Ethynyl-2-fluoro-2'-deoxyadenosine Research Articles

EFdA efficiently suppresses HIV replication in the male genital tract and prevents penile HIV acquisition.

Sexually transmitted HIV infections in heterosexual men are acquired through the penis. Low adherence to condom usage and the fact that 40% of circumcised men are not protected indicate the need for additional prevention strategies. Here, we describe a new approach to evaluate the prevention of penile HIV transmission. We demonstrated that the entire male genital tract (MGT) of bone marrow/liver/thymus (BLT) humanized mice is repopulated with human T and myeloid cells. The majority of the human T cells in the MGT express CD4 and CCR5. Direct penile exposure to HIV leads to systemic infection including all tissues of the MGT. HIV replication throughout the MGT was reduced 100-1,000-fold by treatment with 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), resulting in the restoration of CD4+ T cell levels. Importantly, systemic preexposure prophylaxis with EFdA effectively protects from penile HIV acquisition. IMPORTANCE Over 84.2 million people have been infected by the human immunodeficiency virus type 1 (HIV-1) during the past 40 years, most through sexual transmission. Men comprise approximately half of the HIV-infected population worldwide. Sexually transmitted HIV infections in exclusively heterosexual men are acquired through the penis. However, direct evaluation of HIV infection throughout the human male genital tract (MGT) is not possible. Here, we developed a new in vivo model that permits, for the first time, the detail analysis of HIV infection. Using BLT humanized mice, we showed that productive HIV infection occurs throughout the entire MGT and induces a dramatic reduction in human CD4 T cells compromising immune responses in this organ. Antiretroviral treatment with novel drug EFdA suppresses HIV replication in all tissues of the MGT, restores normal levels of CD4 T cells and is highly efficient at preventing penile transmission.

Co-delivery of an HIV prophylactic and contraceptive using PGSU as a long-acting multipurpose prevention technology

ABSTRACT Objectives Poly(glycerol sebacate) urethane (PGSU) elastomers formulated with 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), levonorgestrel (LNG), or a combination thereof can function as multipurpose prevention technology implants for prophylaxis against HIV and unintended pregnancies. For these public health challenges, long-acting drug delivery technologies may improve patient experience and adherence. Traditional polymers encounter challenges delivering multiple drugs with dissimilar physiochemical properties. PGSU offers an alternative option that successfully delivers hydrophilic EFdA alongside hydrophobic LNG. Methods This article presents the formulation, design, and characterization of PGSU implants, highlighting the impact of API loading, dimensions, and individual- versus combination-loading on release rates. Results Co-delivery of hydrophilic EFdA alongside hydrophobic LNG acted as a porogen to accelerate LNG release. Increasing the surface area of LNG-only implants increased LNG release. All EFdA–LNG, EFdA-only, and LNG-only formulated implants demonstrated low burst release and linear release kinetics over 245 or 122 days studied to date. Conclusion PGSU co-delivers two APIs for HIV prevention and contraception at therapeutically relevant concentrations in vitro from a single bioresorbable, elastomeric implant. A new long-acting polymer technology, PGSU demonstrates linear-release kinetics, dual delivery of APIs with disparate physiochemical properties, and biocompatibility through long-term subcutaneous implantation. PGSU can potentially meet the demands of complex MPT or fixed-dose combination products, where better solutions can serve and empower patients.

Long-acting biodegradable implant for sustained delivery of antiretrovirals (ARVs) and hormones

Women worldwide confront two major reproductive health challenges: the need for contraception and protection from sexually transmitted infections, including Human Immunodeficiency Virus (HIV). Multipurpose Prevention Technologies (MPTs) that simultaneously prevent unintended pregnancy and HIV could address these challenges with a single product. Here, we developed a long-acting (LA) subcutaneously administered and biodegradable implant system that provides sustained delivery of contraceptive and antiretroviral (ARV) with zero-order release kinetics. The MPT system involves two implants comprising an extruded tube of a biodegradable polymer, poly(ε-caprolactone) (PCL). Each implant is filled with a formulation of progestin [levonorgestrel (LNG) or etonogestrel (ENG)], or a formulation of a potent ARV [tenofovir alafenamide (TAF), or 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)]. We demonstrated sustained in-vitro release of LNG, ENG, and EFdA from the implant system for 13–17 months, while maintaining high stability of the drugs (>99%) within the implant reservoirs. We further elucidated the controlled release mechanism of the implant and leveraged several tunable parameters (e.g., type and quantity of the excipient, PCL properties, and implant wall thickness) to tailor the release kinetics and enhance the mechanical integrity of the MPT implant. The optimized MPT showed sustained in-vitro release of ENG and EFdA over 1 year while maintaining a high level of formulation stability and structural integrity. The MPT implant system was further evaluated in a preclinical study using a rodent model and demonstrated sustained release of EFdA (6 months) and ENG (12 months) with high stability of the drug formulation (>95%). This manuscript supports the continued advancement of LA delivery systems for MPTs.

