Epigenetic drug Gar1041 (auranofin) in combination with antiretroviral therapy (ART) reduces the...
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Transcript of Epigenetic drug Gar1041 (auranofin) in combination with antiretroviral therapy (ART) reduces the...
Epigenetic drug Gar1041 (auranofin) in combination with antiretroviral therapy
(ART) reduces the proviral DNA reservoir in SIVmac251-
infected macaques
Andrea Savarino
“Classic” strategies for eradication of virus reservoirs
• Prevent any virus spread by intensified ART– Target multiple sites of viral inhibition
• Induce latently infected cells to replicate virus– Histone deacetylase inhibitors
• Eliminate infected cells– Virus cytotopathogenicity– Immune response– Passive therapies– Drugs Van Lint et al., 2004
Hamer, 2004Archin et al., 2005Savarino et al., 2009Margolis, 2010
“Sabotage”
• Aimed at reducing the capacity of the viral reservoir to expand
• Aimed at decreasing the half-life of the latently infected reservoir persisting during antiretroviral therapy (ART)
Our novel strategy for eradication of virus reservoirs
Auranofin (Gar1041)
1) A gold-based compound,2) Orally available,3) Used for years in the treatment of rheumatoid arthritis
(“drug repositioning”),4) Capable of inducing oxidative stress (generation of
intracellular peroxide through a superoxide dismutase-mimicking reaction),
5) Adopted in the experimental treatment of certain cancers due to its pro-differentiating properties.
Auranofin (Gar1041)
Hydrogen peroxide accumulation plays a major role in cellular differentiation!
Dihydrorhodamine 123 → rhodamine 123 (FLUORESCENT)
Hydrogen peroxide ↓
ACH2 ctrl
0.6 μM auranofin
M1
Reactive oxygen species (ROS)induction
0 103 104 105
<FITC-A>: CD27
0
50K
100K
150K
200K
250K
SS
C-A
18.2
0 103 104 105
<FITC-A>: CD27
0
50K
100K
150K
200K
250KS
SC
-A
28.9
0 103 104 105
<FITC-A>: CD27
0
50K
100K
150K
200K
250K
SS
C-A
94.9
0 103 104 105
<FITC-A>: CD27
0
50K
100K
150K
200K
250K
SS
C-A
94.2
0 103 104 105
<FITC-A>: CD27
0
50K
100K
150K
200K
250KS
SC
-A
4.7
0 103 104 105
<FITC-A>: CD27
0
50K
100K
150K
200K
250K
SS
C-A
10.6
Auranofin induces CD27 downregulation
TCM TTM TEM
Mock treated
50nM auranofin
CD27 expression
Working hypothesis:
CD27 down-modulation in central and transitional memory CD4+ T cells may decrease the half-life of the latently infected viral reservoir and contribute to its depletion.
Response of SIVmac251-infected macaques to antiretroviral therapy
0 10 20 30 40 50101
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P044P249
P252P255
M970
M974
Days
Vir
al lo
ad
(RN
A c
op
ies
ml-1
)
RAL RAL + PMPA + FTC
RAL- 100mg BID, oral
PMPA - 20mg/kg, SQ
FTC – 50mg/kg, SQ
-200 -150 -100 -50 0 500
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P044P249
P252P255
M970
M974
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CD
4+ T
-lym
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l-1
RAL RAL + PMPA + FTC
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P044P249P252P255M970M974
nonhuman primates
Days of treatment
DN
A c
op
ies/
5*1
05 c
ells
Lewis M. Norelli S., et al., Retrovirology 2010.
Auranofin decreases the central memory T cell compartment in vivo but does not affect total CD4 counts
A
0 300
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*
Days of treatmentwith ART + auranofin
% o
f C
D4
+T
CM
lym
ph
oc
yte
s B
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*
Days of treatmentwith ART + auranofin
% o
f C
D4+
TE
M ly
mp
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cy
tes C
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Days of treatmentwith ART + auranofin
tota
l CD
4+ T
-ly
mp
ho
cy
tes
/l o
f b
loo
d
P < 0.05 P < 0.05
Epigenetic drug Gar1041 in combination with antiretroviral therapy (ART) transiently reduces the proviral DNA reservoir in SIVmac251-infected macaques
M. Lewis1, S. Norelli2, N. Chomont3, S. De Fonseca3, M. Sgarbanti2, M. Collins1, B. Chirullo2, J. Yalley Ogunro1, J. Greenhouse1, A.T. Palamara4, E. Garaci2, A. Savarino5
1Bioqual, Rockville, United States, 2Istituto Superiore di Sanità, Rome, Italy, 3VGTI-Florida, Port St. Lucie, United States, 4Università La Sapienza, Rome, Italy, 5Istituto Superiore di Sanita, Dept. of Infectious, Parasitic and Immune Diseases, Rome, Italy
Background: It was recently hypothesised that the lentiviral reservoir in central memory (Tcm) and transitional memory (Ttm) CD4+ cells could be restricted by new therapies targeting pathways downstream of homeostatic proliferation or pathways associated with ´stem cell-ness´, such as those developed for the treatment of leukemias (Chomont et al. Nat Med 2009). Gar1041 is one such epigenetic drug adopted in the experimental treatment of certain types of leukemia. Methods: SIVmac251-infected primates with viral loads stably suppressed by ART (tenofovir/emtricitabine/raltegravir) were administered, for two months, Gar1041 twice daily (a starting dose of 1.5 g in the first week followed by 2 g in the remaining period). ART was continued during Gar1041 treatment. Proviral DNA was quantitated using a Taqman real-time PCR.Results: The proviral DNA content of PBMCs, which had shown no significant changes during 54 days of treatment with ART alone (P > 0.05), fell below the level of detection (2 copies/106 cells) in all study subjects within one month of Gar1041 treatment [P < 0.05; Bonferroni's test following significant (P = 0.0003) repeated-measures ANOVA]. No significant changes were noticed in a control group treated with ART alone (P = 0.49). The decrease in proviral DNA was associated with a significant (P = 0.0156) decrease in the proportions of the Tcm CD4+ cell subpopulation in peripheral blood. However, both proviral DNA and the proportions of Tcm CD4+ rebound after two months of therapy.Conclusions: The present study furnishes proof of concept that pharmacological strategies may impact on the proviral DNA reservoir. Integration with other experimental approaches will be required to prevent the reconstitution of proviral DNA in peripheral blood from the as yet unidentified reservoir associated with renewal of the phenotype Tcm compartment.
