HIV-1 Reverse Transcriptase: Drug
Resistance Mechanisms
HIV Drug Resistance Program
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HIV Drug Resistance Program
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Steps of DNA Polymerization
RT
RT/DNA
thumb
DNAn
E
RT/DNA/dNTP
fingers
PPi
dNTP
E´/DNAn
DNA binding
E´/DNAn/dNTP
dNTP binding
E*/DNAn/dNTP
Conformational change/
catalysis
E´/DNAn+1
E + DNA
Translocation
HIV Drug Resistance Program
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Sarafianos et al. (1999) Chem. & Biol. 6, R137-R145
Nucleoside Analogs and Nonnucleoside RT
Inhibitors
• Nucleoside analogs (NRTIs) that are used to treat HIV-1
infections all lack the normal 3’ OH and act as chain
terminators when incorporated into viral DNA
• Nonnucleoside RT inhibitors (NNRTIs) bind to HIV-1 RT
near, but not at, the polymerase active site distorting the
enzyme and blocking the chemical step of viral DNA
synthesis
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Drug Binding Sites in HIV-1 RT
NRTI
NNRTI
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Sites of Drug Resistance Mutations in HIV-1 RT
NNRTI resistance mutation sites
NRTI resistance mutation sites
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Nonnucleoside RT Resistance Mechanisms
• Mutations that reduce the interactions between RT and
the bound drug (Y181C, Y188L).
• Mutations that cause steric hindrance with the bound drug
(G190A/S).
• Mutation that makes it more difficult for the NNRTI to
enter the binding pocket (K103N).
• Indirect effects (L100I, V106A, V108A).
HIV Drug Resistance Program
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NRTI Resistance
• Resistance to nucleoside analogs (NRTIs) implies that the
mutant reverse transcriptase (RT) has an enhanced
ability to discriminate between the NRTI and the normal
dNTPs.
• This discrimination can occur if the NRTI is incorporated
less efficiently (exclusion) or by enhanced excision of the
NRTI after it has been incorporated (ATP-dependent
pyrophosphorolysis).
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NRTI Resistance Mechanisms
• Exclusion: M184V, 3TC/FTC resistance; steric hinderance
• Exclusion: K65R, Q151M resistance to multiple NRTIs,
altered interactions with the 3’OH of the incoming dNTP
• Excision: TAMs (T215F/Y and friends) cause AZT
resistance by enhanced ATP binding
• Fingers insertions: extend TAMs to cause excision of
many NRTIs
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Steric Hindrance in 3TC Resistance
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AZT Resistance (Excision)
• Excision involves pyrophosphorolysis (the reverse
of polymerization).
• AZT-resistant RTs preferentially excise AZTMP.
• AZT-resistance mutations enhance the ability of RT
to bind ATP, the in vivo pyrophosphate donor.
• AZT is excised easily because the long azido group
interferes with translocation: an AZT terminated
primer preferentially resides at the N (active) site
where it can be excised
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Polymerization
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Closed Complex Reduces Excision
Stable Complex
No Excision
b3-b4 Loop
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AZTMP Excision
Unstable Complex
Excision
AZT MP
b3-b4 Loop
*
*
ATP Binding Mutations
HIV Drug Resistance Program
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Y183
primer
template
D186
P
M184
Y115
D185
D110
AZTMP
g
b
K219E
N
D67N
fingers
a
D113
T215Y
K70R
ATP
M41L
An AZT Terminated Primer in the P Site Causes
Steric Hindrance with a dNTP in the N site
P Site (AZTMP)
N Site (dNTP)
Active Site
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AZT Resistance: Q151M
• Certain NRTI combination therapies (AZT + ddI + ddC)
select Q151M in HIV-1 RT. However, Q151M appears in
only 5% of the treated HIV-1 patients.
• There is less data (fewer treated patients) but HIV-2 prefers
to use the Q151M pathway for AZT resistance
• In HIV-1 (but not HIV-2) the Q151Mutation is usually
accompanied by additional mutations
• Why does HIV-1 predominantly use an ATP-dependent
excision pathway (T215Y/F) while HIV-2 predominantly uses
an AZT exclusion pathway (Q151M)?