Comparative Antiviral Activity of Remdesivir and Anti-HIV Nucleoside Analogs Against Human Coronavirus 229E (HCoV-229E).

Remdesivir is a nucleotide prodrug that is currently undergoing extensive clinical trials for the treatment of COVID-19. The prodrug is metabolized to its active triphosphate form and interferes with the action of RNA-dependent RNA polymerase of SARS-COV-2. Herein, we report the antiviral activity of remdesivir against human coronavirus 229E (HCoV-229E) compared to known anti-HIV agents. These agents included tenofovir (TFV), 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), alovudine (FLT), lamivudine (3TC), and emtricitabine (FTC), known as nucleoside reverse-transcriptase inhibitors (NRTIs), and a number of 5′-O-fatty acylated anti-HIV nucleoside conjugates. The anti-HIV nucleosides interfere with HIV RNA-dependent DNA polymerase and/or act as chain terminators. Normal human fibroblast lung cells (MRC-5) were used to determine the cytotoxicity of the compounds. The study revealed that remdesivir exhibited an EC50 value of 0.07 µM against HCoV-229E with TC50 of > 2.00 µM against MRC-5 cells. Parent NRTIs were found to be inactive against (HCoV-229E) at tested concentrations. Among all the NRTIs and 5′-O-fatty acyl conjugates of NRTIs, 5′-O-tetradecanoyl ester conjugate of FTC showed modest activity with EC50 and TC50 values of 72.8 µM and 87.5 µM, respectively. These data can be used for the design of potential compounds against other coronaviruses.

Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.

Cellular HIV Reservoirs and Viral Rebound from the Lymphoid Compartments of 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine (EFdA)-Suppressed Humanized Mice.

Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine, MK-8591: a novel HIV-1 reverse transcriptase translocation inhibitor.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside reverse transcriptase inhibitor (NRTI) with a novel mechanism of action, unique structure, and amongst NRTIs, unparalleled anti-HIV-1 activity. We will summarize its structure and function, antiviral activity, resistance profile, and potential as an antiretroviral for use in the treatment and preexposure prophylaxis of HIV-1 infection. EFdA is active against wild-type (EC50 as low as 50 pmol/l) and most highly NRTI-resistant viruses. The active metabolite, EFdA-triphosphate, has been shown to have a prolonged intracellular half-life in human and rhesus (Rh) blood cells. As a result, single drug doses tested in simian immunodeficiency virus mac251-infected Rh macaques and HIV-1-infected individuals exhibited robust antiviral activity of 7-10 days duration. Preclinical studies of EFdA as preexposure prophylaxis in the Rh macaque/simian/human immunodeficiency virus low-dose intrarectal challenge model have shown complete protection when given in clinically relevant doses. EFdA is a novel antiretroviral with activity against both wild-type and NRTI-resistant viruses. As a result of the prolonged intracellular half-life of its active moiety, it is amenable to flexibility in dosing of at least daily to weekly and perhaps longer.

