Introduction:
The membranes of mammalian cells consist of a bilayer of a mixture of different phospholipids. The
composition of the phospholipid mixture varies between the inner and outer layers and, in healthy
mammalian cells, anionic species (principally phosphatidylserine, PS) are arranged largely on the
inner layer.1 In some abnormal cells this is not the case and a considerable amount of anionic lipids
are displayed on the outer membrane surface. This phenomenon is especially prevalent in cells
undergoing the early/intermediate stages of apoptosis (programmed cell death),2 tumour
vasculature3, bacteria and viruses.4 The overall aim of the project is to develop new magnetic
resonance imaging (MRI) and positron emission tomography (PET) probes that can be used to detect
apoptotic cells (i.e. cells undergoing programmed cell death) by targeting phosphatidylserine. Such
molecular probes could eventually be used to monitor the extent and speed of onset of apoptosis in
tumours following a treatment. This could therefore be a good indicator of the treatment outcome.5
Figure 1. Simplification of the concept
Summary
During the first year, three bifunctional molecules, two polyaminocarboxylates based molecules and
a molecule based on cyclodecapeptide, were designed and their synthesis started. Details of the
ligands are reported Figure 2. The second year of research was focused on finishing up the synthetic
route to obtain ligands to be tested by biological assays.
Figure 2. Ligands developed during the Fellowship
Results and discussion:
DTPA based ligands, (L1):
The free ligand was obtained after the first year and a molecule containing one gadolinium and four
zinc ions were obtained during the second year (figure 3). This molecule is fully characterised and it is
currently undergoing cell testing (apoptotic vs non apoptotic cells) to validate the efficacy of four
recognition units as compared to two recognition units. If the assays are conclusive the data will be
written up for publication. The molecule L1(Zn)4Gd was presented as a poster during the Eurobic 16
conference last summer.
Figure 3. Ligand L1 with four zincs and one gadolinium
Pyridine based ligands, (L2):
The recognition part was optimised and obtained after the first year. Eight steps out of ten were
optimised for the synthesis route of the imaging part during the second year (Figure 4). The two last
steps should be completed by another member in the group as it is a ligand not previously tested
with gadolinium.
Figure 4. Second synthetic route, dashed arrows represent the step not yet optimized
Cyclodecapeptide ligands, (L3):
Figure 5. Addition of Zn2+
After obtaining the cyclic peptide, four zinc ions were added to be complexed by the DPA motifs
(Figure 5). This molecule was characterized by NMR and UV-Vis (See figure 6).
Figure 6. UV-vis titration of Receptor by PV, Hepes 0.1 M, pH 7.4, top right corner, Job’splot, pH 7.0,
Hepes 0.1 M
Due to the importance of Anionic species6, the selectivity of the receptor L3(Zn)4 were studied on
different anionic phosphate species by using indicator displacement assays (IDAs)7. Pyrocatechol
violet (PV) was chosen as the indicator for the IDAs. The receptor binds to PV changing the colour
from yellow to blue-green, a process which was readily monitored by UV-Vis spectroscopy. Several
ions were tested on the complex receptor: PV = 1 : 4; Adenosine triphosphate (ATP), Adenosine
monophosphate (AMP), Adenosine diphosphate (ADP), pyrophosphate, phosphate, O-phosphoserine, O-phospho-threonine, phosphor-6 Glucose, inositol hexasulfate and phylic acid. This assay
shows selectivity between pyrophosphate and phosphate ions and selectivity between AMP/ADP and
ATP (Figure 7).
Figure 7. Displacement assay of PV by phosphate ions. pH 7.0 hepes buffer 0.1 M
Titrations between the PV/receptor complex and these ions were carried out. They confirmed a high
selectivity for pyrophosphate as only 0.6 equivalents of this ion are added to the complex PV:L3(Zn) 4
to displace PV entirely (Figure 8).
Figure 8. Equivalents of phosphate ions according to absorbance at 595 nm (on the right) and
pyrophosphate ions (on the left) added to PV/receptor complex at pH 7.0 in Hepes 0.1 M. At the
bottom the graph shows a superposition of the two.
The titrations also showed selectivity between ATP/ADP and AMP but a lower affinity compare
pyrophosphate (Figure 9).
Figure 9. Absorbance at 595 nm of Adenosine triphosphate ions (on the right) and Adenosine
diphosphate (on the left) added to PV/receptor complex at pH 7.0 in Hepes 0.1 M. At the bottom the
graph shows a superposition of the two.
The importance of Inositol phospholipids in various diseases10 make them interesting to study. These
particular lipids are recognized in the cell by specific lipid binding domains. In order to determine
whether the receptors can successfully compete with protein domains to bind PPis, competitive
Enzyme Linked Immunosorbent Assays (ELISA)11 were employed. ELISA are routinely used to quantify
the binding of protein domains to their targets. Five PPis where tested with our receptor, Pi(3,4,5)P3,
Pi(4,5)P2, Pi(3,4)P2, Pi3P and Pi4P. Unfortunately for Pi3P, the protein domain FYVE contains a zinc
finger domain, so the results were not conclusive. The ELISA results concerning L3(Zn)4 showed a high
affinity for Pi(4,5)P2, 13.32 ± 1.94 µM (calculated using the method outlined by Orosz and Ovadi12)
compared to Pi(3,4)P2; 243 µM ± 1.5 µM resulting in a difference of ionization state of the phosphate
ions10. The receptor was tested with the tri-phospholipid Pi(3,4,5)P2 and presented no affinity or
recognition for it. To finish, we investigated the affinity between our receptor and a monophosphate
Pi4P. In comparison, an affinity of 74.95 ± 3.89 µM for Pi4P were calculated, lower than Pi(4,5)P2
which can be explained by the presence of the four recognition motif which will have a stronger
affinity for bi-phosphate compared to monophosphate. These results are extremely promising and
are currently being written for publication.
Figure 10. Pi(4,5)P2 1.0 µM, protein domain PLCδ1-PH 10 nM titrated by 0 from 200 µM of receptor;
Pi(3,4)P2, 1.5 µM, protein domain Akt-PH 25 nM titrated by 0 from 200 µM of receptor; Pi(3,4,5)P2
1.0 µM, protein domain GRPI-PH 100 nM titrated by 0 from 200 µM of receptor; Pi4P at 3.0 µM,
protein domain FAPPI-PH 30 nM titrated by 0 from 200 µM of receptor.
Conclusion:
During the two years of the fellowship, three different ligands were developed. The first – L1 – was a
polyaminocarboxylate based ligand with four recognition units which is currently being tested as a
contrast agent for apoptotic cells to validate the importance of increasing the number of recognition
units. L2, the second ligand, is a new polyaminocarboxylate-based ligand containing two recognition
units and requires more time to complete its synthesis. Only two more steps need to be optimized
before complexation and testing. The last ligand – L3 – was designed to be used as contrast agent.
However the preliminary results between phopho-serine and the recognition part has a low affinity
and recognition for this phosphate ion in comparison to pyrophosphate which forced the studies to
different phosphate ion types. Previous and current work in the laboratory study the recognition of
phopho-lipids Phosphatidylinositol. The importance of this lipid is well known and reported. Its four
recognition units has shown high affinity and selectivity for Pi(4,5)P2 compare previous reported
work13 which is the most abundant in cell membrane and also responsible for signalling. These
primary results are really promising for future work in the field of protein-lipid recognition/inhibition.
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