M-238
Differences in Incorporation and Excision of Adenosine Analogues
GS-9148 and Tenofovir by HIV-1 Reverse Transcriptase
1
Scarth ,
2
White ,
2
Miller ,
McGill University
Department of Microbiology & Immunology
Lyman Duff Building (D6)
3775 University Street
Montreal, Quebec
Canada
H3A 2B4
1
Götte
Brian J
Kirsten L
Michael D
Matthias
1McGill University, Montreal, PQ, Canada; 2Gilead Sciences, Foster City, CA, USA
Results
←Tenofovir
←Tenofovir
←GS-9148
←GS-9148
←GS-9148
Primer
←GS-9148
←Tenofovir
←GS-9148
←Tenofovir
←GS-9148
←GS-9148
←GS-9148
←Tenofovir
←Tenofovir
←Tenofovir
←Tenofovir
←GS-9148
←GS-9148
←Tenofovir
←Tenofovir
←GS-9148
←GS-9148
←Tenofovir
←GS-9148
←Tenofovir
-18
-17
Purine
Pyrimidine
Total
GS-9148
10
3
13
Tenofovir
1
8
9
Equal
5
7
12
Incorporation at sites in Figure 1 were determined
to favour either GS-9148 , tenofovir or either NRTI
equally. The nature of the 3’ end of the primer,
either purine or pyrimidine, at each incorporation
site was matched to the NRTI favoured at that site.
A pattern is seen with incorporation of GS-9148
being favoured after the incorporation of purines (A
or G) and tenofovir after pyrimidines (C or T).
Figure 1. Scanning experiments on various randomly selected HIV sequences. 50 nM of each indicated primer/template
was incubated with 250 nM RT, 10 µM dNTP mix and 100 µM of indicated NRTI for 10 minutes. Pausing sites are indicated
on the gel and corresponding template sequences.
We used a variety of complementary biochemical assays,
including enzyme kinetics, dead-end complex (DEC) formation
assays, and site specific footprinting experiments to compare
the effects of GS-9148 and tenofovir on DNA synthesis and NRTI
excision by HIV-1 RT.
Pre translocation
Post translocation
Figure 3. Effect of next nucleotide on translocation. (A) An Fe2+
footprinting reaction was carried out in the presence of increasing
concentrations of dNTP causing a shift from pre to post translocation.
GS-9148 shifts from pre to post translocation under lower concentrations of
dNTP. (B) The experiment was repeated under a narrow range of dNTP to
produce the IC50 values shown in the above graph. PFA (foscarnet) is used as
a control to trap a pre translocation complex.
Conclusions
A
GS-9148
Experimental Scheme
Tenofovir
[dNTPs]
Step 1
Formation of multiple pausing sites
Full length
Pausing sites
3’CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAAT
TAATCATCTTTAAACATGTC + dNTPs and NRTI
B
Single Position Excision Assays
Rate
(min-1)
Rate
(min-1)
Fe2+causes
Addition of
cleavage via
the RNase H active site at template
positions -17 and -18 depending on
the translocation status of RT4.
P N
-17
NRTI can only be excised from RT in the pre translocation conformation
while the binding of the next nucleotide causes the formation of a deadend complex in the post translocation conformation, preventing excision.
DEC can be monitored through inhibition of excision and footprinting5.
C
Step 2
Rescue in presence of physiological
concentration of PPi and increasing
concentrations of dNTP for DEC formation
• GS-9148 and tenofovir, both adenosine analogues, show
sequence-specific differences in incorporation site
preference
• Subtle decreases in the rate of incorporation of GS-9148
as compared to tenofovir are compensated by diminished
rates of GS-9148 excision
3’CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAAT
TAATCATCTTTAAACATGTCTTTN
TAATCATCTTTAAACATGTCTTTACCTTTTCCTTCCCTTTTN
TAATCATCTTTAAACATGTCTTTNCCTTTTCCTTCCCTTTTAN
Fe2+-based site specific footprinting
-18
B
• Rates of incorporation and excision are both major
determinants of the overall effect of NRTIs on DNA
synthesis by HIV-1 RT
Methods
P N
Tenofovir
A
Table 2. Summary of Figure 1
Sites of incorporation in relation to the
nature of the 3’-end of the primer
←Tenofovir
←Tenofovir
←Tenofovir
←GS-9148
←GS-9148
←GS-9148
←GS-9148
←Tenofovir
←Tenofovir
←GS-9148
←Tenofovir
←GS-9148
←GS-9148
←GS-9148
←GS-9148
←GS-9148
←GS-9148
←GS-9148
←GS-9148
Set 4
Set 3
3’CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATA
TAATCATCTTTAAACATGTC
Full length
GS-9148
3’TCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTC
GGTATGTTATGAGGTCATAA
Set 2
Set 1
3’AACAAAATCCAGACATAGTTATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGG
TGTTTTAGGTCTGTATCAAT
GS-9148 is an investigational nucleotide analogue reverse transcriptase
inhibitor (NtRTI). GS-9148 is orally administered as its lymphoid celltargeted amidate prodrug, GS-9131. GS-9148/GS-9131 have a unique
resistance profile including maintained activity against nucleoside reverse
transcriptase (RT) inhibitor resistance mutations M184V, K65R, L74V and
multiple thymidine analog mutations1,2.
