Darunavir

Manufactured by Merck Group

Sourced in Italy

Darunavir is a laboratory equipment product manufactured by Merck Group. It is a protease inhibitor used in the analysis and research of pharmaceutical compounds.

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Darunavir (DRV; (3 citations)

8 protocols using darunavir

Evaluating Antivirals Against SARS-CoV-2

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The following compounds were tested to determine their effects on 3CL_pro activity, cytotoxicity, or inhibition of SARS-CoV-2 replication: cobicistat (sc-500831; Santa Cruz Biotechnology), remdesivir (S8932; Selleckchem Chemicals), tipranavir (sc-220260; Santa Cruz Biotechnology), nelfinavir mesylate hydrate (PZ0013; Sigma-Aldrich), darunavir and lopinavir (both obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID), MG-132 (M8699; Sigma‐Aldrich), and GC376 (BPS Bioscience).

Shytaj I.L., Fares M., Gallucci L., Lucic B., Tolba M.M., Zimmermann L., Adler J.M., Xing N., Bushe J., Gruber A.D., Ambiel I., Taha Ayoub A., Cortese M., Neufeldt C.J., Stolp B., Sobhy M.H., Fathy M., Zhao M., Laketa V., Diaz R.S., Sutton R.E., Chlanda P., Boulant S., Bartenschlager R., Stanifer M.L., Fackler O.T., Trimpert J., Savarino A, & Lusic M. (2022). The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters. mBio, 13(2), e03705-21.

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FRET-labeled HIV-1 Virion Production

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Adherent HEK293T and TZM-bl cells were cultured in Dulbecco’s Modified Eagle’s Medium (Nacalai Tesque) containing 10% Fetal Bovine Serum and 1% Penicillin Streptomycin Glutamine (Invitrogen) (D10) at 37°C with 5% CO₂.
FRET labeled virions were produced by co-transfecting HEK293T cells (3.5 × 10⁶ cells/10 cm dish) with the pHIV-1 Gag-iFRET or iFRETΔPro together with the pNL4-3 or pNL4-3ΔPro parental plasmid respectively at a 1:1, 1:10, or 1:20 ratio using a polyethylenimine transfection reagent (GE Healthcare). The culture medium was replaced with fresh D10 with or without Darunavir (Sigma Aldrich) at a final concentration of 0.1, 1.0, 10, 20, 500, or 1000 nM 3.5 h after transfection. The virus-containing supernatant was harvested 24 h after the medium change, filtered through 0.45 μm pore size sterile polyvinylidene difluoride (PVDF, Millipore) membrane, and concentrated up to 20-fold by ultracentrifugation through a 20% sucrose cushion at 25,000 rpm (112,499 g) for 90 min at 4°C (CP65; Hitachi Koki Co., Ltd.). The virus pellet was resuspended in 500 μl Hank’s Balanced Salt Solution (HBSS) (–) without phenol red (Wako).

Sarca A.D., Sardo L., Fukuda H., Matsui H., Shirakawa K., Horikawa K., Takaori-Kondo A, & Izumi T. (2021). FRET-Based Detection and Quantification of HIV-1 Virion Maturation. Frontiers in Microbiology, 12, 647452.

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Anti-Viral Drug Solubilization Protocol

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Lopinavir, ritonavir, amprenavir, and darunavir were purchased Sigma or Cambridge Biosciences in powder form. The drugs were solubilised in DMSO at a stock concertation of 5 mM concentration and then further diluted to 100 μM in 200 mM ammonium acetate supplemented with 0.5% C₈E₄.

Mehmood S., Marcoux J., Gault J., Quigley A., Michaelis S., Young S.G., Carpenter E.P, & Robinson C.V. (2016). Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24. Nature chemistry, 8(12), 1152-1158.

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Synthesis of Novel HCV Inhibitors

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Glecaprevir [(3aR,7S,10S,12R,21E,24aR)-7-tert-butyl-N-[(1R,2R)-2-(difluoromethyl)-1-{[(1-methylcyclopropyl)sulfonyl] carbamoyl}cyclopropyl]-20,20-difluoro-5,8-dioxo-2,3,3a,5,6,7,8,11,12,20,23,24a-dodecahydro-1H,10H-9,12-methanocyclopenta[18,19][1,10,17,3,6]trioxadiazacyclononadecino[11,12-b]quinoxaline-10-carboxamide; identified by AbbVie and Enanta] (Fig. 1), pibrentasvir (33 (link)), paritaprevir (28 (link)), and lopinavir were synthesized at AbbVie. Grazoprevir (17 ) was purchased from ApexBio (Houston, TX), and darunavir, IFN-α, and RBV were purchased from Sigma-Aldrich (St. Louis, MO).

