Duncan T. Njenda [1,2], Shambhu Prasad Ganeshappa Aralaggupe , Md Shanawazur Rahman  Kamlendra Singh [1,3] Stefan Sarafianos , Anders Sonnerborg , Ujjwal Neogi 
Affiliates:  Karolinska Institutet, Stockholm Sweden.  Stellenbosch University, South Africa.  University of Missouri, USA.
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 and compare the data with first Nevirapine (NVP) and Evafirenz (EFV) and second generation Rilpivirine (RPV) and Etravirine (ETR) non-nucleoside reverse transcriptase inhibitors (NNRTI).
The gag-pol (n=24) from treatment naïve individuals was cloned into pNL4.3Δgag_pol to prepare recombinant replication competent viruses. A panel seven reverse transcriptase inhibitor (RTI) resistant strains (obtained from the NIH AIDS reagent and reference program) together with the two patients’ derived gag-pol clone with DRM and NL43 strain (as control) were used to investigate comparative potency of RTI drugs. Phenotypic drug sensitivity assay (DSA) was performed in TZM_bl cell lines against different drugs- EFdA, NVP, EFV, ETR and RPV respectively. Fold-change in EC50 was calculated against NL43 viruses. Patient derived RTs from HIV-1B, HIV-1C, 01_AE and 02_AG subtypes were cloned, expressed and purified. In vitro inhibition and binding affinity assays were performed using patient derived RTs against all the above-mentioned drugs.
While comparing the recombinant viruses from treatment naïve viruses without any drug resistant mutations (DRMs), EFdA displayed comparable ex vivo potency [median (IQR) EC50 = 1.59 (0.96 – 3.40)] compared to RPV [0.82 (0.51-1.85)], ETR [(1.25 (0.56-1.55)] and EFV [1.15 (0.59 – 1.86)] but better than NVP [68.3 (49.6 – 80.6)]. The EC50 value for EFdA against the RTI strains were below 20nM. In vitro biochemical assays using patient derived RTs identified subtype specific difference in binding affinity among the 1st and 2nd generations NNRTIs. The binding affinity of NVP was significantly low compared to other drugs, while EFdA has higher binding efficacy.
Our combined in vitro biochemical data and ex vivo virological data suggests that EFdA inhibits both wild type and RTI-resistant viruses efficiently in subtype independent manner. No subtype specific effect was observed in in vitro DSA despite difference in in vitro binding affinity on RTs without any DRM was observed across subtypes with different classes of RTIs. Therefore, EFdA can be a good choice drug for clinical trials involving both therapy naïve and therapy failure individuals.