Poster 497
Bictegravir Dissociation Half-life from HIV-1 G140S/Q148H Integrase-DNA Complexes
Kirsten White*, Anita Niedziela-Majka, Nikolai Novikov, Michael Miller, Haolun Jin, Scott Lazerwith, and Manuel Tsiang
Gilead Sciences, Foster City, CA
BIC, DTG, EVG, and RAL were measured using wild-type or G140S/Q148H
HIV-1 integrase-DNA complexes and a scintillation proximity assay at 37ºC
as previously described (Figure 2)5. Data were analyzed using the published
exponential decay method. During the long time course required for these
assays, gradual sedimentation of the SPA beads resulted in overestimates of
dissociation half-lives of the INSTIs. Therefore, an equilibrium binding model
was developed here and was used for the analysis of the G140S/Q148H
mutant IN-DNA complexes.
Equilibrium Binding Model Analysis: The equilibrium binding model
incorporates a correction for the gradual sedimentation of the SPA beads
by replacing kon and koff with decreasing hyperbolic functions of time (zon
and zoff) where tc is the time of cold competitor addition and r and n are
correction factors.
zon = kon
zoff = koff
(for t < tc )
(for t < tc)
k (r − 1)
k
+ on
zon = on
n(t − tc ) + r r
zoff =
koff (r − 1)
n(t − tc ) + r
+
koff
r
(for t > tc )
By Exponential Decay
BIC+WT IN/DNA
80
60
40
20
0
-20
Apparent t1/2
(hr) [**]
p-value vs BIC
BIC
135 ± 20 [na]
--
38 ± 19
--
DTG
79 ± 13 [71]
< 0.0001
16 ± 9
0.017
RAL
14 ± 3
[8.8]
< 0.0001
5.2 ± 0.6
0.003
EVG
3.6 ± 0.7 [2.7]
< 0.0001
1.5 ± 0.2
0.0006
t1/2 (hr)
120
p-value vs BIC
4000
5000
Time
(min)
TIME(hr)
6000
7000
8000
60
40
20
0
♦♦ The Exponential Decay Method of analysis results in an overestimation of
the dissociation half-lives due to an experimental artifact; the Equilibrium
Binding Method more accurately describes the kinetics
Presented at the Conference on Retroviruses and Opportunistic Infections (CROI), February 13-16, 2017, Seattle, WA
2000
300 0
4000
500 0
6000
Time
(min)
TIME(hr)
7000
800 0
9000
♦♦ Phase 3 clinical studies of the single tablet regimen of
BIC/FTC/TAF are ongoing.
1. Lazerwith et al., “Discovery of Bictegravir (GS-9883), a Novel, Unboosted,
Once-Daily HIV-1 Integrase Strand Transfer Inhibitor (INSTI) with Improved
Pharmacokinetics and In Vitro Resistance Profile.” ASM Microbe, June 19, 2016,
Boston, MA, Poster #414.
40
20
Time (hr)
120
DTG+140/148 IN/DNA
100
80
40
20
-20
2. Tsiang et al., “Antiviral Activity of Bictegravir (GS-9883), a Potent Next Generation
HIV-1 Integrase Strand Transfer Inhibitor.” ASM Microbe, June 19, 2016, Boston,
MA, Poster #416.
3. Gallant et al., “Novel Integrase Strand Transfer Inhibitor Bictegravir 10 Day
Monotherapy in HIV-1-Infected Patients.” ASM Microbe, June 19, 2016, Boston,
MA, Poster #415.
4. Jones et al., “Bictegravir (GS-9883), a Novel HIV-1 Integrase Strand Transfer
Inhibitor (INSTI) with Optimized In Vitro Resistance profile.” ASM Microbe, June 19,
2016, Boston, MA, Poster #413.
60
0
1000
♦♦ Long residence times of INSTIs on the integrase-DNA
complex have been correlated with potent antiretroviral
activity and a high barrier to resistance in vitro5
60
9000
80
0
life (i.e. actual dissociation t1/2 = (ln2)/koff ) and a terminal half-life (i.e.
a prolonged t1/2 = r(ln2)/koff due to the gradual sedimentation of the SPA
beads).
