Differences in Incorporation and Excision of Adenosine Analogues GS9148 and Tenofovir by HIV1 Revers

1
M-238
McGill University Department of Microbiology
Immunology Lyman Duff Building (D6) 3775
University Street Montreal, QuebecCanada H3A
2B4 brian.scarth_at_mail.mcgill.camatthias.gotte_at_mc
gill.ca p. 514.398.1317 f. 514.398.7052
Differences in Incorporation and Excision of
Adenosine Analogues GS-9148 and Tenofovir by
HIV-1 Reverse Transcriptase
Brian J Scarth1, Kirsten L White2, Michael D
Miller2, Matthias Götte1 1McGill University,
Montreal, PQ, Canada 2Gilead Sciences, Foster
City, CA, USA
Background
Results
GS-9148 is an investigational nucleotide
analogue reverse transcriptase inhibitor (NtRTI).
GS-9148 is orally administered as its lymphoid
cell-targeted 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 GS-9148, we compared the incorporation and
excision of these nucleotide analogues.
Tenofovir
GS-9148
Set 2
Set 3
Set 4
Set 1
Mg(-) NRTI(-) Tenofovir GS-9148
Mg(-) NRTI(-) Tenofovir GS-9148
Mg(-) NRTI(-) Tenofovir GS-9148
Mg(-) NRTI(-) Tenofovir GS-9148
A
Table 2. Summary of Figure 1 Sites of
incorporation in relation to the nature of the
3-end of the primer
Full length


?GS-9148

?Tenofovir
?Tenofovir

?Tenofovir
?Tenofovir
?GS-9148
-18
?Tenofovir
?GS-9148
?Tenofovir
-17

?GS-9148
?GS-9148
?Tenofovir


?GS-9148
?Tenofovir
?Tenofovir

?Tenofovir



B
?GS-9148
?GS-9148

?GS-9148
?Tenofovir
?GS-9148

?GS-9148




?GS-9148
?GS-9148
?Tenofovir
?GS-9148

?GS-9148
?GS-9148


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).
?GS-9148
3AGAGGAGCTGAGACAACATCTGTTGAGGTGGGGACTTACCACACCAG
ACAAAAAACATCAGAAAGAACCT
3TCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAA
ATGGAGAAAATTAGTAGATTTC
?GS-9148
3AACAAAATCCAGACATAGTTATCTATCAATACATGGATGATTTGTATG
TAGGATCTGACTTAGAAATAGG
3CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAA
AAATTGGGCCTGAAAATCCATA
?Tenofovir
?Tenofovir

?GS-9148
?Tenofovir

?Tenofovir
?GS-9148
?GS-9148
?GS-9148
?GS-9148

?Tenofovir
?Tenofovir
?GS-9148
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.
?GS-9148
?GS-9148
GGTATGTTATGAGGTCATAA
TGTTTTAGGTCTGTATCAAT
TAATCATCTTTAAACATGTC

CTCCTCGACTCTGTTGTAGA
Primer
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.
Conclusions

• Rates of incorporation and excision are both
  major determinants of the overall effect of NRTIs
  on DNA synthesis by HIV-1 RT
• 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
• 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

Methods
Mg(-) PPi(-) 12µM 25µM 50µM
100µM 200µM
Mg(-) PPi(-) 12µM 25µM 50uµM
100µM 200µM
A
Tenofovir
GS-9148
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.
Experimental Scheme
B
dNTPs
Single Position Excision Assays
Step 1 Formation of multiple pausing sites
Full length
Rate (min-1)
Rate (min-1)
Pausing sites
3CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAA
AAAT
TAATCATCTTTAAACATGTC dNTPs and NRTI
? position 2
3CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAA
AAAT
? position 1
TAATCATCTTTAAACATGTCTTTN
Fe2-based site specific footprinting
TAATCATCTTTAAACATGTCTTTACCTTTTCCTTCCCTTTTN
TAATCATCTTTAAACATGTCTTTNCCTTTTCCTTCCCTTTTAN
C
Single Position Dead End Complex Formation Assays
using excision as a readout
Step 2 Rescue in presence of physiological
concentration of PPi and increasing
concentrations of dNTP for DEC formation
Pre translocation
P N
Addition of Fe2causes cleavage via the RNase H
active site at template positions -17 and -18
depending on the translocation status of RT4.
N.D.
-18
3CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAA
AAAT
Post translocation
TAATCATCTTTAAACATGTCTTTACCTTTTCCTTCCCTTTTAAAGTTTTT
A
TAATCATCTTTAAACATGTCTTTACCTTTTCCTTCCCTTTTN
TAATCATCTTTAAACATGTCTTTNCCTTTTCCTTCCCTTTTAAAGTTTTT
A
References
P N
Some positions will be rescued while some will be
subject to DEC formation
1 Cihlar, T. et al. Design and profiling of
GS-9148, a novel nucleotide analog active against
nucleoside-resistant 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, 35362-72 (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).
Primer
-17
Set 3
3CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGGAAAATTTCAA
AAATTGGGCCTGAAAATCCATA
TAATCATCTTTAAACATGTC
?
?
2
1
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.
NRTI can only be excised from RT in the pre
translocation conformation while the binding of
the next nucleotide causes the formation of a
dead-end complex in the post translocation
conformation, preventing excision. DEC can be
monitored through inhibition of excision and
footprinting5.
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.