The overall aim of this application is to develop methods to image the expression and activity of phospholipases in vivo. A multi-modality approach with sequential magnetic resonance imaging/spectroscopy (MRI/MRS) and optical imaging using near infrared (NIR) fluorophores will be employed to obtain information that is not available from either technique separately. The key hypothesis to be addressed is that in vivo activation of phospholipases can be measured by determining the changes in MR-visible lipid metabolite levels together with measuring fluorescence released by enzymatic hydrolysis of a self- quenching NIR phospholipid fluorophore. We will compare and validate the MR spectroscopy with results obtained using NIR optical imaging in tumor models in which changes in choline metabolite levels associated with tumor progression and response to therapy are well known. Since phospholipases have been implicated in a wide range of pathologies including cancer, arthritis and Alzheimer's disease, this study will supply critical in vivo data on the specific biochemical pathways involved in these diseases. The hypothesis will be tested by the achievement of the following specific aims:
Aim 1) : Design and synthesis of chiral lipid-based self-quenching fluorophores containing a stable pyropheophorbide NIR fluorophore, coupled to a dark quencher. The fluorophore will be attached to different positions of the glycerol backbone to interrogate the activity of different phospholipase isoforms. The quencher linkage will be attached at different points on the glycerol backbone to distinguish between phospholipase types. Structure-activity studies will be performed by synthetically inserting linkers of various length to determine the effects of steric hindrance on probe activity.
Aim 2) : In vitro characterization of the synthesized probes. This will include test-tube demonstration of the sensitivity and specificity of the probes to various phospholipase types and isoform and detailed measurement of kinetics of catalysis and inhibition. We will further characterize the delivery and activation of these self- quenching fluorophores in cultured cell lines.
Aim 3) : Demonstration of in vivo delivery, bio-distribution and subsequent activation of phospholipases in tumor xenograft models of prostate cancer and non-Hodgkin's lymphoma, followed by dual modality molecular imaging using 1H MR spectroscopy and optical imaging. These data will supply critical information on the specific lipid catabolic pathways involved in the onset and subsequent treatment of disease.
To develop near-infrared fluorescent beacons for the in vivo detection of phospholipase activity via dual optical imaging and MR spectroscopy.
|Arlauckas, Sean P; Popov, Anatoliy V; Delikatny, Edward J (2014) Direct inhibition of choline kinase by a near-infrared fluorescent carbocyanine. Mol Cancer Ther 13:2149-58|
|Mawn, Theresa M; Popov, Anatoliy V; Delikatny, E James (2012) A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging. Anal Biochem 422:96-102|
|Delikatny, E James; Chawla, Sanjeev; Leung, Daniel-Joseph et al. (2011) MR-visible lipids and the tumor microenvironment. NMR Biomed 24:592-611|
|Popov, Anatoliy V; Mawn, Theresa M; Kim, Soungkyoo et al. (2010) Design and synthesis of phospholipase C and A2-activatable near-infrared fluorescent smart probes. Bioconjug Chem 21:1724-7|