THE DEVELOPMENT AND APPLICATION OF CLICKABLE LIPID ANALOGS FOR ELUCIDATING AND HARNESSING LIPID FUNCTIONS - Click Chemistry in Glycoscience: New Developments and Strategies - page 93

transfer of PS to the outer leaflet of the bilayer during apoptosis, which triggers cell
surface recognition events that result in cell death. In the design for probes of type 37 ,
both a benzophenone photoaffinity and an alkyne secondary tag were included within
the sn -1 acyl chain. These probes were studied for the labeling of prothrombin-1,
which was assessed through CuAAC followed by in-gel fluorescence detection. Here,
it was found that a series of PS probes, including those with inverted stereochemistry,
all labeled prothrombin-1 in a concentration-dependent manner, suggesting that the
stereochemistry does not affect recognition.
Our group has been interested in developing activity probes corresponding to
lipids such as PA ( 20g , Fig. 4.5) and the phosphatidylinositol polyphosphates (PIP n s)
[77]. We recently reported the application of activity probe 38 corresponding to the
headgroup of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3, Figure 4.9b)
[78]. The PIPns were targeted since these lipids bind and activate a litany of protein
targets, and these interactions are commonly aberrant in various diseases [79-83].
We first sought to demonstrate the efficacy of this compound for labeling a known
protein target based on a specific binding interaction. In these studies, probe 38
was effective for the labeling of the PH domain of Akt (Akt-PH), as judged in-
gel fluorescence scanning. Control studies were next undertaken to show that this
labeling event results from a specific binding interaction. First, heat denaturation of
the protein target was observed to abrogate in-gel labeling due to the loss of protein
structure. Additionally, co-incubation of Akt-PH with activity probe 38 along with
an unlabeled headgroup analog as a competing ligand also led to blockage of the
labeling event. Following validation of specific labeling, probe 38 was exploited for
the characterization of target proteins in cancer cell extracts, followed by enrichment
of probe-labeled proteins and mass spectrometry-based proteomics. This enabled
the identification of 265 putative protein targets that were labeled by this probe,
including at least 44 known PI(3,4,5)P3-binding proteins in addition to numerous
novel candidate targets. From these early examples, it is clear that lipid activity
probes provide an efficient means for the collective characterization of protein targets
from complex samples.
4.6 PROBING COVALENT PROTEIN LIPIDATION
An area in which clickable lipid probes have been exploited for extensive biological
investigations involves the elucidation of covalent protein lipidation [3]. A large num-
ber of eukaryotic proteins undergo the covalent addition of lipid units, which plays a
key role in regulating both the function and subcellular localization of the modified
protein, generally by promoting membrane anchoring or cycling between different
membrane compartments. A common modification is the addition of saturated fatty
acid chains, including the cotranslational N -myristoylation of glycine, producing an
amide linkage, and the post-translational S -palmitoylation of cysteine, resulting in a
thioester attachment. The latter case differs among examples of covalent lipidation
as palmitoylation is reversible, allowing for dynamic regulation of lipidation. Prenyl
groups are also added to a number of proteins through the post-translational addition