GS9148Diphosphate GS9148DP, an Active Metabolite of a Novel Adenine Nucleotide Analogue, is an Effec

1
GS-9148-Diphosphate (GS-9148-DP), an Active
Metabolite of a Novel Adenine Nucleotide
Analogue, is an Effective Inhibitor of HIV-1
Reverse Transcriptase (RT)

• KL White, JY Feng, AS Ray, G Laflamme, F Yu, M
  Tsiang, R Wang, M McDermott, MD Miller, R
  Mackman,
• and T Cihlar
• Gilead Sciences, Inc., Foster City, CA, USA

2
Introduction

• GS-9148 (phosphonomethoxy-2-fluoro-2,3-dideoxyd
  idehydroadenosine, Fig. 1) is a novel nucleotide
  HIV-1 reverse transcriptase (RT) inhibitor
  (NtRTI) with a favorable in vitro resistance
  profile against a spectrum of HIV-1 variants with
  all classes of NRTI mutations1
• GS-9131, a mono-phosphonoamidate prodrug of
  GS-9148 (Fig. 1), exhibits good oral
  bioavailablity in vivo and effectively delivers
  GS-9148 and the active diphosphate metabolite
  (GS-9148-DP) into lymphocytes2

3
Objectives

• Define the enzymatic mechanism of action of
  GS-9148-DP against HIV-1 RT
• Determine the potency of GS-9148-DP against HIV-1
  RT and its selectivity with respect to host DNA
  polymerases in vitro

4
Structures of GS-9131, GS-9148, and the Active
Metabolite GS-9148 - Diphosphate
Intracellular enzymatic hydrolysis
O
N
H
N
H
N
H
2
2
2
Phosphorylation
N
N
N
N
E
t
O
N
N
O
O
O
O
O
H
N
N
N
O
P
O
P
N
P
P
N
N
O
P
N
O
O
O
O
H
O
H
O
O
OH
O
H
OH
OH
Ph
O
F
F
F
GS-9148 Parent NtRTI
GS-9148-diphosphate Active metabolite
GS-9131 Prodrug
5
Methods

• HIV-1 reverse transcriptase assays
• Steady state incorporation assay. Wild-type HIV-1
  reverse transcriptase heterodimer was expressed
  and purified as previously described3. Steady
  state inhibition constants (Ki) were determined
  as previously described3 using activated calf
  thymus DNA as a template
• Pre-steady state kinetics. Transient kinetic
  experiments were performed at 37ºC using a KinTek
  Instrument Model RQF-3 rapid-quench-flow
  apparatus as described4
• Gel-based chain-termination assay. A 31-nt
  33P-DNA primer was annealed to a 50-nt DNA
  template. 6 pM primer/template was incubated
  with 60 pM HIV-1 RT in RT buffer, 10 µM of the 3
  non-competing dNTP and varying concentrations of
  the competing dNTP and N(t)RTI for 10 min at 37C
  
• ATP-mediated excision assay. ATP-dependent
  nucleotide excision assays used a 19-mer DNA
  primer chain-terminated with either GS-9148 or
  tenofovir pre-annealed to a D36-mer template and
  HIV RT 4-TAM (AZT-resistant) mutant with 1 mM
  dATP, various concentrations of dCTP (0.2, 2 or
  20 µM) and 3 mM ATP

6
Methods (contd)

• Host DNA polymerase assays
• Polymerase alpha and beta assay. A
  heteropolymeric template (78-mer)/primer
  (18-mer), DNA pol alpha or beta (Replizyme, UK)
  and various concentrations of inhibitor were
  incubated at 37C and used to measure
  incorporation of 3HdATP at 30 and 60 min
• Polymerase gamma assay. Activated calf thymus
  DNA, human recombinant DNA polymerase gamma
  expressed in baculovirus (Provided by William
  Copeland, NIEHS, Research Triangle Park, NC), and
  various concentrations of inhibitor were
  incubated at 30C and used to measure
  incorporation of 3HdATP at 10 and 20 min

7
ResultsGS-9148-DP is a Competitive Inhibitor of
HIV-1 RT with Respect to dATP by Double
Reciprocal Plot Analysis
Tenofovir-DP
GS-9148-DP
0
.
00
8
0
.
02
4
I 2 µM
I
1
.
2
µ
M
I 1 µM
0
.
02
0
0
.
00
6
I
0
.
5
µ
M
0
.
01
6
I
0
.
6
µ
M
0
.
00
4
0
.
01
2
I
0
I
0
.
3
µ
M
1/
V
m
a
x
0
.
00
8
1/
V
v
v
m
a
x
0
.
00
2
1/
1/
0
.
00
4
I
0
0
.
00
0
0
.
00
0
-0
.
004
-0
.
002
0
.
00
0
0
.
002
0
.
00
4
0
.
006
-0
.
002
0
.
00
0
0
.
002
0
.
00
4
0
.
006
1
/
S

