinium with intact B16 melanoma cells could lead to improved chemotherapy for cutaneous lesions.
