Study Of New Biotinylated Analogue Of Dequalinium For Protein Kinase C??Inhibition And Identification Of The Binding Sites

2027 words - 8 pages

Specific Aims

Protein Kinase Cα (PKCα) is well known for its role in cellular signaling pathways that govern normal cell growth and differentiation. Due to its role in tumor formation and metastasis, PKC is being studied as a target for new chemotherapeutic agents. From 1987, Dequalinium (DECA) (quinolinium, 1,1’-(1,10-decanediyl)-bis(4-amino-2-methyl diiodie) has been studied as potent antitumor agent that recognizes several isoforms of PKC in vitro and in cells.

C10-DECA consists of two quinolinium moieties linked by a ten-carbon methylene bridge. Previous studies have shown that, PKC activity in vitro can be irreversibly inhibited when treated with 250nM of DECA and irradiated with 366nm of light. DECA directly interacts with catalytic domain of PKCα such that ring moieties bind at two distinct sites in a trans-oid fashion. The identity of these two sites remains unknown. Previous studies show that PKCαthe linker length of DECA. Studies have shown that maximal inhibition is achieved in vitro with a C-14 alkyl linker. In this project we are using biotinylated analogue of C14-DECA to study its PKC inhibition and identify the binding sites. We hypothesize that this DECA analogue will bind the catalytic sites of PKCα and thus inhibit its activation. Following binding, PKCα-DECA complex will be pulled down and further analyzed for the identification of binding sites.

The specific aims of this project are:

1. To identify the catalytic binding site
a. To isolate PKCα and biotinylated C14-DECA complex.
After photoinduced inactivation of PKCα with biotinylated C14-DECA, the complex will be isolated using streptavidin-agarose resin.
b. To identify the ligand bound peptide fragment.
The purified PKCαDECA complex will be trypsinized and the fragments will be identified by MALTI-TOF.

2. To do a comparative study of PKCα inhibition by new biotinylated analogue of C14-DECA vs conventional C14-DECA.
The biotinylated DECA analogue should have similar PKC inhibition compared to C14-DECA analogue. It will be studied using phosphotransferase activity of PKCα.

3. To determine cell motility inhibition by new biotinylated analogue of C14-DECA.
Since DECA is a potent PKCα inhibitor, inhibition of this enzyme should limit motility of cancerous cells. Wound healing assay will be performed to measure the motility of cells under influence of DECA drugs.

The long-term goal of this project is to develop better PKCα inhibitors. From our experiments we will determine if the biotinylated C14-DECA analogue is as much efficient as conventional C14-DECA analogue for PKCα inhibition. The biotin tag will assist in pulling down and isolating probed PKCα and thus utilize it for identification of binding sites. The identification of binding site will provide important information on further modification of DECA and development of new drugs for PKCα inhibition.

Background and Significance

PKCαand Cancer: ...

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