P21. Subtype-independent broad inhibitory activity of EFdA with or without drug resistance mutations in HIV-1 reverse transcriptase

Duncan T. Njenda1, Md Shanawazur Rahman1, Kamalendra Singh2, Stefan Sarafianos2, Anders Sonnerborg1, Ujjwal Neogi1
Affiliates: 1Karolinska Institutet, Stockholm Sweden 2University of Missouri, US

Background
EFdA (4’-Ethnyl-2’-Fluoro-2’ deoxyadenosine) is a novel translocation-defective reverse transcriptase inhibitor (TDRTI). In contrast to other approved NRTIs, EFdA contains 3’OH group that improves the phosphorylation potential of this drug in vivo, making it a strong competitor with natural (dATP) substrate during HIV-1 cDNA synthesis. EFdA has been shown to have low toxicity higher in vitro potency to therapy naïve viruses. Our study investigates 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) and protease inhibitor resistant strains.

Methods
Patient derived gag-pol (n=16) was cloned into pNL4.3Δgag_pol to prepare recombinant replication competent viruses. A panel of six protease inhibitors (PIs) and eight reverse transcriptase inhibitor (RTI) resistant strains (obtained from the NIH AIDS reagent and reference program) together with the NL43 strain (as control) were used to investigate efficacy of EFdA. TZM_bl cells were infected with viruses at an M.O.I of 0.005 in the presence of serial EFdA dilutions and then assayed indirectly after 48 hours post infection using luciferase activity as a surrogate marker of infectivity. Fold-change in EC50 was calculated against NL43 viruses. RTs from HIV-1B, HIV-1C and 01_AE subtypes were cloned into the heterodimer expression plasmid pRT6H-PROT and the catalytic efficiency of polymerization of HIV-1B and HIV-1C RTs was determined by pre-steady state kinetic analysis.

Results
After excluding two viruses with low replication fitness, the therapy naïve chimeric viruses (n=14) representing HIV-1C, B, 01_AE and 02_AG, 12 (85%) gave an EC50 fold change ≤1 compared to NL4-3. One 01_AE chimera virus with E138A mutation showed fold change of EC50 1.4 while one wild type 02_AG virus also showed fold change of EC50 2.2. Seven out of eight major RTI-DRM (both NRTI and NNRTI mutations) and six out of seven PI-DRM containing strains also showed fold change of EC50 ≤1 compared to NL4-3 (Table 1). The pre-steady kinetics of nucleotide incorporation indicated that EFdA has greater efficiency compared to the natural substrate dATP with fold change 2.9, 3.7 and 2.7 for HIV-1B, HIV-1C and 01_AE respectively.

Conclusion
Our combined in vitro virological and biochemical data suggests that EFdA inhibits both wild type and RTI-resistant viruses efficiently in subtype independent manner. Therefore this drug makes a strong choice drug for clinical trials involving both therapy naïve and therapy failure patients.

P21-Duncan_Fig_1