1
/
S

Wild-type HIV-1 RT DNA polymerase activity was
assayed by quantification of 3H-dATP
incorporation using a heteropolymeric DNA
template. The mode of inhibition of GS-9148-DP
and tenofovir-DP with respect to dATP
incorporation was assayed with increasing
concentrations of inhibitors and showed a
constant Vmax, demonstrating competitive
inhibition
8
Steady State Inhibition Constants for Wild-type
HIV-1 RT
NRTI
K
(µM)
a
i
GS-9148-DP
0.80 0.07
T
enofovir-DP
0.18 0.03
dd
A
TP
0.021 0.004
b
c
Carbovir-TP
b
0.
1
1 0.03
c
AZ
T
-TP
0.038 0.006
c
d4
T
-TP
0.06 0.02
c
3TC-TP
2.0 0.4
c
FTC-TP
1.2 0.4
c
a. Ki values are means from 3 experiments
standard deviation b. ddATP is the active
metabolite of ddI carbovir-TP is the active
metabolite of abacavir c. Ki values were
obtained from reference 3
9
Pre-Steady State Incorporation by HIV-1 RT
Wild-type HI
V
-1
R
T
Substrate
k
(s
)
K
(µM)
k
/K
(µM
s
)
-1
a
a
-1
-1
a
pol
d
pol
d
d
A
TP
183 9
41 6
4.4 0.7
GS-9148-DP
22 2
194 32
0.
1
1 0.02
T
enofovir-DP
25 1
42 4
0.58 0.07

• kpol maximum rate of incorporation
• Kd dissociation constant
• kpol/Kd incorporation efficiency
• Values are mean standard deviation

10
GS-9148-DP Functions as a DNA Chain-Terminator
s
s
s
s
s
s
P
P
P
P
T
T
T
T
No dNTPs dATP(10), dNTPs No dATP,
dNTPs GS-9148-DP(10), No dATP, dNTPs
, dN
, dN
AZT
-
T
P
AZT
-
T
P
TF
V
-
D
P
TF
V
-
D
P
, dN
, dN
dATP, dNTP
P
GS-9148-DP (10 µM)
dATP, dNTP
P
o
o
T
T
(10 µM
)
(10 µM
)
(10 µM
)
(10 µM
)
s
N
s
N
s
s
TTP(10), dNTPs No TTP, dNTPs AZT-TP(10), No
TTP, dNTPs
s
TTP
s
TTP
P
s
P
s
P
P
T
o
T
o
0),
0),
TFV-DP(10), No dATP, dNTPs
TP
TP
N
1
N
N
1
N
TT
P
(
µ
M
)
TT
P
(
µ
M
)
(
(
dATP (µM)
dATP (µM)
,
,
P
P
Chain-termination activity of N(t)RTIs was
assayed with wild-type HIV-1 RT using a
heteropolymeric DNA template. Distinct
chain-termination products were observed that
corresponded to sites of dATP incorporation for
GS-9148-DP and tenofovir-DP (TFV-DP) and TTP
incorporation for AZT-TP. For all N(t)RTIs,
chain-termination was most efficient with low
concentrations of the competing dNTP
dN
dN


0.4 2 10 50
0.4 2 10 50
0.4 2 10 50
,
,
(10)
(10)
10), dNTP
TP
10), dNTP
TP
(
(
-
-
-DP(10), No dA
TTP
-DP(10), No dA
TTP
-9148-D
-9148-D
V
V
o
o
N
N
No dNTPs
No dATP, dNT
TTP
AZT
No dNTPs
No dATP, dNT
TTP
AZT
dATP(10), d
dATP(10), d
GS
GS
TF
TF
50-mer
C
C
T
T
C
C
A
A
C
C
G
G
A
A
C
C
T
T
C
C
C
C
A
A
G
G
A
A
A
A
G
G
31-mer
T
T
A
A
A
A
11
Excision of GS-9148 by AZT-Resistant HIV-1 RT
0
.
7
Tenofovir
0
.
6
GS-9148
)
1
-
Values are mean standard deviation
n
0
.
5
mi
(
Excision Rate ( min-1 )
0
.
4
ATP-mediated excision of incorporated NtRTIs was
assayed using AZT-resistant HIV-1 RT and
increasing concentrations of the next
complementary nucleotide, dCTP. The excision of
NtRTIs was decreased when the concentration of
dCTP was increased
e
Rat
0
.
3
0
.
2
0
.
1
Excision
0
0
.
2
2
2
0

d
C
TP

(
µ
M
)
12
Inhibition of Host DNA Polymerases by GS-9148-DP
a. Mean standard deviation
13
Conclusions

• The active metabolite of GS-9148 is an effective
  competitive inhibitor of HIV-1 RT with respect to
  dATP, acting as an obligatory DNA chain
  terminator
• Once incorporated into an elongating DNA strand,
  GS-9148 appears to be poorly excised by HIV-1 RT
  with multiple AZT resistance mutations
• GS-9148-DP exhibits a favorable selectivity
  against host DNA polymerases including DNA
  polymerase gamma
• In addition to previously presented favorable in
  vitro pharmacological profile1,2, these data
  further support the development of the GS-9148
  oral prodrug (GS-9131) for the treatment of HIV-1
  infection

14
References

• T Cihlar, A Ray, D Boojamra, L Zhang, H Hui, D
  Grant, K White, M Desai, N Parkin, and R Mackman.
  Abstr. 45, CROI 2006, Denver.
• A Ray, J Vela, R Mackman, L Zhang, H Hui, R
  Pakdaman, A Carey, M Wright, G Rhodes, and T
  Cihlar. Abstr. 498, CROI 2006, Denver.
• K White, N Margot, J Ly, J Chen, A Ray, M
  Pavelko, R Wang, M McDermott, S Swaminathan, and
  M Miller, AIDS 2005, 191751-1760.
• W Kati, K Johnson, L Jerva, K Anderson. J. Biol.
  Chem. 1992. 26725988-25997