structural basis for hiv-1 reverse transcriptase drug resistance to zidovudine (azt) and tenofovir
DESCRIPTION
Structural Basis for HIV-1 Reverse Transcriptase Drug Resistance to Zidovudine (AZT) and Tenofovir. Kalyan Das CABM & Rutgers University, NJ, USA. NRTI - Inhibition and Resistance. NRTI Inhibition Nucleoside/nucleotide analog - PowerPoint PPT PresentationTRANSCRIPT
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Structural Basis for HIV-1 Reverse Transcriptase Drug
Resistance to Zidovudine (AZT) and Tenofovir
Kalyan Das
CABM & Rutgers University, NJ, USA
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NRTI - Inhibition and Resistance
• NRTI Inhibition
– Nucleoside/nucleotide analog– Gets incorporated at the DNA-primer terminus by
RT and acts as a chain terminator
• NRTI Resistance
– Different RT mutations or sets of mutations emerge in response to different NRTIs
– A mutant RT has the ability to discriminate the drug from normal nucleotides
– Discrimination can occur @» Binding» Incorporation» Excision
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AZT-resistance Mutations
• AZT-MP gets incorporated
• RT removes AZT-MP by excision
• Excision is reverse of polymerization
• ATP is the primary excision substrate in vivo
• ATP excises AZT-MP to form AZTppppA
Meyer et al. 1998, PNAS 95:1347 Meyer et al. 1999, Mol. Cell 4:35 Boyer et al. 2001. J. Virol. 75:4832
HNO
O
N
ON3
O
P
O
O-O
P
O
O-O
P
O
O-O
P O-O
O
OHO
HON N
N
N
NH2
ATP’
AZTTP
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Methods
Five crystal structures were determined
– wt RT/dsDNA/AZTppppA (3.1 Å resolution)
– AZTr RT/dsDNA/AZTppppA (3.2 Å)
– AZTr RT/dsDNA terminated with AZTMP at N-site (3.6 Å)
– AZTr RT/dsDNA terminated with AZTMP at P-site (2.9 Å)
– apo AZTr RT (2.6 Å)
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primer
template
palm
fingers
dTTP
primer
template
palm
fingers
T215Y
K219Q
D67N
K70RM41L
dTTP
primer
template
palm
fingers
T215Y
K219Q
D67N
K70RM41L
AZTppppA
primer
template
palm
fingers
dNTP Incorporation and AZT-Resistance
Mutations
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T215YK70RK70R
R72R72
K65K65
PrimerPrimer
Appp
AppppAZT
pAZT
Binding of AZTppppA to AZTr RT/dsDNA Complex
K70R and T215Y are Excision Enhancing Mutations (EEMs)
T215YK70RK70R
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T/Y215
K/R70
K65
R72
YMDD
'
'
DNA primer
template
AZTppppA (I)
AZTppppA (II)
Y215
R70
Site I
Site II
ATP’
The ATP as an excision substrate binds differently to wild-type RT and EEM/TAM RT
The mutations create a new ATP-specific binding site
Wild-type RT does not have high specificity for ATP binding
ATP binds differently to wild-type and EEM RT
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K65R Background
• K65R is an NRTI resistance mutation in HIV-1 RT:
– Selected by TDF, ABC, ddI, and occasionally d4T
– Observed in 2-5% of antiretroviral-experienced patients
– Low-level resistance to all NRTIs, with the exception of AZT which remains susceptible
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K65R Background
• K65R biochemical functions:
– Decreases incorporation rate (kpol) of dNTPs and NRTIs
– Decreases NRTI excision
– Increases fidelity
– Decreases viral replication capacity
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p51p66
fingers
thumbRNase H
palm
DNA primer
DNA template
TFV-DP/dATP
Structures of K65R RT/dsDNA/TFV-DP (3.0 Å; R 0.251; R-free 0.284) K65R RT/dsDNA/dATP (3.3 Å; R 0.254; R-free 0.286)
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K65
dNTP
primer :
template
fingers
palm
dNTP Binding Site
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Y115 Y115Q151 Q151
R72 R72
R65 R65
K65R and R72 form a Molecular Platform
Like K65 in wt RT structures, R65 also interacts with the-phosphate.
The guanidinium planes of arginines at positions 65 and 72 stack to form a Molecular Platform.
R72 is highly conserved; mutations at R72 impair RT polymerization.
How does the platform discriminate TFV-DP from dATP?
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Y115
R65
R721.7
Binding of dATP and TFV-DP to K65R RT
N
N
NN
H2N
O
OH
O
P
O
O-O
P
O
O-O
P
O-
O-O
N
N
NN
H2N
P
O
O-O
P
O
O-O
P
O-
O-O
O
Q151
dATP TFV-DP
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dATP and TFV-DP show alternate R65/R72 rotameric conformations
N
NN
NR65
R72
Y115
dATP
N
Y115
R65
N
N
R72
TFV-DP
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K65R mutation:
1. Does not significantly alter interaction of residue 65 with dNTP
2. Forms a Molecular Platform with R72 that may work as a “Check Point”
- Reduces dNTP incorporation- Reduces NRTI excision- Increases fidelity
3. The platform has alternate rotameric conformations when TFV-DP vs. dATP binds
- Causes discrimination of TFV-DP from dATP
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K65R and Excision Enhancing Mutations
• K65R:– Decreases excision– Increases AZT susceptibility
• K65R and EEMs (TAMs): – Antagonistic for mutation
development
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K65R and M184V
• M184V is a primary mutation emerges against 3TC and FTC
• M184V with K65R– Increases resistance to ABC
and ddI– Partial re-sensitization to TFV
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V184
R65
R72
R70
Mg2+
Y215
Y115
'
'
'
ATP’
dATP/AZT3TC/FTC resistance site
TFV resistance site
Excision Enhancing Mutations
HNO
O
N
ON3
O
P
O
O-O
P
O
O-O
P
O
O-O
P O-O
O
OHO
HON N
N
N
NH2
ATP’
AZT- TP
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Conclusions
• EEMs create a site for binding ATP as excision substrate – K70R and T215Y help ATP binding
• K65R forms a molecular platform that is responsible for– selective NRTI resistance, reduced dNTP
incorporation, reduced excision and reduced viral fitness
• The K65R/R72 platform cross-talks with other NRTI resistance mutations– With M184V across the substrate ribose ring– Negatively with L74V through the templating
base– Negatively with EEMs (K70R and T215Y)
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Acknowledgements
HIV DRPPaul L. BoyerStephen H. Hughes
Rutgers University
CABMXiongying TuRajiv BandwarArthur D. Clark, Jr.Joseph BaumanStefan SarafianosSteve TuskeEddy Arnold
ChemistryQianwei HanBarbara L. GaffneyRoger A. Jones
Gilead Sciences, Inc.Kirsten WhiteJoy FengMichael Miller
NIH funded