Resistance Mechanisms of HIV-1 Reverse Transcriptase Mutants K65R, M184V, and K65R M184V to NRTIs

1
Resistance Mechanisms of HIV-1 Reverse
Transcriptase Mutants K65R, M184V, and
K65RM184Vto NRTIs

• JK Ly, NA Margot, HL MacArthur, M Hung,
• MD Miller, KL White
• Gilead Sciences Inc., Foster City, CA

2
NtRTI and NRTI Resistance Mutation Background

• 8 currently approved NtRTI and NRTIs
• TDF, ddI, ABC, FTC, 3TC, ddC, AZT, d4T
• K65R
• Selected by TDF, ABC, ddI, and occasionally d4T
• Observed in 2-5 of antiretroviral-experienced
  patients
• M184V
• Selected by FTC, 3TC, ABC
• Observed in gt50 of antiretroviral-experienced
  patients
• K65RM184V
• Selected by TDF/FTC, ABC, other drug combinations
• 50 of patients with K65R also have M184V

3
Study Objective

• To measure the biochemical resistance mechanisms
  for K65R, M184V, and K65RM184V mutant RT and
  determine their contributions to resistance.

N(t)RTI Susceptibility


Binding or Incorporation
Excision
Steady State Ki / Km
Excision Rate
4
Susceptibility of Mutant Viruses to N(t)RTIsa
a. PhenoSense Assay (Monogram Biosciences). b.
Mean fold change for K65R alone ( n gt 110)
M184V/I alone (n gt 1930) K65RM184V/I (n
70). Yellow bars represent lower clinical
cut-offs.
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Altered Binding or Incorporation of N(t)RTIs
Fold increase in Ki / Km compared to wild-typea Fold increase in Ki / Km compared to wild-typea Fold increase in Ki / Km compared to wild-typea
K65R M184V K65RM184V
TFV-DP 7.8 1.1 5.6
ddATP 6.8 1.6 12.8
FTC-TP 3.9 gt15 gt15
3TC-TP 2.1 gt10 gt10
CBV-TP 4.8 3.1 11.6
d4T-TP 5.7 0.8 4.8
AZT-TP 4.9 1.9 6.1
a. Mean fold increase were determined from 3 or
more independent experiments.
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Altered Binding or Incorporation of N(t)RTIs
Fold increase in Ki / Km compared to wild-typea Fold increase in Ki / Km compared to wild-typea Fold increase in Ki / Km compared to wild-typea
K65R M184V K65RM184V
TFV-DP 7.8 1.1 5.6
ddATP 6.8 1.6 12.8
FTC-TP 3.9 gt15 gt15
3TC-TP 2.1 gt10 gt10
CBV-TP 4.8 3.1 11.6
d4T-TP 5.7 0.8 4.8
AZT-TP 4.9 1.9 6.1
a. Mean fold increase were determined from 3 or
more independent experiments.
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Excision Assay Methodology

• Rescue of polymerization assay
• Primer-NRTI Template
• HIV-1 RT (excess)
• Physiological ATP and dNTPs
• Time course, RT inactivation
• dNTP Klenow elongation

ATP
ATP-T
T
-5CTACTAGTTTTCTCCATCTAGACGATACCAGA
3GATGATCAAAAGAGGTAGATCTGCTATGGTCTAACTTCTGGAGTCGTG
AG
HIV-1 RT

• Factors affecting excision
• RT mutations
• N(t)RTI translocation
• Next nucleotide inhibition
• Primer/template sequence context

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Altered Excision of N(t)RTIs (ATP-mediated)
TFV
AZT
14
14
12
12
10
10
Primer Rescued ()
8
8
Primer Rescued ()
6
6
4
4
2
2
0.1
1
10
100
1000
0.1
1
10
100
1000
dATP (mM)
dATP (mM)

• K65R and K65RM184V mutants showed significantly
  reduced removal of TFV and AZT mediated by ATP.
• For all other NRTIs tested, ATP-mediated excision
  was minimal by WT and mutants.

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Altered Excision of N(t)RTIs (PPi-mediated)
TFV
AZT
100
40
80
30
60
Primer Rescued ()
Primer Rescued ()
20
40
10
20
1
10
100
1
10
100
dATP (mM)
dATP (mM)

• K65R and K65RM184V mutants showed reduced
  removal of TFV and AZT by pyrophosphate.
• M184V showed reduced excision of AZT.

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Conclusions

• In cells, both mechanisms of resistancebinding
  or incorporation (discrimination) and
  excisionmay contribute to altered drug
  susceptibility
• K65R shows increased drug discrimination
  (increased Ki/Km) for all N(t)RTIs
• Counteracted by decreased excision for most
  N(t)RTIs, resulting in full susceptibility to AZT
• M184V substrate discrimination correlates with
  N(t)RTI susceptibility
• For AZT, decreased excision may also contribute
  to sensitization
• K65RM184V results in additive resistance for ddI
  and ABC at the level of discrimination, but
  increased sensitivity relative to K65R for TDF,
  d4T and AZT
• Sensitization to AZT may be due to decreased
  excision
• Sensitization to d4T and TDF is likely at the
  level of substrate discrimination

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Acknowledgements
Clinical Virology, Foster City, CA Kirsten
White Nicolas Margot Damian McColl Rebecca
Ledford Michael Miller Clinical Virology,
Durham, NC Joy Feng Jenny Svarovskaia Josh
Waters Katyna Borroto-Esoda
Biology Holly MacArthur Magdeleine Hung Ruth
Wang Martin McDermott Manuel Tsiang Computationa
l Chemistry James Chen S. Swaminathan