Synthesis and Evaluation of Cytosolic Phospholipase A₂ Activatable Fluorophores for Cancer Imaging

Abstract

Activatable fluorophores selective to cytosolic phospholipase A2 (cPLA2) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA2 was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA2 when compared to esterification with palmitic acid. In vitro analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a non-selective phospholipase (sPLA2 group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally in vitro and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA2. In contrast to the in vitro analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA2 enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA2 resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA2 selective activatable fluorophore capable of detecting cPLA2 in triple negative breast cancer cells.

Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study

“…The aromatic hydroxyl group can be converted into an ester (or ether) masking fluorescence. The cleavage by an appropriate enzyme or active species (reactive oxygen or nitrogen species) would restore fluorescence [49].…”

Focus on the glycerophosphocholine pathway in choline phospholipid metabolism of cancer

“…For this purpose, the Delikatny group recently synthesized and preclinically tested several phospholipase‐activatable “smart” fluorescent imaging probes, which are specifically activated by PLC only or by cPLA2 only . Recently, the same group developed a second series of cPLA2‐based imaging agents with improved activity . In these smart imaging agents, AA was coupled to red‐shifted fluorophores, which maximized light tissue penetration, and which achieved cPLA2 specificity by employing AA, because cPLA2 is the only phospholipase with specificity for AA‐containing phospholipids .…”

“…Because of their deep tissue penetration capabilities, these novel near‐infrared fluorescent smart probes can be used in vivo. One of the agents has shown the potential to be imaged in vivo in triple‐negative breast cancer mouse models in which cPLA2 was overexpressed . Continuing the development of cPLA2‐activatable imaging probes to determine in vivo cPLA2 activity will help with future translation of cPLA2 inhibitors as therapeutic targets for cancer treatment.…”

Imaging of cancer lipid metabolism in response to therapy

“…Previous studies had shown that arachidonic acid esterified to visible fluorophores such as 7‐hydroxycoumarin were highly specific for cPLA2 and displayed hydrolysis kinetics similar to those of native substrates . We adapted this caged fluorescence protocol using fluorophores that emit in the red wavelength range (660–680 nm) to create cPLA2 sensitive probes suitable for in vivo imaging . Probe selectivity could be modulated by choice of fatty acids and fluorophores, with DDAO 6,8‐dichloro‐7‐hydoxy‐9,9‐dimethylacridin‐2( 9H )‐one arachidonate performing the best in vitro, in cells, and in tumors.…”