Antiviral Activity of EFdA Against NRTI-Sensitive and -Resistant Strains of HIV-2Vincent H. Wu1, Robert A. Smith1, Sara Masoum1, Dana N. Raugi1, Selly Ba2, Moussa Seydi2, Jay Grobler3, and Geoffrey S. Gottlieb1,4

for the University of Washington-Dakar HIV-2 Study Group

1Department of Medicine, Division of Allergy and Infectious Diseases and 4Department of Global Health, University of Washington, Seattle, Washington, USA2Clinique des Maladies Infectieuses Ibrahima DIOP Mar, Centre Hospitalier Universitaire de Fann, Universite Cheikh Anta Diop de Dakar, Dakar, Senegal

3Merck & Co., Inc., West Point, Pennsylvania, USA

CROI 2017Seattle, WA

correspondence: smithra@uw.edu

AimTo evaluate the activity of EFdA against HIV-2 isolates

and NRTI-resistant HIV-2 mutants in culture.

Approach• Single-cycle drug susceptibility assays using HeLa-CD4 indicator cells

(MAGIC-5A) and a colorimetric readout for quantifying viral infection (CPRG substrate conversion).

• Resistance testing with site-directed and patient-derived RT mutants.

HIV-2 is more sensitive than HIV-1 to EFdA

Results

ResultsEFdA is active against NRTI-resistant HIV-2 mutantsBackground

HIV-2 infection is a significant public health problem in West Africa and has been reported in other countries with ties to the region.

Historically, clinical outcomes of antiretroviral therapy in HIV-2 and HIV-1/HIV-2 dually-positive patients have been poor, with high rates of immunovirologic failure and multidrug resistance. Newer antivirals with improved safety, efficacy, and resistance profiles are needed for HIV-2– infected individuals.

Background

ConclusionsEFdA a potent inhibitor of HIV-1 and HIV-2 in cell culture with mean EC50 values of 2.6 ± 0.6 nM and 0.53 ± 0.15 nM, respectively. The observation that HIV-2 is 5-fold more sensitive to EFdA can be explained by structural features that affect the 4'-ethynyl group.

As observed for HIV-1, K65R mutants of HIV-2ROD9 are hypersusceptible to EFdA. K65R+Q151M mutants of HIV-2 are also hypersusceptible to the drug.

The M184V change in HIV-2ROD9 confers a 20-fold shift in the potency of EFdA, but the EC50 for the mutant virus is only 3-fold higher than the mean EC50 for HIV-1. Addition of other NRTI resistance changes in combination with M184V does not increase the level of EFdA resistance in HIV-2.

EFdA should be further evaluated in clinical studies involving HIV-2–infected individuals.

AcknowledgmentsWe thank Merck & Co., Inc. for providing EFdA (MK-8591). VW recieved a monetary award for this project from the Mary Gates Undergraduate Research Program. These studies were also supported by grants to GSG from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID; AI060466 & AI120765) and the University of Washington Center for AIDS Research. The UW-Dakar HIV-2 Study Group includes Moussa Seydi,Papa Salif Sow, Selly Ba, Fatima Sall, Fatou Traore, Khadim Faye, Ousseynou Ndiaye, Marie Pierre Sy, Bintou Diaw, Mbaye Ndoye, Amadou Bale Diop, Marianne Fadam Diome (Service des Maladies Infectieuses Ibrahima Diop Mar, Centre Hospitalier Universitaire de Fann, Universite Cheikh Anta Diop de Dakar, Dakar, Senegal); Alas-sane Niang, El Hadji Ibrahima Sall, Ousseynou Cisse, Ibrahima Tito Tamba, Jean Phillippe Diatta, Raphael Bakhoum, Jacque Francois Sambou, Juliette Gomis (Region Medicale de Ziguinchor, Ziguinchor, Casamance, Senegal); Stephen Hawes, Noelle Benzekri, Robert Coombs, Ming Chang, John Lin, Nancy Kiviat, and James Mullins (Univ. of Washington).

440

EFdA is a novel, 4´-modified nucleoside analog inhibitor

dA(2´-deoxyadenosine)

EFdA (MK-8591)(4´-ethynyl-2-fluoro-2´-deoxyadenosine)

EFdA is a nucleoside reverse transcriptase translocation inhibitor (NRTTI). It is highly active against HIV-1 in culture, with EC50 values in the low- nanomolar to picomolar range and negligible cytotoxicity.

EFdA is converted to EFdA-5´-triphosphate (EFdATP) by cellular kinases and binds to the polymerase active site of HIV-1 reverse transcriptase (RT) with an affinity ≥ that of dATP.

