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
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 Fellow
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
Anionic species are involved in various chemical and biological processes where phosphate groups
are residue commonly involved in half of biological protein interaction reactions6. We studied the
selectivity of the receptor L3(Zn)4 on different anionic phosphate species by using indicator
displacement assays (IDAs) are a simple and effective method of determining binding affinity
between receptors and analytes7. 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-phospho-serine, 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.
We believe that the formation of intramolecular hydrogen bonds stabilize the pyrophosphate in
between two DPA-Zn units8 (see Figure 9).
Figure 9 - Possible binding modes for R1-pyrophosphate
The titrations also showed selectivity between ATP/ADP and AMP which can be explained by the
number of four recognition units on L3(Zn)4 which have tendency to recognise multi-phosphate (see
Figure 10). However, the high selectivity for pyrophosphate over ATP was expected and can be
attributed in part to the difference in the total anionic charge density involved in the binding. O-P
oxygen atoms have a relatively smaller total negative charge in ATP to complex Zn2+ compared to
those of PPi8. This results in the reduction of the binding affinity. Another factor is the rigidity of the
peptide scaffold, the presence of two β-turns9 gives little flexibility, which can interfere with the
larger ATP and ADP binding.
Figure 10. 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.
Inositol phospholipids have emerged as key players in a wide variety of cellular functions.
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is by far the most abundant of all
phosphoinositides and it has attracted much attention in recent years due to its important role in
numerous cellular signaling events and regulations, which in turn impact several human diseases10.
This particular lipid is 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, the affinity of Pi4P and Pi(4,5)P2 is lower with our receptor which can be
explained by the presence of the four recognition motif which will have a stronger affinity for biphosphate compared to monophosphate. However, the affinity is strong (74.95 ± 3.89 µM). These
results are extremely promising and are currently being written for publication.
Figure 11. 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 my fellowship, I developed three different ligands. The first – L1 – was a
polyaminocarboxylate based ligand with four recognition units is currently being tested as a contrast
agent for apoptotic cells to validate the importance of increasing the number of recognition units.
The second ligand – L3 – with 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 inhibition. L2, the third 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.
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