M184I/V substitutions and E138K/M184I/V double substitutions in HIV reverse transcriptase do not significantly affect the antiviral activity of EFdA

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside analogue inhibitor of HIV that has an unusually long half-life. Cell culture selections with either EFdA or lamivudine using both subtype B and non-B clinical isolates selected the M184I/V substitutions in reverse transcriptase (RT). Unlike lamivudine, however, EFdA retained significant activity against viruses containing the M184I/V substitutions. In other clinical trials that evaluated rilpivirine together with emtricitabine in first-line therapy, the emergence of both the M184I/V and E138K mutations in RT was demonstrated. Moreover, the M184I/V and E138K substitutions were shown to be compensatory for each other with regard to both efficiency of RT activity and viral replicative capacity. This creates concern that E138K might emerge as a compensatory mutation for M184I/V in the aftermath of the use of EFdA. We wished to determine whether the E138K mutation in HIV RT together with M184I/V would compromise the activity of EFdA. Recombinant viruses containing the M184I/V and/or E138K substitutions were generated by site-directed mutagenesis and evaluated in tissue culture for susceptibility to various nucleoside compounds, including EFdA. Susceptibility to EFdA was retained in M184I/V viruses that also contained the E138K substitution. Moreover, the E138K substitution was not generated in these studies under selection pressure with EFdA. These findings help to alleviate concern that EFdA may not be active against viruses that contain both the M184I/V and E138K substitutions in RT.

Enantioselective Synthesis of 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) via Enzymatic Desymmetrization.

An enantioselective synthesis of the potent anti-HIV nucleoside EFdA is presented. Key features of stereocontrol include construction of the fully substituted 4'-carbon via a biocatalytic desymmetrization of 2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate and a Noyori-type asymmetric transfer hydrogenation to control the stereochemistry of the 3'-hydroxyl bearing carbon. The discovery of a selective crystallization of an N-silyl nucleoside intermediate enabled isolation of the desired β-anomer from the glycosylation step.

Physiological Mg2+ Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro.

Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50 values) more effective at low than at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MP(P)• dT-MP(N) ), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MP(P)• EFdA-MP(N) ); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MP(P)• EFdA-MP(*N) ). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4'-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

Efficient Inhibition of HIV Replication in the Gastrointestinal and Female Reproductive Tracts of Humanized BLT Mice by EFdA.

BackgroundThe nucleoside reverse transcriptase inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) in preclinical development exhibits improved safety and antiviral activity profiles with minimal drug resistance compared to approved NRTIs. However, the systemic antiviral efficacy of EFdA has not been fully evaluated. In this study, we utilized bone marrow/liver/thymus (BLT) humanized mice to investigate the systemic effect of EFdA treatment on HIV replication and CD4+ T cell depletion in the peripheral blood (PB) and tissues. In particular, we performed a comprehensive analysis of the female reproductive tract (FRT) and gastrointestinal (GI) tract, major sites of transmission, viral replication, and CD4+ T cell depletion and where some current antiretroviral drugs have a sub-optimal effect.ResultsEFdA treatment resulted in reduction of HIV-RNA in PB to undetectable levels in the majority of treated mice by 3 weeks post-treatment. HIV-RNA levels in cervicovaginal lavage of EFdA-treated BLT mice also declined to undetectable levels demonstrating strong penetration of EFdA into the FRT. Our results also demonstrate a strong systemic suppression of HIV replication in all tissues analyzed. In particular, we observed more than a 2-log difference in HIV-RNA levels in the GI tract and FRT of EFdA-treated BLT mice compared to untreated HIV-infected control mice. In addition, HIV-RNA was also significantly lower in the lymph nodes, liver, lung, spleen of EFdA-treated BLT mice compared to untreated HIV-infected control mice. Furthermore, EFdA treatment prevented the depletion of CD4+ T cells in the PB, mucosal tissues and lymphoid tissues.ConclusionOur findings indicate that EFdA is highly effective in controlling viral replication and preserving CD4+ T cells in particular with high efficiency in the GI and FRT tract. Thus, EFdA represents a strong potential candidate for further development as a part of antiretroviral therapy regimens.

Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) Inhibits HIV-1 Reverse Transcriptase with Multiple Mechanisms