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AZTTP Inhibition of Polymerization of HIV-1RT, HIV-2
RT, and Their Q151M Mutants
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HIV-1 RT (WT and Mutants) Are Better at AZTMP
Excision than HIV-2 RT (WT and Mutants)
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ATP Binding sites in HIV-1 RT and HIV-2 RT
P
185
215
112
215 214 ATP 113
115
112
214
ATP
118
117
113
44
1
4
2
46
5
41
4
115
116
116
117
211
AZTMP
AZTMP
N
209
P
185
41
44
46
3
HIV-1 RT
HIV Drug Resistance Program
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HIV-2 RT
N
HIV-1 and HIV-2 AZT Resistance
• Each virus prefers a resistance pathway that is best suited
to extend the properties of their respective wild-type RTs.
• Viewed in this light, the ability of HIV-1 to develop AZT
(and multi-NRTI) resistance using ATP-mediated excision
is an unfortunate coincidence based on the existence of a
nascent ATP binding site that appears to have no normal
function.
HIV Drug Resistance Program
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Delayed Chain Terminators
• HIV-1 excision works efficiently because the NRTI
remains at the end of the primer strand where it can be
excised.
• Delayed chain terminators block DNA synthesis several
nucleotides after they have been incorporated.
• As expected, delayed chain terminators block the excision
reaction and inhibit the growth of viruses that replicate
using excision proficient RTs.
HIV Drug Resistance Program
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Conclusions
• There are a number of distinct mechanisms for
NRTI and NNRTI resistance.
• Resistance mechanisms evolve as logical
extensions of the properties of wild type RT.
• Understanding the mechanism(s) of RT
resistance makes it possible to develop
strategies (and drugs) that counteract these
mechanisms .
HIV Drug Resistance Program
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Acknowledgments
NCI-Frederick
Paul L. Boyer
Pat Clark
John Julias
CABM/Rutgers
Stefan Sarafianos*
Kalyan Das
Eddy Arnold
Victor Marquez
Tel Aviv
Amnon Hizi
HIV Drug Resistance Program
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HIV Drug Resistance Program
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North-Methanocarbathymidine (N-MCT)
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N-MCdATP Is a Delayed Chain Terminator
T
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N-MCT Inhibits HIV-1 Viruses that Replicate Using
Excision-Proficient RTs in HSV TK+ Cells
WT
AZT-R
SSGR/T215Y
Δ67 Complex
Relative Infectivity
100
80
60
40
20
0
0
HIV Drug Resistance Program
NCI-Frederick
0.05
0.1
µM North-MCT
0.5
HIV Infection/Mortality
•
Over 3 million deaths in 2003; about 7 per minute
•
40-50 million infected worldwide
•
In 2003, about 5 million new infections, mostly in
Africa, South and Southeast Asia
•
About 1 million already infected in the US; about
45,000 new infections and 15,000 deaths in 2003
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HIV Evolution
•
HIV replication is error prone: Error rate ca. 1 per
genome per replication cycle
•
The rapid replication of the virus, together with the
error rate and high viral load causes the virus to
evolve rapidly
•
The rapid evolution of the virus makes the
problems of vaccine development and drug
therapy particularly difficult
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Anti-HIV Drugs and Drug Targets
•
•
•
•
Target should be essential and conserved; enzymes
are better targets (RT, PR, IN)
HIV develops resistance to all drugs
RT has at least two separate drug targets (there may
be more); PR and IN have one
Understand resistance; develop drugs that are
effective against resistant viruses
HIV Drug Resistance Program
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Anti-HIV Drugs and Drug Resistance
•
Drugs do not cure an HIV infection
•
Drugs need to be taken (regularly) for the life of
the patient
•
For drugs to be effective replication must be
