A cell free phosphorylation method to assess the utility of new
nucleotides as Nucleotide Reverse Transcriptase Inhibitors
Phiyani J. Lebea, Moira L. Bode, Kgama Mathiba and Dean Brady
CSIR Biosciences, Ardeer Rd., Modderfontein, 1645, South Africa
Background and Objectives
The acyclic nucleoside phosphonates such as tenofovir have proved to be effective against a wide variety of DNA virus and retrovirus infections1. Generally, phosphonates need to be converted into a
prodrug to improve their bioavailability. Secondly, they have to be recognised by the host cell kinases for subsequent phosphorylation into triphosphates. Only after both of these two steps will they be
effective as HIV-1 reverse transcriptase inhibitors. Therefore, the development of assays that can separate bioavailability from phosphorylation and enzyme inhibition are critical for high throughput screening
of potential nucleotide reverse Transcriptase Inhibitors (NtRTIs). The objective of this study was to evaluate the cell-free phosphorylation of tenofovir and UMP, as test cases, using mammalian cell extracted
nucleotide kinases. Further studies would then concentrate on cell-free phosphorylation of new compounds prepared as possible reverse transcriptase inhibitors to assess their capacity for phosphorylation
as well as linking this directly to testing HIV-1 reverse transcriptase enzyme inhibition potential in a single assay.
Figure 3
β-NADH depletion as a function of nucleotide monophosphate phosphorylation to its triphosphate product
Methods
Control (No enzym e) vs Assay control (UMP)
Intact Caco2 and HeLa cells were fractionated into cytosolic and membrane proteins and the
total compartmentalized native proteins were quantified. Substrate (monophosphate and
phosphonate) phosphorylation was initiated by addition of the isolated total protein mixture with
pyruvate kinase as the secondary phosphorylation catalyst. Lactate dehydrogenase was used in
the reaction to catalyse conversion of the resultant pyruvate to lactate with concomitant
oxidation of β-NADH cofactor2. The reaction scheme is shown in figure 1. The amounts of mono-,
di- and triphosphate present in the reaction mixture were quantified using HPLC.
1.22
1.2
Optical density
1.18
Figure 1
Reaction scheme of the phosphorylation of phosphonates and natural nucleoside monophosphates
y = -0.0044x + 1.206
R2 = 0.9676
1.16
1.14
1.12
y = -0.0231x + 1.1935
R2 = 0.9986
1.1
1.08
ATP + NMP
NMP kinase (cellular protein mixture)
ADP + NDP
(1)
1.06
0
1
2
3
4
5
6
Time (minutes)
Control
ADP + NDP + 2PEP
Pyruvate kinase
ATP + NTP + 2Pyruvate
UMP
Linear (Control)
Linear (UMP)
(2)
Figure 4
2Pyruvate + (2NADH + H+)
2Lactate + 2NAD+
LDH
β-NADH depletion as a function of nucleotide monophosphate (UMP) and phosphonate (tenofovir)
phosphorylation to their respective triphosphate products
(3)
Beta-NADH depletion as a function of nucleoside phosphorylation
1.2
1.18
Figure 2
1.16
Optical density
Wavelength scan (beta-NADH)
5
4.5
y = -0.0146x + 1.1925
R2 = 0.9844
1.14
1.12
1.1
y = -0.0191x + 1.1754
R2 = 0.9919
4
Optical density
1.08
3.5
1.06
3
0
1
2
3
2.5
4
5
6
Time (minutes)
Tenofovir
2
UMP
Linear (Tenofovir)
Linear (UMP)
1.5
1
Figure 5
0.5
0
0
50
100
150
200
250
300
350
400
450
HPLC quantification chromatograms of the phosphorylation products of tenofovir and UMP (insert). The
left panel indicates diphosphate formation from tenofovir and UMP. The right panel shows triphosphate
product yields of tenofovir and UMP.
7.680 - ATP
mAU
160
120
140
5.109
Tenofovir diphosphate
7.682 ATP
100
100
40
References
40
20
0
0
7.696 - ATP
60
7.725 ATP
80
20
20
The first experiment was conducted using only the extracted total cellular proteins to confirm the
phosphorylation of the primary substrates (UMP and tenofovir) to their diphosphates (reaction 1,
figure1). Figure 5 shows the presence of tenofovir diphosphate and uridine diphosphate (dotted
lines) in significant quantities when compared to the blank sample (solid lines). In the second
experiment pyruvate kinase (reactions 1 & 2, figure 1) was added together with the cellular total
protein mixture to convert the diphosphate into the triphosphate form. Although UMP is converted to
UTP, tenofovir is mainly converted to its diphosphate and not to the triphosphate as shown in figure
5. The lack of triphosphorylation of tenofovir is due to competition with ADP as a substrate which is
preferentially phosphorylated over the artificial nucleoside analogue.
100
5.112 - ADP
40
120
7.191 - UTP
5.071 - ADP
20
60
7.701 ATP
80
140
4.831 - UDP
100
mAU
3.391 - UMP
60
5.108 - ADP
40
4.660 - Tenofovir diphosphate
3.392 - UMP
60
mAU
120
80
7.683 - ATP
80
120
5.067 - ADP
The optimal wavelength with which to monitor β-NADH oxidation was determined to be 340 nm as
shown in figure 2. The β-NADH was monitored at 340 nm throughout the initial 5 minutes of the
reaction to compute the reaction turnover and thus calculate the activity of the nucleotide
monophosphate kinase. The phosphorylation of both the natural substrate (UMP) and tenofovir
using the extracted total cytoplasmic kinases as well as utilising membrane bound total proteins
was shown to be possible since there was considerable oxidation of β-NADH compared to the
control sample (figure 3 and 4). The cytoplasmic reaction rate of substrate phosphorylation was
found to be 2.7 x 10-2 for the natural substrate uridine monophosphate (UMP) while that of tenofovir
was 1.7 x 10-2 units/ml of enzyme. Furthermore the cytosolic conversion rate of the natural substrate
(UMP) was 20% faster than that of the same amount of protein extract from the membrane bound
total protein.
7.709 ATP
3.362 - Tenofovir
mAU
7.676 - ATP
Results and Discussion
3.363 - Tenofovir
Wavelength (nm)
0
2
4
6
8
10
12
14 min
0
0
2
4
6
8
10
12
14 min
0
0
2
4
6
8
10
12
14
min
0
2
4
6
8
10
12
14
Conclusions
Cell free phosphorylation of mono- and diphosphate nucleotides as well as phosphonates has been
shown to be possible, providing a method for assessing the suitability of newly designed compounds as
possible NtRTIs in a cell free system. Relative rates of phosphorylation between the cytosol and
mitochondria can be determined and used as a means of evaluating the mitochondrial capacity to
process the inhibitor compounds. Furthermore, even before the newly synthesized phosphonates are
evaluated as possible inhibitors of the HIV-1 reverse transcriptase, their potential phosphorylation in the
cells of choice can be tested...
1. De Clercq, E. (1997) Acyclic nucleoside phosphonates in the chemotherapy of DNA virus and retrovirus infection. Intervirology 40 (5 – 6): 295 – 303
2. Blondin, C., Serina, L., Wiesmuller, L., Gilles, A.-M. and Barzu, O. (1994). Improved spectrometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase /lactate dehydrogenase coupling system. Anal Biochem. 220:219-221
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