| With the aim of being able to detect disregulated choline lipid metabolism in cancer and elucidate the pathways involved in the malignant transformation, our group is designing and synthesizing a series of phospholipase-specific NIR molecular beacons. The focus of my dissertation has been to characterizing these NIR enzyme-activated fluorescent phospholipids and to assess their potential as suitable in vivo phospholipase molecular beacons. Several key criteria were necessary to be determined a successful in vivo phospholipase probe: (1) sensitivity and specificity to phospholipase activity, (2) adequate signal amplification, (3) high enzyme-substrate affinity allowing for sufficient in vivo activation, (4) ability to overcome biological delivery barriers, and (5) in vivo pharmacokinetics that allows for sufficient tumor absorption and activation.;These criteria have been explored here for the first enzyme-activated fluorescent phospholipid to be synthesized by our group, Pyro-PL-BHQ. Pyro-PL-BHQ is highly specific to phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine (PC). Incubation of Pyro-PL-BHQ with PC-PLC in solution demonstrated a 150-fold amplification. Enzyme kinetics determined an apparent Km of 1.4--1.9 muM and Vmax of 160.7 +/- 29.2 nmol/min/mg. The PC-PLC inhibitor, tricyclodecan-9-yl xanthogenate (D609), inhibited probe activation with an IC50 of 34 +/- 8 muM. Pyro-PL-BHQ was internalized by DU145 human prostate cells, and subsequently activated. Tumor-bearing mice injected with Pyro-PL-BHQ, followed by in vivo NIR imaging, resulted in a 4-fold tumor-specific increase in radiance over background. Tumor probe activation was inhibited with administration of D609. As Pyro-PL-BHQ has satisfied the above prerequisites, we present Pyro-PL-BHQ as the first NIR phospholipase-activated molecular beacon.;Preliminary characterization has been performed on the second enzyme-activated phospholipid synthesized, Pyro-C12-PL-BHQ. This substrate displays high specificity for secreted phospholipase A2 type IB (sPLA2 IB). Enzyme kinetics determined an apparent Km of 2 muM. Tumor-bearing mice injected with Pyro-C12-PL-BHQ presented activation specifically in the gut, an expected result given that sPLA2 IB is primarily a digestive enzyme.;These results demonstrate the feasibility of designing a phospholipase-specific molecular imaging probe capable of directly, sensitively and quantitatively measuring phospholipase activity in vivo, an important contribution to the study of lipid metabolism as it pertains to cancer. |