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside analog that, unlike approved anti-human immunodeficiency virus type 1 (HIV-1) nucleoside reverse transcriptase inhibitors, has a 3'-OH and exhibits remarkable potency against wild-type and drug-resistant HIVs. EFdA triphosphate (EFdA-TP) is unique among nucleoside reverse transcriptase inhibitors because it inhibits HIV-1 reverse transcriptase (RT) with multiple mechanisms. (a) EFdA-TP can block RT as a translocation-defective RT inhibitor that dramatically slows DNA synthesis, acting as a de facto immediate chain terminator. Although non-translocated EFdA-MP-terminated primers can be unblocked, they can be efficiently converted back to the EFdA-MP-terminated form. (b) EFdA-TP can function as a delayed chain terminator, allowing incorporation of an additional dNTP before blocking DNA synthesis. In such cases, EFdA-MP-terminated primers are protected from excision. (c) EFdA-MP can be efficiently misincorporated by RT, leading to mismatched primers that are extremely hard to extend and are also protected from excision. The context of template sequence defines the relative contribution of each mechanism and affects the affinity of EFdA-MP for potential incorporation sites, explaining in part the lack of antagonism between EFdA and tenofovir. Changes in the type of nucleotide before EFdA-MP incorporation can alter its mechanism of inhibition from delayed chain terminator to immediate chain terminator. The versatility of EFdA in inhibiting HIV replication by multiple mechanisms may explain why resistance to EFdA is more difficult to emerge.

In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a novel nucleoside reverse transcriptase inhibitor with a unique mechanism of action and highly potent activity against both wild-type and clinically relevant drug resistant HIV-1 variants. Furthermore, in vivo efficacy and safety evaluations have shown EFdA to be a promising therapeutic candidate for use in the treatment of HIV infection. However, little is known about the pharmacokinetic and biopharmaceutical properties of EFdA. In this study, we evaluated cellular EFdA transport using Caco-2 and Madin-Darby Canine Kidney II (MDCKII) in vitro cell models. Studies using Caco-2 cell monolayers showed that EFdA efflux ratios were >2.0, suggesting that active drug transport mechanisms may play a role in EFdA flux. ABCB1 transporter (PGP1) inhibition was assessed using the acetomethoxy derivate of calcein (calcein-AM) as a fluorescent probe in both wild-type MDCKII and PGP1 overexpressing MDCKII cells. Nonetheless, our data showed that EFdA is not a substrate of PGP1. Additionally, comparative bidirectional flux of EFdA and Lucifer yellow (LY, a well-known paracellular marker) was studied over a range of EFdA concentrations. In MDCKII monolayers, EFdA had an apparent permeability coefficient (Papp) (a–b) of <1×10−6cm/s. The Papp values significantly increased in the presence of the paracellular permeability enhancer, indicating that EFdA primarily permeates via the paracellular route.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) using pre-steady-state kinetics

The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed.

Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4'-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2'-exo/C-3'-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4'-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4'-ethynyl and cyano substitutions decrease the susceptibility of adenosine-based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pKa of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites.

Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a novel nucleoside analog of great interest because of its superior activity against wild-type and multidrug-resistant HIV-1 strains, and favorable safety profiles in vitro and in vivo. The aim of this work was to provide preformulation information of EFdA important for delivery system development. A simple, accurate and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed for quantification of EFdA. In addition, physicochemical characterizations including pH solubility profile, octanol/water partition coefficient (Log Po/w), DSC analysis, field emission scanning electron microscopy, and stability studies under various conditions were conducted. EFdA existed in planar or flake shape, with a melting point of ∼130 °C, and had a pH dependent solubility. The log Po/w value of EFdA was −1.19. The compound was stable upon exposure to pH levels from 3 to 9 and showed good stability at elevated temperature (65 °C). In vitro cytotoxicity assessments were performed in two different epithelial cell lines. In cell-based studies, the EFdA selectivity index (50% cytotoxic concentration [CC50] values/50% effective concentration [EC50]) was found to be greater than 1 × 103. Permeability studies using cell- and tissue-based models showed that EFdA had an apparent permeability coefficient (Papp) < 1×10−6 cm/s and that the paracelluar pathway was the dominant transport route for EFdA. Overall, EFdA possesses favorable characteristics for further formulation development.

Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA

BackgroundThe K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.ResultsHere we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3’ primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.ConclusionWe have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.

Evaluation of Combinations of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine with Clinically Used Antiretroviral Drugs

Drug combination studies of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) with FDA-approved drugs were evaluated by two different methods, MacSynergy II and CalcuSyn. Most of the combinations, including the combination of the two adenosine analogs EFdA and tenofovir, were essentially additive, without substantial antagonism or synergism. The combination of EFdA and rilpivirine showed apparent synergism. These studies provide information that may be useful for the design of EFdA combination regimens for initial and salvage therapy assessment.