HIV-1 reverse transcriptase (RT). EFdA-TP binds to the active site of HIV‑1 RT and incorporates into the growing viral DNA chain, causing chain termination either immediately (ICT) or after the incorporation of a few additional nucleotides (DCT).

EFdA-TP tetralithium (0.05-10 μM; duration 15 min) inhibits RT-catalyzed DNA synthesis as an ICT or DCT[1]. EFdA-TP tetralithium can block RT as a translocation-defective RT inhibitor, thereby significantly slowing DNA synthesis, acting as a de facto direct chain terminator[1]. EFdA-TP tetralithium can act as a delayed chain terminator, allowing the incorporation of additional dNTPs before blocking DNA synthesis[1].

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In cell‑free assays, EFdA-TP (0.05‑10 uM) inhibits RT‑catalyzed DNA synthesis as an effective ICT or DCT. It inhibits HIV‑1 RT through multiple mechanisms. It has potent antiviral activity against a broad range of HIV‑1 isolates, including those resistant to other NRTIs. The EC50 is in the low nanomolar range.

In vivo, EFdA-TP is the active intracellular anabolite of the prodrug EFdA. It is not typically administered directly due to its triphosphate form, which is negatively charged and cell‑impermeable. However, when delivered as a prodrug, the active EFdA-TP is generated inside cells, where it potently inhibits HIV‑1 replication.

A general cell‑free protocol for assessing reverse transcriptase inhibition: Recombinant HIV‑1 RT is incubated with a synthetic RNA/DNA primer/template, dNTPs, and varying concentrations of EFdA-TP. The reaction is allowed to proceed for 15‑60 minutes at 37degC. The extension products are separated by denaturing PAGE, and the degree of chain termination is quantified by phosphorimaging. The IC50 is calculated from the inhibition curve.

A general cellular protocol for assessing antiviral activity: MT‑4 cells (human T‑lymphocytes) are infected with HIV‑1. The cells are then incubated with a prodrug that is converted to EFdA-TP or with EFdA-TP directly (using a transfection reagent). After 5‑7 days, viral replication is measured by a p24 antigen capture ELISA or by a cytopathic effect (CPE) reduction assay. The EC50 is calculated.

General animal protocol for a prodrug that generates EFdA-TP: Humanized mice (e.g., hu‑PBMC‑NSG mice) are infected with HIV‑1. The EFdA prodrug is administered orally at doses of 1, 5, and 25 mg/kg once daily. Plasma viral RNA is measured by qRT-PCR at regular intervals. At the end of the study, the concentration of the active EFdA-TP is measured in peripheral blood mononuclear cells (PBMCs) by LC‑MS/MS.

General PK protocol for an EFdA prodrug: A typical PK study in rats would involve oral (PO, 10 mg/kg) and intravenous (IV, 2 mg/kg) administration. Blood samples would be collected over 24 hours. The concentration of the prodrug in plasma would be measured by LC‑MS/MS, while the active anabolite, EFdA-TP, would be measured in PBMC lysates.

General toxicity protocol for EFdA-TP: The toxicity of the EFdA prodrug, not the triphosphate salt, would be evaluated. A 28‑day repeat‑dose oral toxicity study in rats would be performed at doses of 10, 30, and 100 mg/kg/day. Standard parameters would include clinical signs, body weight, food consumption, hematology (with special attention to reticulocyte and platelet counts), serum chemistry, and histopathology.

[1]. 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) inhibits HIV-1 reverse transcriptase with multiple mechanisms. J Biol Chem. 2014 Aug 29;289(35):24533-48.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside analog. Unlike approved nucleoside reverse transcriptase inhibitors against human immunodeficiency virus type 1 (HIV-1), EFdA has a 3'-OH group and exhibits significant activity against both wild-type and drug-resistant HIV. EFdA triphosphate (EFdA-TP) is unique among nucleoside reverse transcriptase inhibitors because it inhibits HIV-1 reverse transcriptase (RT) through multiple mechanisms. (a) EFdA-TP can act as a translocation-deficient RT inhibitor, blocking RT and thus significantly slowing DNA synthesis, effectively acting as an immediate chain terminator. Although untranslocated EFdA-MP terminated primers can be unblocked, they can be efficiently converted back to their EFdA-MP terminated form. (b) EFdA-TP can act as a delayed chain terminator, allowing the incorporation of additional dNTPs before blocking DNA synthesis. In this case, EFdA-MP terminated primers are spared excision. (c) EFdA-MP can be efficiently mismatched and incorporated by reverse transcriptase, making it extremely difficult for mismatched primers to extend and also exempt from excision. The background of the template sequence determines the relative contribution of each mechanism and affects the affinity of EFdA-MP for potential incorporation sites, which partly explains the lack of antagonism between EFdA and tenofovir. Changes in the nucleotide type before EFdA-MP incorporation can change its inhibitory mechanism from a delayed chain terminator to an immediate chain terminator. The multifunctionality of EFdA in inhibiting HIV replication through multiple mechanisms may explain why resistance to EFdA is more difficult to develop.

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EFdA-TP tetralithium is a potent HIV‑1 reverse transcriptase inhibitor. It is unique among NRTIs because it inhibits RT through multiple mechanisms, including acting as an immediate and delayed chain terminator. The tetralithium salt form enhances its solubility and stability for biochemical research. The parent compound, EFdA, is an investigational anti‑HIV drug. This active triphosphate is a critical tool for studying the mechanism of HIV‑1 replication and resistance.

C12H14FLI4N5O12P3

559.95

Solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 100 mg/mL (178.59 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)