Sensitivity of a novel translocation defective reverse transcriptase inhibitor, EFdA in comparison to second generation NNRTI, Etravirine and Rilpivirines on diverse HIV-1 subtypes

Duncan T. Njenda1, Leonard Rogers,3 Shambhu G. Aralaggupe1 Md Shanawazur

Rahman,1 Kamalendra Singh,1,3 Stefan Sarafianos,3 Anders Sonnerborg,1

Ujjwal Neogi1

1Karolinska Institutet, Stockholm Sweden 2 Stellenbosch University, South Africa 3University of Missouri, USA

Global Distribution of HIV-1 subtypes

Hemelaar 2012

Subtype in treatment response

Treatment response does not differs between subtypes

EFdA (4′-ethynyl-2-fluoro-2′-deoxyadenosine)

• EFdA is a nucleoside reverse transcriptase translocation inhibitor (NRTTI)

• Prolonged persistence of triphosphate form in PBMC and macrophages

• Potential for once weekly dosing

• Long-acting formulations under development

1Michailidis et al J Biol Chem 284: 35681-91; 2009 2Murphey-Corb et al AAC 56:4707-12; 2012

Zhe Li Salie, PNAS, 9274–9279, Kamalendra Singh personal communication

Tight binding

OCT (obligate chain terminator)

N site P site

EFdA-TP

Pre-translocation complex

EFdA-MP

N site

P site EFdA-MP

Translocation Defective RT Inhibitor (TDRTI)

DCT (Delayed chain terminator)

HIV RT

Post-translocation complex

N site

P site

115

160

184

185

114

EFdA-MP

183

Enhanced Misincorporation T/PEFdA-MP

T/PEFdA-MP+dTMP

Time (min)

More likely at OCT sites

Mode of action is sequence dependent

Decreased excision

Aim

To evaluate the virological and biochemical inhibitory potentials of EFdA

against a broad spectrum of subtype-specific viruses and a panel of known

reverse transcriptase inhibitor (RTI) resistant strains and compare the data

with first and second generation non-nucleoside reverse transcriptase

inhibitors (NNRTI).

Virological Assay Biochemical Assay M

eth

od

s

HIV-1 subtypes and anti-retrovirals

Reverse Transcriptase Inhibitors

Efavirenz (EFV)

Rilpivirine (RPV)

Etravirine (ETR)

Nevirapine (NVP)

EFdA

Tenofovir Alafenamide (TAF) C

B

02_AG

01_AG

Binding affinity of EFdA

Subtype

kpol (s-1)

Binding Affinity Kd.dNTP (µM)

kpol/ Kd.dNTP (µM-1 s-1) Fold Change

EFdA-TP/dATP

dATP EFdA-TP dATP EFdA-TP dATP EFdA-

TP

HIV-1B 5.9 ± 0.2 4.5 ± 0.1 1.2 ± 0.1 0.17 ± 0.02 4.9 26.5 5.4

HIV-1C 3.0 ± 0.3 0.5 ± 0.03 4.8 ± 0.2 0.23 ± 0.02 0.63 2.2 3.5

01_AE 10.8 ± 0.4 10.7 ± 0.5 2.0 ± 0.1 0.95 ± 0.04 5.4 11.3 2.1

02_AG 10.4 ± 0.4 3.6 ± 0.2 2.1 ± 0.3 0.20 ± 0.01 5.0 18 3.6

In vitro DSA

Data from 21 patient-derived recombinant clones without any primary DRM, triplicate assay in two independent experiments

RPV

ETR

NVP

EFV

EFdA

TAF

0

2

4

6

8

10

100

200

£

£

££

££

£

££££££

££££

£

£££

¤

¤¤¤

¤¤ ¤¤

¤

¤¤

¤¤

¤¤

¤

¤¤

¤¤

¤

lllll

ll

ll

l

ll

l

lll

lllll

¥ ¥

¥

¥¥¥¥

¥

¥

¥¥¥

¥

¥¥

¥

¥

¥

¥¥

¥

p

p

pp

pppp p

p

p

p

p

p

p p

p

p

p

p

p

[[

[

[

[

[

[[

[[

[

[[

[

[

[

[

[

[

[

[

Drugs

EC

50 (n

M)

HIV-1B

HIV-1C

01_AE/02_AG

In vitro DSA

RPV

ETR

NVP

EFV

EFdA

TAF

0

50

100

50010001500

Drugs

EC

50 (n

M)

Subtype DRMs

B M41L, D67N, T69D, L74I, L210W, T215Y, K101E, Y181C,G190A

B K70R, M184V, T215F, K101E, Y181V

B M41L, T215D, K101E, E138K, Y181C

B K101E, G190S

B M41L, T215Y, K101P, K103N

B M41L, L74V, M184V, T215Y,L100I, K103N

B M41L, D67G, L74I, M184V, T215Y, L100I, M230L

B Y181C, G190A

D K65R, T69del, L100I, Y181C, Y188L, H221Y

C K65R, M184V, V108I, Y181C, G190S

Conclusion • Better efficiency of EFdA-TP incorporation is due to its tighter

binding (lower Kd.EFdA-TP) to RTs than dATP (higher Kd.dATP)

• HIV-1C RT has lowest efficiencies for both dATP and EFdA-TP; however, this RT prefers EFdA-TP over dATP by about 3.4 – fold.

• EFdA inhibits both wild type and RTI-resistant viruses efficiently in subtype independent manner (<10nM).

• There is one wild type 02_AG recombinant virus without any known RTI DRM which showed higher EC50 (>10nM).

• EFdA is an attractive drug for clinical trials with therapy naïve and therapy failure individuals.

Acknowledgements

Jonas Söderquist’s Stipendium for Experimental Virology and Immunology Research-2016

Collaborations:

Prof. Anders Sonnerborg, Karolinska Institutet, Sweden

Prof. Susan Engelbrecht, Stellenbosch University, South Africa

Prof. Stefan Sarafianos, Univ. of Missouri, US

Swedish National InfCare HIV cohort

Conference Participation Grant