Lamivudine 3tc

Manufactured by Merck Group

Sourced in United States

Lamivudine (3TC) is a nucleoside reverse transcriptase inhibitor (NRTI) used in the treatment of HIV infection and chronic hepatitis B. It works by interfering with the reverse transcriptase enzyme, which is necessary for the viral replication of HIV and hepatitis B virus. Lamivudine is an important component in various antiretroviral therapy regimens.

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8 protocols using lamivudine 3tc

HepG2 Hepatocyte Cell Culture Protocol

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Human liver carcinoma cell line HepG2 and its derivative HBV reporter line HepG2.2.15 (kind gift of Dr. S. Jameel, International Center for Genetic Engineering & Biotechnology, New Delhi, India). Cells were grown and maintained in RPMI-1640 medium (Gibco, USA), supplemented with 10% heat-inactivated bovine serum (Gibco, USA), 1xpenicillin-streptomycin mix, and 1x sodium pyruvate (HyClone Laboratories, USA) at 37 ⁰C in a humified chamber with 5% CO₂ supply. 2,7-Dichlorofluorescein (DCFH; Sigma, USA) was used as an inducer of in vitro hepatotoxicity. The approved nucleoside analog-based anti-HBV drug, lamivudine (3TC; Sigma, USA), was used as standard.

Parvez M.K., Al-Dosari M.S., Arbab A.H, & Niyazi S. (2019). The in vitro and in vivo anti-hepatotoxic, anti-hepatitis B virus and hepatic CYP450 modulating potential of Cyperus rotundus. Saudi Pharmaceutical Journal : SPJ, 27(4), 558-564.

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Hepatoma Cell Line Protocol

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Human hepatoma cells (HepG2) and HBV cell line, HepG2.2.2.15 (derivative of HepG2) (kind gift of Dr. S. Jameel, International Center for Genetic Engineering & Biotechnology, New Delhi, India), were grown in RPMI-1640 medium (Invitrogen, USA), supplemented with 10% heat-inactivated bovine serum (Gibco, USA), 1x penicillin-streptomycin (Invitrogen, USA), and 1x sodium pyruvate (HyClone Laboratories, USA) at 37°C in a humidified chamber with 5% CO₂ supply. 2,7-Dichlorofluorescein (DCFH; Sigma, USA) was used as cytotoxin on cultured HepG2 cells. The approved nucleoside analog-based anti-HBV drug, Lamivudine (3TC; Sigma, USA), was used as standard.

Arbab A.H., Parvez M.K., Al-Dosari M.S., Al-Rehaily A.J., Al-Sohaibani M., Zaroug E.E., AlSaid M.S, & Rafatullah S. (2015). Hepatoprotective and Antiviral Efficacy of Acacia mellifera Leaves Fractions against Hepatitis B Virus. BioMed Research International, 2015, 929131.

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Culturing Human Embryonic Stem Cells

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WT-hESCs (Line H9, from WiCell Research) and SIRT7^−/− hESCs were maintained on mitomycin C-inactivated MEFs in hESC culture medium (Cheng et al., 2019 (link)): 80% DMEM/F12 (Gibco), 20% Knockout Serum Replacement (Gibco), 2 mmol/L GlutaMAX (Gibco), 0.1 mmol/L non-essential amino acids (NEAA, Gibco), 55 μmol/L β-mercaptoethanol (Invitrogen), and 10 ng/mL bFGF (Joint Protein Central); hESCs were also cultured on Matrigel (BD Biosciences) with mTeSR medium (STEMCELL Technologies, Vancouver). All hMSCs were cultured in MSC culture medium: 90% α-MEM with GlutaMAX (Gibco), 10% fetal bovine serum (FBS, Lot A77E01F, Gemcell), 1% penicillin/streptomycin (Gibco) and 1 ng/mL bFGF (Joint Protein Central). 10 μmol/L Lamivudine (3TC) (L1295-50mg, Sigma-Aldrich) was used to treat with hMSCs.

Bi S., Liu Z., Wu Z., Wang Z., Liu X., Wang S., Ren J., Yao Y., Zhang W., Song M., Liu G.H, & Qu J. (2020). SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer. Protein & Cell, 11(7), 483-504.

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Screening Natural Antiviral Compounds

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Twelve natural antiviral compounds of plant origin were selected on the basis of their published in vitro or vivo antiviral activities. All cell culture grade natural compounds viz. rutin, quercetin, menisdaurin, β-sitosterol, hesperidin, psoralen, bergenin, azadirachtin, baccatin III, lupeol, embelin and naringenin were purchased (Sigma-Aldrich, Germany). The approved anti-HBV nucleoside analog, lamivudine (3TC; Sigma-Aldrich, Germany) served as standard (positive control).

Parvez M.K., Tabish Rehman M., Alam P., Al-Dosari M.S., Alqasoumi S.I, & Alajmi M.F. (2018). Plant-derived antiviral drugs as novel hepatitis B virus inhibitors: Cell culture and molecular docking study. Saudi Pharmaceutical Journal : SPJ, 27(3), 389-400.