Efforts to evaluate the activity of EFdA against HIV-2 in culture are limited; a single report showed that a group B strain (HIV-2EHO) was sensitive to the drug in spreading infections of MT-4 cells*. The ability of EFdA to inhibit HIV-2 mutants that are resistant to other NRTI is unknown.

A single 10-mg dose of EFdA demonstrated potent antiviral activity for 10 days in a phase 1b proof-of-concept clinical trial.

Median plasma viral load reduction through d10 = 1.78 Log10 copies/ml.

Median half life of EFdA in plasma = 60 h.

EFdA inhibits HIV-1 RT via multiple mechanisms

92U

G02

9 (M

/A)

NL4

-3 (M

/B)

LAI (

M/B

)

89.6

(M/B

)

MJ4

(M/C

)

92U

G00

1 (M

/D)

94U

G11

4.1.

6 (M

/D)

93B

R02

0 (M

/F)

MV

P51

80-9

1 (O

)

BC

F01

(O)

RO

D9

(A)

7924

A (A

)

MVP

1513

2 (A

)

6041

5K (A

)

CB

L-20

(A)

CB

L-23

(A)

CD

C77

618

(A)

ST

(A)

CD

C31

0072

(B)

CD

C31

0319

(B)

EH

O (B

)

CO

U (B

)

BER

(B)

7312

A (A

/B)

0

2

4

6

EC50

(nM

)

HIV-1

HIV-2

Summary: EFdA resistance profile for HIV-2

HIV-1 HIV-20

1

2

3

4

EC50

(nM

)

p < 0.0001,Mann-Whitney

test

4.9X

G

A

C

T

G

C

T

G

G

A

C

T

G

C

T

G

EFdATP

G

A

C

T

G

C

T

G

E

G

A

C

T

G

C

T

G

E

G

A

C

T

G

C

T

G

E

G

A

C

T

G

C

T

G

E

CdCTP

G

A

C

T

G

C

T

G

A

EFdATP

G

A

C

T

G

C

T

G

A

E

... ... ...... ... ...

polymeraseactive site

Immediatechaintermination

Delayedchaintermination

Mispair-mediatedtermination

...

...

CLINICAL STUDIES

HIV-1 and HIV-2 RTs show subtle differences in the area surrounding the 4´-ethynyl group

0.01 0.1 1 10 100 10000

10

20

30

40

50

60

70

80

90

100

110

120

[EFdA] (nM)

% o

f no-

drug

con

trol

HIV-1 NL4-3HIV-2 ROD9

HIV-1NL4-3

HIV-2ROD9

0.0

0.5

1.0

1.5

2.0

2.5

EC50

(nM

)

Results of 9 independentside-by-side assays

Example: dose-response profilesfrom a single assay run

HIV-2group A

HIV-2group B

0.0

0.2

0.4

0.6

0.8

EC50

(nM

)

n = 8 6

HIV-2 group A vs. group B

p = 0.43,Mann-Whitney

test

0.1

1

10

100

EC

50 (n

M)

2-fold

13–20-fold

2-fold

0.1

1

10

100

9-fold

HIV-2 HIV-1

K65RQ151M

WT K65R Q151M M184VK65R Q151M K65R

Q151MK65RK70R

Q151M

K65RY115FQ151M

PD RT(4-7a)*

M184V+ WT M184V

Adopted from Michailidis et al., J. Biol. Chem. 289(35):24533

Friedman et al. , CROI 2016 Abstr. 437 LB

* Kawamoto et al., Int. J. Biochem. Cell Biol. 40:24102´,3´-didehydro-3´-deoxy-4´-ethynylthymidine is shown as the incoming nucleotide (Smith et al. Antimicrob. Agents Chemother. 59:7437).

PubChem CID: 6483431

Data for AZT, FTC and TDF are from Smith et al. J. Infect. Dis. 199:1323 and unpublished results.

*Values for single and double mutants were obtained using unmodified tenofovir.

*Patient-derived clone encodingK65R, N69S, V111I, Q151M and M184V.

*Mutant AZT FTC EFdA TDF

K65R 1 85 0.4 1Q151M 43 5 0.9 0.3K65R+Q151M 56 250 0.5 2M184V 1 >200 20 1Q151M+M184V 29 >200 16 1K65R+Q151M+M184V 66 >200 15 3K65R+K70R+Q151M+M184V 116 >100 17 2K65R+Y115F+Q151M+M184V 139 >100 15 4

Fold Resistance to:

CC50 for EFdA in MAGIC-5A cells: >100 nM (CellTiter Glo® Assay)