Transcript Structural Basis for K65R Function: Tenofovir Resistance

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
– 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
AZT-resistance Mutations
O
•
AZT-MP gets incorporated
AZTTP
HN
O
N
O
•
N3
RT removes AZT-MP by excision
O
O P O-
•
Excision is reverse of polymerization
O
O P O-
•
O
O P O-
ATP is the primary excision substrate in vivo
O
•
O P O-
ATP excises AZT-MP to form AZTppppA
O
ATP’
HO
O
HO
Meyer et al. 1998, PNAS 95:1347
Meyer et al. 1999, Mol. Cell 4:35
Boyer et al. 2001. J. Virol. 75:4832
N
N
NH2
N
N
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)
dNTP Incorporation and
AZT-Resistance Mutations
dTTP
T215Y
palm
K219Q
D67N
M41L K70R
AZTppppA
fingers
Binding of AZTppppA to AZTr RT/dsDNA Complex
Primer
R72
K65
T215Y
K70R
K70R and T215Y are Excision Enhancing Mutations (EEMs)
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
Site I
Wild-type RT does not
have high specificity for
ATP binding
Site II
The mutations create a
new ATP-specific
binding site
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
K65R Background
• K65R biochemical functions:
– Decreases incorporation rate (kpol) of dNTPs and
NRTIs
– Decreases NRTI excision
– Increases fidelity
– Decreases viral replication capacity
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
fingers
RNase H
TFV-DP/
dATP
palm
p66
p51
dNTP Binding Site
dNTP
b3
K65

b
b
palm

fingers
K65R and R72 form a
Molecular Platform
Y115
Y115
Like K65 in wt RT structures,
Q151
R65 also interacts
with the
-phosphate.
Q151
R72
The guanidinium planes of R72
arginines at positions 65 and
72 stack to form a
Molecular Platform.
R72 is highly conserved;
mutations at R72 impair
RT polymerization.
R65
How does the platform
R65
discriminate TFV-DP from dATP?
Binding of dATP and TFV-DP to K65R RT
Q151
N
H2N
N
N
N
H2N
N
N
N
Y115
N
b
O
O
OH
O
O
O
P
O-
O
O
P
O P
P
O-
O-
O
O P
O
b3
O-
O
O
O-
P
R72
1.7
O-
O-
O-
R65
dATP
TFV-DP
dATP and TFV-DP show alternate R65/R72
rotameric conformations
Y115
Y115
R72
R72
N
N
N

b
N

N
b
N
N

R65
dATP

R65
TFV-DP
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
K65R and Excision Enhancing Mutations
• K65R:
– Decreases excision
– Increases AZT susceptibility
• K65R and EEMs (TAMs):
– Antagonistic for mutation
development
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
3TC/FTC
resistance
site
dATP/AZT
Y115
O
HN
O
N
O
N3
AZTTP
V184
O
R72

O P OO
O P OO
O P O-
Mg2+ b'
O
O P
O-
O
ATP’
R65
HO
O
HO
N
b'
N
NH2
N
N
Y215
ATP’
'
R70
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)
Acknowledgements
Rutgers University
CABM
Xiongying Tu
Rajiv Bandwar
Arthur D. Clark, Jr.
Joseph Bauman
Stefan Sarafianos
Steve Tuske
Eddy Arnold
Chemistry
Qianwei 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