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Pluripotent Stem Cell Differentiation to Neurons

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Pluripotent stem cell medium (MT): mTeSR1 (Stem Cell Technologies).
Differentiation medium for embryoid Bodies (N2): Dulbecco’s Modified
Eagle’s Medium/Ham’s F12 (DMEM/F12 50/50; Corning Cellgro)
with 1× HEPES, 1× penicillin-streptomycin, Glutamax (Life
Technologies), and N2 NeuroPlex (Gemini Bio-products), supplemented with
1μM dorsomorphin (Tocris) and 10μM Stemolecule SB431542
(StemGent). Neural progenitor cell medium (NGF): DMEM/F12 50/50 with
1× HEPES, 1× penicillin-streptomycin, Glutamax (Life
Technologies), N2 NeuroPlex (Gemini Bio-products), Gem21 NeuroPlex (Gemini
Bio-products), supplemented with 20ng/mL basic fibroblast growth factor
(bFGF; Life Technologies). Neuronal medium (NG): DMEM/F12 50/50 with 1x
HEPES, 1x penicillin-streptomycin, Glutamax (Life Technologies), N2
NeuroPlex (Gemini Bio-products), Gem21 NeuroPlex (Gemini Bio-products);
cAMP, GDNF, and BDNF were added to the neuronal medium for purified neurons.
Astrocytes were cultured in Astrocyte Growth Medium (Lonza). For treatment
with reverse-transcriptase inhibitors 1μM Stavudine (D4T),
10μM Lamivudine (3TC) (Sigma-Aldrich), and 400nM Nevirapine (NVP)
were used. Astrocyte-conditioned media were generated by overlaying
Neurobasal with B27 and 5% FBS over astrocytes for 48 hours.

Thomas C.A., Tejwani L., Trujillo C.A., Negraes P.D., Herai R.H., Mesci P., Macia A., Crow Y.J, & Muotri A.R. (2017). Modeling of TREX1-dependent autoimmune disease using human stem cells highlights L1 accumulation as a source of neuroinflammation. Cell stem cell, 21(3), 319-331.e8.

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Dissolution and Storage of NRTI Drugs

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NRTI drugs: zidovudine (AZT), lamivudine (3TC) and stavudine (D4T) were purchased from Sigma (Sigma-Aldrich). NRTIs were dissolved in PBS to a final concentration of AZT (8 mg/ ml), 3TC (4 mg/ml) and D4T (0.8 mg/ml) and stored at −20 °C.

Wu T., Zhang J., Geng M., Tang S.J., Zhang W, & Shu J. (2017). Nucleoside reverse transcriptase inhibitors (NRTIs) induce proinflammatory cytokines in the CNS via Wnt5a signaling. Scientific Reports, 7, 4117.

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Retinoic Acid Induces HIV-1 Infection in PBMCs

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HIV-1_BaL was generated in CD8-depleted PBMCs in the presence of 10 nM retinoic acid (Sigma-Aldrich, St Louis, MO), 20 U/ml IL2 (NIH) and 50 ng/ml of anti-CD3 monoclonal antibodies (clone OKT3; e-Bioscience, San Diego, CA) and titered in TZMbl cells as previously described^{14 (link)}. Lamivudine (3TC) (Sigma-Aldrich) was used as a control for inhibiting HIV-1_BaL infection. PBMCs and endocervical tissues were incubated with E2 (Sigma Aldrich). The following inhibitors or activators of E2 signaling were used: selective estrogen receptor modulator (SERM) Raloxifene (Sigma-Aldrich), and LIMKi3 (LIMK1/2 inhibitor blocking CFL1 phosphorylation; Tocris, Minneapolis, MN).
Antibodies (Abs) used in Western Blot were rabbit monoclonal anti-human CFL1 (D3F9), pCFL1 (77G2), β-Actin (D6A8); AKT (pan) (C67E7) and pAKT (D9E) (Cell Signaling Technology, Danvers, MA); HPRT1 (Sigma-Aldrich); and HRP-conjugated goat anti-rabbit IgG (secondary Ab) (Abcam, Cambridge, MA).
Antibodies used for Immunofluorescence staining (IF) were rabbit monoclonal anti-human CFL1 (EP6376) (Abcam); mouse monoclonal HIV-1-p24 (24-4)-AlexaFluor 488 (AF488) (Santa Cruz Biotechnology); mouse monoclonal anti-human CD3 (OKT3)-AF488, IgG-AF488 (eBM2a), goat anti-rabbit secondary Ab AF568, IgG isotype controls (Invitrogen, Carlsbad, CA) and DAPI (Sigma-Aldrich). FVS 780-APC-Cy7 (FVS780) (Live/Dead dye) was used for flow cytometry.

Verma N., Mukhopadhyay S., Barnable P., Plagianos M.G, & Teleshova N. (2022). Estradiol inhibits HIV-1BaL infection and induces CFL1 expression in peripheral blood mononuclear cells and endocervical mucosa. Scientific Reports, 12, 6165.

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Synthesis and Testing of Modified NRTIs

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The NRTI lamivudine (3TC), as well as the NonNRTIs EFV, DLV, NVP, and ATP, were purchased from Sigma-Aldrich. LPS was purchased from InvivoGen. A trimethyl-modified version of 3TC (TM-3TC) and a diethyl-modified version of AZT (DE-AZT) were synthesized as previously described (27 ).

Fukuda S., Narendran S., Varshney A., Nagasaka Y., Wang S.B., Ambati K., Apicella I., Pereira F., Fowler B.J., Yasuma T., Hirahara S., Yasuma R., Huang P., Yerramothu P., Makin R.D., Wang M., Baker K.L., Marion K.M., Huang X., Baghdasaryan E., Ambati M., Ambati V.L., Banerjee D., Bonilha V.L., Tolstonog G.V., Held U., Ogura Y., Terasaki H., Oshika T., Bhattarai D., Kim K.B., Feldman S.H., Aguirre J.I., Hinton D.R., Kerur N., Sadda S.R., Schumann G.G., Gelfand B.D, & Ambati J. (2021). Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity. Science Advances, 7(40), eabj3658.

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