3000
BIC+140/148 IN/DNA
100
-20
♦♦ This deviation correction, enabled the determination of an initial half-
2000
By Equilibrium Binding Model
*Average ± standard deviation from 5 to 7 experiments
**Published t1/2 values from Hightower et al., Antimicrobial Agents and Chemotherapy. (2011) 55(10):4552-4559.
(for t > tc)
1000
–– RAL and EVG did not bind efficiently enough for
dissociation measurements
References
80
0
0
♦♦ Bictegravir also has the longest measured dissociation
half-life from mutant G140S/Q148H HIV-1 IN-DNA
complexes compared to DTG DTG+WT IN/DNA
100
150
Dissociation of INSTI from Wild-type IN-DNA Complexes*
120
♦♦ Bictegravir has the longest measured dissociation halflife from wild-type HIV-1 IN-DNA complexes compared
to DTG, RAL, and EVG
–– The long plasma half-life and high Cmin of BIC in vivo
should also contribute to a high resistance barrier3
Figure 3. Association and Dissociation Time Course of BIC and DTG from WT HIV-1 Integrase-DNA Complexes and Equilibrium
Binding Model Fit
100
Table 3. Dissociation Half-lives of INSTIs from WT HIV-1 Integrase-DNA
Complexes
INSTI
DTG, RAL, and EVG
––Significantly longer from wild-type HIV-1 IN-DNA complexes
––Significantly longer from G140S/Q148H HIV-1-DNA complexes
0
1000
2000
3000
4000
5000
Time
(hr)
TIME (min)
6000
7000
8000
150
♦♦ SPA Assay: The apparent association and dissociation kinetics of 3H-labelled
where E = integrase-donor DNA complex, L = 3H-INSTI, C = cold (nonradioactive) INSTI, EL = 3H- intermediate complex, ELf = 3H final complex,
EC = cold intermediate complex, ECf = cold final complex, kon and koff =
on- and off-rate constants for L and C, k2 & k-2 = kforward and kreverse for EL and
EC, i = rate of addition of C and Q = dosing function for C, (0 or 1)
♦♦ Bictegravir has the longest measured dissociation half-life compared to
120
♦♦ BIC and DTG have similar association rates to IN-DNA complexes
at time = t c
*Average ± standard deviation from 2 to 7 experiments
**BIC t1/2 with G140S/Q148H mutant IN-DNA complexes was statistically longer than the EVG dissociation t1/2
with wild-type IN-DNA complexes.
***DTG t1/2 with G140S/Q148H mutant IN-DNA complexes was statistically shorter than the EVG dissociation t1/2
with wild-type IN-DNA complexes.
125
patient-derived HIV-1 isolates with high-level INSTI resistance were profiled
for susceptibility to BIC, DTG, EVG, and RAL using the PhenoSenseIN
assay conducted by Monogram Biosciences (South San Francisco, CA). The
panel was chosen from the available Monogram HIV-1 library and included
all available isolates with >2.5-fold reduced susceptibility to DTG (n=24) as
well as representative isolates with EVG and/or RAL resistance mutations
(n=23). Here, the 16 isolates with G140S + Q148H ± other INSTI resistanceassociated substitutions are described.
*Average ± standard deviation from 2 to 7 experiments
nd = No data available due to low binding of INSTI to the mutant IN-DNA complexes
ND
125
♦♦ Clinically-derived HIV-1 Isolate Phenotyping: A subset of a panel of 47
(scheme A)
-0.1050
nd
nd
ND
100
Metal-Chelating Core: Oxygen atoms chelate a pair of Mg2+ ions and bind the integrase catalytic active site
Halogenated Phenyl: Interacts with the integrase pocket that is normally occupied by the terminal 3’ base of viral DNA
k–2
ELf
37 ± 4
24 ± 5
nd
nd
0.0006
100
Qi
koff
EL
k2
-0.648
0.020
0.00003
1.5 ± 0.2
75
kon
C + E+ L
kon
35 ± 5
36 ± 4
24 ± 8
15 ± 6
EVG
75
k2
EC
koff
p-value vs BIC
ND
30
40
50
ECf
k-2
Association
t1/2 (min)*
ND
20
an equilibrium binding model (scheme A) simultaneously to both the
binding and competition binding phases. The actual dissociation t1/2 was
calculated as (ln 2)/koff
BIC
DTG
RAL
EVG
p-value vs BIC
0.003
20
30
40
50
♦♦ The kon and koff values of the compounds were determined by curve fitting
Association
t1/2 (min)*
5.2 ± 0.6
5
10
