Structural Basis for HIV-1 Reverse Transcriptase Drug Resistance to Zidovudine (AZT) and Tenofovir

1
Structural Basis for HIV-1 Reverse Transcriptase
Drug Resistance to Zidovudine (AZT) and Tenofovir

• Kalyan Das
• CABM Rutgers University, NJ, USA

2
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 _at_
• Binding
• Incorporation
• Excision

3
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 951347
• Meyer et al. 1999, Mol. Cell 435
• Boyer et al. 2001. J. Virol. 754832

4
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)

5
dNTP Incorporation and AZT-Resistance Mutations
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Binding of AZTppppA to AZTr RT/dsDNA Complex
Primer
AppppAZT
R72
K65
T215Y
K70R
K70R and T215Y are Excision Enhancing Mutations
(EEMs)
7
ATP binds differently to wild-type and EEM RT
The ATP as an excision substrate binds
differently to wild-type RT and EEM/TAM RT
Wild-type RT does not have high specificity for
ATP binding
The mutations create a new ATP-specific binding
site
8
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

9
K65R Background

• K65R biochemical functions
• Decreases incorporation rate (kpol) of dNTPs and
  NRTIs
• Decreases NRTI excision
• Increases fidelity
• Decreases viral replication capacity

10
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)
thumb
RNase H
fingers
TFV-DP/ dATP
DNA template
DNA primer
palm
p51
p66
11
dNTP Binding Site
primer template
dNTP
K65
b3
?
?
??
?
palm
fingers
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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?
13
Binding of dATP and TFV-DP to K65R RT
Q151
Y115
??
b3
R72
1.7
R65
dATP
TFV-DP
14
dATP and TFV-DP show alternate R65/R72 rotameric
conformations
Y115
R72
N?
N?
?
?
N?
R65
?
TFV-DP
15

• K65R mutation
• Does not significantly alter interaction of
  residue 65 with dNTP
• 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

16
K65R and Excision Enhancing Mutations

• K65R
• Decreases excision
• Increases AZT susceptibility
• K65R and EEMs (TAMs)
• Antagonistic for mutation development

17
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

18
dATP/AZT
Y115
V184
AZT- TP
R72
?
Mg2
???'
ATP
R65
???'
Y215
ATP
?'
R70
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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)

20
Acknowledgements
Rutgers University CABM Xiongying Tu Rajiv
Bandwar Arthur D. Clark, Jr. Joseph Bauman Stefan
Sarafianos Steve Tuske Eddy Arnold Chemistry Qian
wei Han Barbara L. Gaffney Roger A. Jones
Gilead Sciences, Inc. Kirsten White Joy
Feng Michael Miller
HIV DRP Paul L. Boyer Stephen H. Hughes
NIH funded