Like tenofovir, GS-9148 is an adenosine analogue, although in this case,
the base and phosphonate moieties are linked with a unique cyclic sugar
ring, while tenofovir is acyclic (see structures below). The effects of the
chemical nature of the linker on both efficiency of nucleotide incorporation
and its excision at multiple sites remain elusive. It has previously been
reported using a single incorporation site that GS-9148 displays
approximately 5-fold reduced incorporation efficiency relative to dATP and
tenofovir-DP under pre-steady state conditions3.
To further understand the effects of these structural differences and to
better elucidate the mechanism for the improved resistance profile of GS9148, we compared the incorporation and excision of these nucleotide
analogues.
p. 514.398.1317
f. 514.398.7052
3’AGAGGAGCTGAGACAACATCTGTTGAGGTGGGGACTTACCACACCAGACAAAAAACATCAGAAAGAACCT
CTCCTCGACTCTGTTGTAGA
Background
[email protected]
[email protected]
Single Position Dead End Complex Formation Assays using
excision as a readout
N.D.
3’CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAAT
TAATCATCTTTAAACATGTCTTTACCTTTTCCTTCCCTTTTAAAGTTTTTA
TAATCATCTTTAAACATGTCTTTACCTTTTCCTTCCCTTTTN
TAATCATCTTTAAACATGTCTTTNCCTTTTCCTTCCCTTTTAAAGTTTTTA
• GS-9148 more efficiently prompted DEC formation in
both excision and footprinting experiments
• Rapid DEC formation provides a higher degree of
protection for GS-9148 against a background of TAMs by
slowing the rate of primer unblocking
References
Some positions will be rescued while some will
be subject to DEC formation
Primer
Set 3
3’CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATA
TAATCATCTTTAAACATGTC
Figure 2. Excision and Dead-end complex formation susceptibility assay. (A) 50 nM primer is incubated with 10 µM NRTI and 1 µM dNTP mix for 10 minutes to establish multiple pausing
sites (PPi minus lane). A solution of 50 µM PPi and varying concentrations of dNTP mix are then added for 10 additional minutes allowing for excision (seen as the disappearance of pausing
sites). Increasing concentrations of dNTP block excision by dead-end complex formation (seen as persistence of pausing sites). Two sites of interest are indicated, these sites were examined in
time course experiments for rate of excision (B) and single position DEC susceptibility (C) using TAMs (M41L/T215Y/L210W/D67N) containing RT and with ATP as a pyrophosphate donor.
1 Cihlar, T. et al. Design and profiling of GS-9148, a novel nucleotide analog active against nucleosideresistant variants of human immunodeficiency virus type 1, and its orally bioavailable phosphonoamidate
prodrug, GS-9131. Antimicrob Agents Chemother52, 655-65 (2008).
2 White et al., SADR 2008 poster 38
3 White et al., ICAAC 2006 poster H-0251
4 Marchand, B. & Gotte, M. Site-specific footprinting reveals differences in the translocation status of HIV-1
reverse transcriptase. Implications for polymerase translocation and drug resistance. J Biol Chem278, 3536272 (2003).
5 Marchand, B. et al. Effects of the translocation status of human immunodeficiency virus type 1 reverse
transcriptase on the efficiency of excision of tenofovir. Antimicrob Agents Chemother51, 2911-9 (2007).
Acknowledgements
We would like to thank Suzanne McCormick for excellent technical support.
This work was funded in part by the Canadian Institute for Health Research and Gilead Sciences.