blocked completely (stop evolution of resistance)
•
It takes at least three drugs in combination to fully
block viral replication/evolution
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Retroviral Life Cycle
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Mechanism of K103N Resistance
Rigid
inhibitor
Steric hindrance
Flexible
inhibitor
Torsional changes
(wiggling)
Reorientation and
repositioning (jiggling)
Subdomains of the Subunits of HIV-1 RT
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Two NRTI Drug Resistance Paradigms:
Reduced Incorporation and Enhanced Excision
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M814V Causes Steric Hindrance with 3TCTP
Clash Between Incorporated AZT and Active Site
Aspartic Acid Prevents Translocation
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WT Complex N superposed on WT Complex P
template
primer
P site
D186
D185
AZTMP
D110
N site
AZTMP
Q151
R72
fingers
P site
Primer
(priming site)
dNTP
N site
3’OH
(nucleotide binding site)
a
Catalytic
carboxylates
b
g
Released
as
pyrophosphate
Y183
primer
template
D186
P
M184
Y115
D185
D110
N
AZTMP
g
b
D67N
fingers
K219E
D113
T215Y
K70R
PPi
M41L
NRTI Excision
Unstable Complex
Excision
b3-b4 Loop
Fingers Mutations
*
*
ATP Binding Mutations
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Excision/Extension Assay
+ATP, dNTPs, NRTITPs
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HIV-1 RT and HIV-2 RT Sequences
HIV-1
(BH10)
HIV-2 (ROD)
---PGIRYQYNVLPQGWKGSPAIFQ--|| || | ||||||||||||||
---PGKRYIYKVLPQGWKGSPAIFQ--151
---IYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTPDKKHQKEPP--—
| |||||• ••||
|
• |• |
| •||| | ||•||
---IIQYMDDILIASDRTDLEHDRVVLQLKELLNGLGFSTPDEKFQKDPP--185
215 219
HIV Drug Resistance Program
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ATP-Mediated AZTMP Excision/Extension
Deblock and Extend
100 uM each dNTP
vary ATP
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PPi-Mediated AZTMP Excision/Extension
Deblock and extend
100 uM each dNTP
vary PPi
HIV Drug Resistance Program
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Q151 in HIV-1 and HIV-2 RT
74
115
151
73
HIV-1 /DNA/dNTP
HIV-1
116
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HIV-2
Template Sequence Affects
Delayed Chain Termination
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Relative Infectivity
North and South MCT Block HIV Replication
in HSV-TK+ Cells
100
90
80
70
60
50
40
30
20
10
0
N-MCT
S-MCT
AZT
0
0.1
µM Drug
HIV Drug Resistance Program
NCI-Frederick
1
10
North-Methanocarbathymidine (N-MCT)
A-form DNA
HIV Drug Resistance Program
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South-Methanocarbathymidine (S-MCT)
B-form DNA
AZT Excision/Extension
WT
D67 Complex
AZT-R
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100.0 µM each dNTP
10.0 µM AZTTP
PMPA Excision/Extension
100.0 µM each dNTP
10.0 µM PMPA PP,
WT
D67 Complex
AZT-R
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ATP-Mediated Excision of N-MCTMP
N-MCTMP Blocked Primer
10 mM each dNTP
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N-MCdAMP Inhibits AZT-R RT Excision
Simplified Excision/
Extension Assay
3´ MC-dAMP
+ dNTPs, ATP
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N Site AZTMP Complex Compared to
the Ternary Complex
P site
AZTMP
D185
D186
dNTP
~2.5 Å
D110
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N site
Q151
P Site Complex Compared to Binary Complex
primer
P site
N site
D185
D110
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thumb
template
primer
K103N
dNTP
L74I
D67
NNRTI
Binding Pocket
b
fingers
g
T69G
palm
K219Q
K70R
M41L
T215Y
ATP
Binding Pocket
HIV Drug Resistance Program
NCI-Frederick
AA219 in HIV-1 RT and HIV-2 RT
Q151 in HIV-1 and HIV-2 RT
186
HIV-1/DNA/dTTP
185
HIV-1/DNA/TNV-DP
TNV-DP
219
HIV-2 unliganded
HIV Drug Resistance Program
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AA214 in HIV-1 RT and HIV-2 RT
110
185
219
HIV-1
HIV-2
115
116
214
215
N-ter
N-ter
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