(2) where M is the starting value and
G140S/Q148H IN
RAL
5
10
y = M (e )
t1/ 2 = (ln 2) / k
Wild-Type IN
0.0076
0
− kt
INSTI
0.65 ± 0.2***
0
competition binding phase using equation (2):
Apparent Association t1/2 of INSTI from IN-DNA Complexes
0.017
% Maximum Bound
♦♦ The apparent dissociation t1/2 was determined by curve fitting the
16 ± 9
LBound,
#DTG_Set1AM
%
Maximum
Bound
y = M (1 − e − kt ) (1) where M is the plateau and t1/ 2 = (ln 2) / k
DTG
150
Figure 1. Structure of Bictegravir (BIC) and other INSTIs
Table 2. Apparent Association Half-life of INSTIs from HIV-1 Integrase
DNA Complexes
--
150
determined by curve fitting the binding phase using equation (1):
2.5 ± 0.07**
125
♦♦ Exponential Decay Analysis: The apparent association t1/2 was
--
125
Light
38 ± 19
100
a. PhenoSense IN assay (Monogram Biosciences)
b. Mean ± SD
c. The 2.5-fold cutoff is a standard biological cut-off in the Monogram assay; 4-fold is the lower clinical cut-off
for DTG in the PhenoSense IN assay
BIC
100
Biotin/Streptavidin
interaction
p-value vs BIC
75
particles
1.9
2.8
7.1
2.3
t1/2 (hr)*
75
BIC
DTG
RAL
EVG
p-value vs BIC
30
40
50
3’-processed
donor DNA
G140S/Q148H Mutant IN
t1/2 (hr)*
20
IntegraseD imer
Wild-Type IN
INSTI
20
30
40
50
EC50 (nM)
♦♦ Bictegravir is a potent INSTI with a high barrier to
resistance and improved activity compared to DTG, RAL,
and EVG against HIV-1 with INSTI resistance mutations
in vitro6
Dissociation t1/2 of INSTI from IN-DNA Complexes
5
10
INSTI
Wild-Type
5
10
IN/DNA
Assembly
Susceptibility of HIV-1 to INSTIsa
G140S/Q148H ± Other Mutants (n=16)
Sensitivity (% of isolates
Foldp-value
below cut-offs)c
Change
vs BIC
vs WTb
<2.5-fold
<4-fold
3.4 ± 1.7
50%
75%
-7.6 ± 4.3
0%
25%
< 0.001
>143
0%
0%
< 0.001
>150
0%
0%
< 0.001
0
Bound
Radiolabeled
INSTI
Table 4. Dissociation Half-lives of INSTIs from WT and G140S/Q148H
HIV-1 Integrase-DNA Complexes by the Equilibrium Binding
Model
0
kon determined from direct binding of H-INSTI
koff determined from competition binding of 3H-INSTI with cold/non-radioactive INSTI
3
SPA Bead Surface
Methods
Table 1. Susceptibility of INSTIs to Clinically-derived Isolates of HIV-1
Containing G140S + Q148H ± other INSTI-R Substitutions
% Maximum Bound
Figure 2. SPA-Based INSTI Binding Assay
Conclusions
%LBound,
Maximum
Bound
#BIC_Set1AM
upon existing INSTIs and is part of a complete single tablet regimen with the
F/TAF backbone1, 2,3
♦♦ Bictegravir has an improved resistance profile compared to elvitegravir
(EVG), raltegravir (RAL), and dolutegravir (DTG), particularly for patient
isolates with high-level INSTI resistance containing combinations of mutations
such as E92Q+N155H or G140C/S+Q148R/H/K (Table 1)4,6
♦♦ In biochemical studies, the apparent dissociation half-life of DTG from
integrase-DNA complexes was shown to be longer than RAL or EVG and was
predicted to correlate with potent antiretroviral activity and a higher genetic
barrier to resistance5
♦♦ In this study, we report the dissociation kinetics of bictegravir and other INSTIs
from wild-type and G140S/Q148H HIV-1 integrase-DNA complexes in vitro
Results
3’
♦♦ Bictegravir (BIC; GS-9883) (Figure 1) is a novel, potent INSTI that improves
Methods (cont'd)
3’
Introduction
Kirsten White, PhD
Gilead Sciences, Inc.
333 Lakeside Drive
Foster City, CA 94404
Tel: (650) 522-5162
E-mail: [email protected]
9000
5. Hightower et al., “Dolutegravir (S/GSK1349572) Exhibits Significantly Slower
Dissociation then Raltegravir and Elvitegravir from Wild-Type and Integrase
Inhibitor-Resistance HIV-1 Integrase-DNA Complexes.” Antimicrobial Agents and
Chemotherapy. (2011) 55(10):4552-4559.
6. Tsiang et al., “Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Stand
Transfer Inhibitor with an Improved Resistance Profile.” Antimicrobial Agents and
Chemotherapy. (2016) 60(12):7086-7097.
© 2017 Gilead Sciences, Inc. All rights reserved.