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HIV Latency Approaches
Daria Hazuda
March 2014
Why ARV treatment doesn’t cure HIV:
2
Activated CD4+ T-cell
HIV
Activated CD4+ T-cell
Apoptosis
HIV
HIV HIV
HIV
Fate of Latently Infected Cells
HIV
Activated CD4+ T-cell
HIV
Resting Memory CD4+ T-cell
HIV
Myeloid cell
Minority of cells become latently
infected
Majority of cells die or are eliminate
Establishment of Latency
HIV PD1
HIV
PD1 and other inhibitory molecules contribute to
T-cell dysfunction
Immune dysfunction reduces the clearance of infected cells
Exhausted T-cell
Dysfunctional B-cell
Immune dysfunction Prevents Clearance of latently infected cells
HIV Particle
• A latent reservoir of long-lived T-cells with integrated HIV DNA is seeded early during acute infection
• Viral persistence results in immune dysfunction
“Flush” and “Kill” Strategy for HIV Eradication
“
HIV genome Memory
CD4+ T cell
DNA
HIV particles
HIV proteins HIV RNA
Dying infected cell Uninfected cell
Antiretroviral therapy
FLUSH Kill
Title Box HIV DNA transcription prevented by restricted
access to needed host enzymes, chromatin remodeling and transcriptional interference
HDAC inhibitors (eg TSA, SAHA) Methyltransferase inhibitors NF-kB activators (Prostratin, PMA) Akt/HEXIM-1 modulators, (HMBA)
nuc-0 nuc-1
HIV TSS
P-TEFb
Latency at the HIV Promoter: Epigenetic Silencing and Transcription
HDAC EZH2
CDK9 HEXIM1
CyclinT1
7S RNA
+
NFAT NFκB
Chromatin Structure
HAT
IκB
Transcription Regulation
Phosphorylation
+
Reactivation Step 1: Relaxation of chromatin
Reactivation Step 2: Stimulate transcription
Oncology HDACIs Being Explored for HIV Latency
Vorinostat (SAHA – Merck): Pan HDACI, oral, AMES positive Latency clinical POC (Margolis, Lewin)
Increase in HIV RNA in resting T-cells Increase in viremia (some pts), no effect vDNA
Ames(+) = potential safety liabilities?
Panobinostat (Novartis): Pan-HDACI; potent/reversible, oral Latency clinical POC (Rasmussen; Lichterfeld CROI ‘14)
Increase in HIV RNA in resting cells Increase in viremia and decrease in vDNA (some pts)
Romidepsin (Gilead): Pan HDACI, potent/irreversible, IV, DDIs Clinical POC in progress
HN
ONH
OOH
L-001079038
Zolinza (Vorinostat)
NH
OOH
HN
HN MeL-001306168
Panobinostat (LBH-589)Phase III - Novartis
HN
HN
HN
O
NH
Me
O
O
OMe
Me
O
O Me
Me
SS
L-001302455Romidepsin
(Istodax)
Can HDACIs be optimized for HIV latency?
Potential Approaches to Improve Efficacy and Therapeutic Window of HDACIs
►Optimize HDAC activity for HIV latency – Increase potency and selectivity profile – PK, understand dose/response relationship
• Differs between reversible and irreversible binders?
► Identify other mechanisms to be used in combination – Enhance overall effect and/or – Reduce HDACI dose
Criteria SAHA Next gen
HDACi Isozyme
Selectivity HDAC3 required 1, 2, 3, 6, 8 1, 2, 3
HIV Latency in Jurkat T-cell
Model EC50 (nM)
Is the cellular activity
≤ SAHA? 1000nM 200 nM
PK profile To be determined in PK/PD expts
very high clearance
Can be tuned
Next Generation HDAC inhibitors with Improved Selectivity/PK Profile for HIV Latency
8
Next gen HDACIs: Historical chemical matter Structure-guided design + modeling Screening
Potential Approaches to Improve Efficacy and Therapeutic Window of HDACIs
►Optimize HDAC activity for HIV latency – Increase potency and selectivity profile – PK, understand dose/response relationship
• Differs between reversible and irreversible binders?