Ng T.I., Tripathi R., Reisch T., Lu L., Middleton T., Hopkins T.A., Pithawalla R., Irvin M., Dekhtyar T., Krishnan P., Schnell G., Beyer J., McDaniel K.F., Ma J., Wang G., Jiang L.J., Or Y.S., Kempf D., Pilot-Matias T, & Collins C. (2017). In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS3/4A Protease Inhibitor Glecaprevir. Antimicrobial Agents and Chemotherapy, 62(1), e01620-17.

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OATP1B Inhibitory Potential of Drugs

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RST, E 2 17bG, and CCK8 (Sigma) were used as substrates in this study. For radiolabeled tracing, [ 3 H]-E 2 17bG and [ 3 H]-CCK8 were used, which were both purchased from PerkinElmer Life and Analytical Sciences. OATP1B inhibitory potential of 22 drugs was investigated. Sixteen of these 22 drugs (i.e., baicalin, cyclosporine A, darunavir, digoxin, erythromycin, ezetimibe, fluconazole, gemfibrozil, grazoprevir, ketoconazole, lopinavir, metformin, rifampicin, ritonavir, telmisartan, and valsartan) were purchased from Sigma; three (i.e., atazanavir, fimasartan, and velpatasvir) were purchased from Selleckchem; and asunaprevir, eltrombopag, and eluxadoline were purchased from Carbosynth, SeqChem, and Toronto Research Chemicals, respectively.

Sane R., Cheung K.W.K., Kovács P., Farasyn T., Li R., Bui A., Musib L., Kis E., Plise E, & Gáborik Z. (2020). Calibrating the In Vitro-In Vivo Correlation for OATP-Mediated Drug-Drug Interactions with Rosuvastatin Using Static and PBPK Models. Drug metabolism and disposition: the biological fate of chemicals, 48(12).

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Cell Viability Assay with DMEM and MTT

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Dulbecco's Modified Eagle Medium (DMEM), dimethyl sulfoxide (DMSO), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Darunavir were purchased from Sigma Aldrich (Milan, Italy). Trypsin-EDTA solution, Fetal Bovine Serum (FBS), glutamine, penicillin-streptomycin and Phosphate Buffered Saline (PBS) were purchased from Euroclone (Milan, Italy). QuikChange XL Site-Directed Mutagenesis Kit was purchased from Agilent Technologies.

Tramutola F., Armentano M.F., Berti F., Chiummiento L., Lupattelli P., D'Orsi R., Miglionico R., Milella L., Bisaccia F, & Funicello M. (2019). New heteroaryl carbamates: Synthesis and biological screening in vitro and in mammalian cells of wild-type and mutant HIV-protease inhibitors. Bioorganic & medicinal chemistry, 27(9).

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Anti-Viral Drug Solubilization Protocol

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Isolation and Infection of MDM with HIV

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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors at the NIH Blood Bank (Bethesda, MD). Monocytes were isolated by adherence and differentiated into macrophages (MDM) by culture in RPMI 1640 (Thermo Fisher; Waltham, MA) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic-antimycotic for ≥ 7 days. MDM were infected with HIV_SF162 (>1 ng p24 per million cells) using polybrene (5 μg/ mL) (Sigma-Aldrich; St. Louis, MO) for four hours, then cells were washed with PBS and medium was replaced with complete RPMI containing dimethyl sulfoxide (DMSO) or darunavir (1 μM in DMSO). darunavir was obtained through the NIH AIDS Reagent Program (cat #11447), Division of AIDS, NIAID, NIH from Tibotec, Inc. Supernatants and cells were collected after seven days for product enhanced reverse transcriptase (PERT) assay ^{[31 (link)]} or Tat ELISA, respectively.

HENDERSON L.J., JOHNSON T.P., SMITH B.R., REOMA L.B., SANTAMARIA U.A., BACHANI M., DEMARINO C., BARCLAY R.A., SNOW J., SACKTOR N., MCARTHUR J., LETENDRE S., STEINER J., KASHANCHI F, & NATH A. (2019). Presence of Tat and Trans-activation response (TAR) element in spinal fluid despite antiretroviral therapy. AIDS (London, England), 33(Suppl 2), S145-S157.

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