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

• 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

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)

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

T215YK70RK70R

R72R72

K65K65

PrimerPrimer

Appp

AppppAZT

pAZT

Binding of AZTppppA to AZTr RT/dsDNA Complex

K70R and T215Y are Excision Enhancing Mutations (EEMs)

T215YK70RK70R

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

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

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)

K65

dNTP

primer :

template

fingers

palm

dNTP Binding Site

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?

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

dATP and TFV-DP show alternate R65/R72 rotameric conformations

N

NN

NR65

R72

Y115

dATP

N

Y115

R65

N

N

R72

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

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

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

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