► Identify other mechanisms to be used in combination – Enhance overall effect and/or – Reduce HDACI dose
nuc-0 nuc-1
HIV TSS
P-TEFb
Latency at the HIV Promoter: Epigenetic Silencing and Transcription
HDAC EZH2
CDK9 HEXIM1
CyclinT1
7S RNA
+
NFAT NFκB
Chromatin Structure
HAT
IκB
Transcription Regulation
Phosphorylation
+
Reactivation Step 1: Relaxation of chromatin
Reactivation Step 2: Stimulate transcription
Novel Ultra High Throughput Screen to Identify New Mechanisms and Combinations
“Other"
HDAC Inhibitors
Farnesyltransferase Inhibitors
66.5%
16.1%
17.4%
Latent Jurkat T-cell model (Jon Karn) Screened 2.9 million compounds in the presence of 250 nM SAHA All hits titrated plus and minus 250 nM SAHA Identified HDACIs plus 2400 unique hits
FTi’s with Differential Binding Modes and Structures
MRK-16 MRK-17
From: Bell, I. J. Med. Chem. 2004, 47, 1869-1878.
X-ray Crystallographic images of different farnesyl-transferase inhibitor binding modalities. (A) Zinc Binding, (B) Exit Groove Blocker
A B
Correlation Between Farnesyl Transferase Inhibition and HIV-Latency Activation
13
• Strong Positive correlation between FTi potency (IC50 enzyme assay) and HIV latency activation in the presence of 250nM SAHA (EC50 in Jurkat T-cell model system)
• Knock-down of FTi-beta subunit leads to activation of HIV LTR in a Jurkat model system
ADD si RNA KD data
Scrambled siRNA
shRNA knockdown of FTβ Subunit
Untreated
3 Days 10 Days
Farn
esyl
-Tra
nsfe
rase
E
nzym
atic
Ass
ay IC
50 (µ
M)
Jurkat T-cell Induction (EC50, µM)
FTIs Increase Emax and Lower SAHA EC50
1
SAHA FTi #1
FTIs Enhance HDACI Effectiveness in Primary T-cells by Stimulating Transcription (J.Karn)
eGFP
Ac
tive
PTEF
-b
0 101 102 103 104 105
R670-A
010
110
210
310
410
5B
530-
A
58.75% 41.25%
0.00% 0.00%
R2: 58.75%57.24% Unstimulated
35.86% Stimulated
NEF
0 101 102 103 104 105
YG610-A
010
110
210
310
410
5R
780-
A0.90% 45.52%
50.90% 2.69%
R6: 0.90%
50.37% Unstimulated
47.21% Stimulated
Cyclin D3 0 101 102 103 104 105
YG610-A
010
110
210
310
410
5R
780-
A
0.72% 0.90%
95.87% 2.51%
R6: 0.72%
95.25% Unstimulated
0.54% Stimulated
Cyclin D3
0 101 102 103 104 105
R670-A
010
110
210
310
410
5B
530-
A
96.39% 3.61%
0.00% 0.00%
R2: 96.39%92.99% Unstimulated
2.62% Stimulated
NEF
0 101 102 103 104 105
YG610-A
010
110
210
310
410
5R
780-
A
0.34% 4.68%
89.75% 5.24%
R6: 0.34%
88.49% Unstimulated
5.46% Stimulated
Cyclin D3
0 101 102 103 104 105
R670-A
010
110
210
310
410
5B
530-
A74.00% 26.00%
0.00% 0.00%
R2: 74.00%71.88% Unstimulated
22.20% Stimulated
NEF
No Stimulation 500nM SAHA 10µM FTi 10µM FTi + 500nM SAHA
0 101 102 103 104 105
YG610-A
010
110
210
310
410
5R
780-
A
1.00% 49.44%
46.70% 2.86%
R6: 1.00%
46.14% Unstimulated
51.62% Stimulated
Cyclin D3
0 101 102 103 104 105
R670-A
010
110
210
310
410
5B
530-
A
23.54% 76.46%
0.00% 0.00%
R2: 23.54%22.37% Unstimulated
67.32% Stimulated
NEF
Validation of FTIs as Inducers of Latent HIV Expression: Summary of data to date
► Farnesyl Transferase (FT) Inhibitors from different structural classes induce latent HIV expression
► Effect on latent HIV expression is reproduced by siRNA knockdown
► Positive correlation between FT enzymatic activity and HIV latency activation in a Jurkat T-cell model system
► FTIs synergize with SAHA in a primary T-cell model and in latently infected memory T-cells isolated from HIV infected patients
► FTIs may increase active pTEFb levels in resting cells
16
Per
cent
Act
ivat
ion
(Nor
mal
ized
) to
SA
HA
)
MRK 23 (HDACi) Prostratin TNFα No Enhancer
Log Compound (M)
FTIs Synergize with other HIV Latency Activation Mechanisms
Identifying Compounds which Synergize with other Established Mechanisms
Jurkat HIV Latency T-cell Model (Luc
reporter)
Cytotoxicity of Compounds +/- EC20 of known HIV Activators
N=3
~2000 HIV Latency uHTS hits (non-HDACi) Complete Dose Response,
+/- EC20 of SAHA, HDACi (1,2,3), TNFα, JQ1, HMBA, Prostratin
Complete Dose Response of uHTS hits +/- EC20 of known HIV
Activators