SIVmac251 inhibition in CEMx174cells
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EC50 = 6.6 nMP = 0.02
DRV conc. [nM]
% in
hib
itio
n
Darunavir (DRV) complexed with HIV-2 protease DRV complexed with SIVmac251 protease
Barreca ML, Norelli S, and Savarino A,unpublished
Kovalevsky et al., 2008
Darunavir as a tool for ART intensification in SIV+ macaques
iART alone
0 20 40 60 80 1000
102030405060708090
100110
44234416Mean
Days of ART intensificationp
rov
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l DN
A c
op
ies
/ 5*1
05 c
ells
iART + Gar1041
0 50 100 1500
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Days of ART intensification
pro
vira
l DN
A c
op
ies
/ 5*
105 c
ells
iART/Gar1041 (average trend over time: P = 0.0207; t-test for regression). iART alone (average trend over time: P = 0.8878; t-test for regression).
Auranofin (Gar1041) significantly decreases proviral DNA in PBMC from SIV+ macaques treated with in ART intensified with ritonavir-boosted
darunavir (iART)
Stimulation of viral replication by histone deacetylase inhibitor, SAHA in SIVmac251-infected monkeys treated with intensified ART alone but not in
monkeys SIVmac251-infected monkeys treated with iART and auranofin
-20 -10 10 20 30 40 50 60 70 8010
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**
* *
P044P249P252P255M970M972
therapysuspesion
Monkeys:
Days from SAHA start
Vir
al l
oa
d(R
NA
co
pie
s m
l-1) intensified ART alone
intensified ART plus auranofin
*P < 0.05 **P < 0.01
0 2 4 6 8 100
25
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75
100iART plus auranofiniART alone
A
weeks following treatment interruption
per
cen
tag
e o
f an
imal
s sh
ow
ing
un
det
ecta
ble
vir
al lo
ads
Addition of auranofin to intensified ART delayed re-appearance of viral load following treatment suspension
*
Intensified ART aloneIntensified ART plus auranofin
*P < 0.05; Gehan-Breslow-Wilcoxon test for survival
*P < 0.05; paired Student’s t-test
Upon re-appearance of viral load following therapy suspension, auranofin-treated monkeys, but not intensified ART (iART)-only controls,
acquire the ability to control viral load
iART alone
iART plus Gar1041
peakWhen viral load re-appeared in monkeys that had been treated with auranofin, an immediate peak was observed that was followed by progressive declines in viral RNA
…viral RNA eventually declined to levels lower than those presented before ART initiation
decrease
Drug free remission… eventually?
iART alone
iART plus Gar1041
Upon re-appearance of viral load following therapy suspension, auranofin-treated monkeys, but not intensified ART (iART)-only controls, maintain high CD4 counts
Conclusions
Our findings following ART intensification indicate that a drug-free remission is possible, at least for the follow up period adopted in this study, and represent a milestone towards the identification of future drugs capable of curing HIV/AIDS or inducing a drug-free remission.
Our results also highlight the need for ART intensification when adopting drugs capable of inducing HIV-1 replication.
Oxidative stress inducing agent auranofin impacts the proviral DNA reservoir in SIVmac251-infected macaques under treatment with ART, likely acting by an entirely novel mechanism (CD28 downmodulation in long-lived and proliferation-competent central and transitional memory T cells → generation of short-lived phenotypes)
AcknowledgmentsBioqualMatt CollinsJake Yalley-Ogunro University of Rome “Tor Vergata” Jack Greenhouse Enrico GaraciWendy Wagner
ICGEBIstituto Superiore di Sanità, Rome Marina LusicSandro NorelliBarbara Chirullo University of Rome, La
SapienzaMarco Sgarbanti Rossella SgarbantiAndrea Savarino Anna Teresa Palamara
VTGI, FloridaNicolas ChomontSandrina DaFonsecaRafick-Pierre Sékaly