Re-test of compounds with decreased EC50 and/or increased Emax in the presence of known HIV activators
N=3
Synergy Profile of uHTS Hits of Unknown Mechanism
EC50 Synergy Emax Synergy EC50 and Emax Synergy
HMBA 0 0 0
JQ1 12% 72% 7%
PKC 15% 81% 11%
HDACi (1,2,3) 14% 78% 10%
TNFa 13% 80% 9%
19
Some Key Questions in HIV Latency Drug Discovery ►Can we improve the efficacy/safety of
latency activating agents ►Can we increase the extent and spectrum
(breadth) of activation through combinations ►Will the same agents/combinations work in
all cells ►If we increase HIV expression will it lead to
cell death or will another modality be required to eliminate these cells
►How much expression is “enough”
Jurkat 2C4 Cells (Luciferase) Compounds Synergistic
Jurkat Population Cells (eGFP) No Synergy Detected
Differential Activity of FTIs in Two Different Jurkat T-cell Models
Some Key Questions in HIV Latency Drug Discovery ►Can we improve the efficacy/safety of
latency activating agents ►Can we increase the extent and spectrum
(breadth) of activation through combinations ►Will the same agents/combinations work in
all cells ►If we increase HIV expression will it lead to
cell death or will another modality be required to eliminate these cells
►How much expression is “enough”
gp120 is Expressed on Latently Infected Jurkats After Stimulation w/ TNF or SAHA
TNF alpha
SAHA
% p
ositi
ve GFP +
gp120 +
GFP + gp120 +
% p
ositi
ve
Untreated cells: 2G12
TNF treated: 2G12
ng/ml
nM *KARN cells were treated with TNF or SAHA for 24 hrs. GFP and gp120 expression were determined at by imaging and flow cytometry.
23
“
Approaches for the “Kill”
DNA
HDACi with Optimal Selectivity/PK
+ Synergistic Agent
FLUSH
• Ab-mediated Killing • IMR/Resurrection of
immune response +/- • Therapeutic Vaccine
KILL
HIV Antibody Conjugates can Eliminate HIV-infected Cells in vivo Platform mAb MOA POC
(Model) Pros Cons
Bispecific mAb (BsAb)
αgp120/αCD3
Redirected T-Cell killing (CD3)
Yes (Rhesus Macaque)
Highly specific Does not require high antigen expression/ internalization Clinical precedence for MOA
Uncertainty around cis/trans effect with CD3 expression Clinically significant immune activation?
Antibody Drug Conjugate (ADC)
αgp120 Intracellular cytotoxin delivery (Doxorubicin, PE, ricin)
Yes (BLT Mouse)
Highly specific Intracellular toxicity Clinical precedence
Requires internalization May require high antigen expression
Antibody Radioisotope Conjugate
αgp41 Localized radiation induced damage (Bi213 )
Yes (BLT Mouse)
Highly specific Does not require high antigen expression or internalization Clinical precedence
Manufacturing and regulatory hurdles Short t1/2 of isotope and commercial impact
25
Why ARV treatment doesn’t cure HIV:
26
Activated CD4+ T-cell
HIV
Activated CD4+ T-cell
Apoptosis
HIV
HIV HIV
HIV
Fate of Latently Infected Cells
HIV
Activated CD4+ T-cell
HIV
Resting Memory CD4+ T-cell
HIV
Myeloid cell
Minority of cells become latently
infected
Majority of cells die or are eliminate
Establishment of Latency
HIV PD1
HIV
PD1 and other inhibitory molecules contribute to
T-cell dysfunction
Immune dysfunction reduces the clearance of infected cells
Exhausted T-cell
Dysfunctional B-cell
Immune dysfunction Prevents Clearance of latently infected cells
HIV Particle
• A latent reservoir of long-lived T-cells with integrated HIV DNA is seeded early during acute infection
• Viral persistence results in immune dysfunction
The Latent Reservoir Established Early in Infection….may be Maintained by
Multiple Mechanisms
HIV/CMV persistence
Immune activation/ Inflammation
Immune dysfunction
Homeostatic proliferation
Addressing Latency Through Multiple Interventions…
HIV/CMV persistence
Immune activation/ Inflammation
Immune dysfunction
Homeostatic proliferation
Therapy intensification
Anti- inflammatory?
Immune reconstitution?
Shock and Kill
Acknowledgements
The Merck HIV
Team
Many collaborators..
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