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The Lupane-type Triterpene 30-Oxo-calenduladiol Is a CCR5 Antagonist with Anti-HIV-1 and Anti-chemotactic Activities

2009; Elsevier BV; Volume: 284; Issue: 24 Linguagem: Inglês

10.1074/jbc.m109.005835

ISSN

1083-351X

Autores

Jonathan Barroso-González, Nabil el Jaber-Vazdekis, Laura García-Expósito, José David Machado, Rafael Zárate, Ángel G. Ravelo, Ana Estévez‐Braun, Agustı́n Valenzuela-Fernández,

Tópico(s)

Natural product bioactivities and synthesis

Resumo

The existence of drug-resistant human immunodeficiency virus (HIV) viruses in patients receiving antiretroviral treatment urgently requires the characterization and development of new antiretroviral drugs designed to inhibit resistant viruses and to complement the existing antiretroviral strategies against AIDS. We assayed several natural or semi-synthetic lupane-type pentacyclic triterpenes in their ability to inhibit HIV-1 infection in permissive cells. We observed that the 30-oxo-calenduladiol triterpene, compound 1, specifically impaired R5-tropic HIV-1 envelope-mediated viral infection and cell fusion in permissive cells, without affecting X4-tropic virus. This lupane derivative competed for the binding of a specific anti-CCR5 monoclonal antibody or the natural CCL5 chemokine to the CCR5 viral coreceptor with high affinity. 30-Oxo-calenduladiol seems not to interact with the CD4 antigen, the main HIV receptor, or the CXCR4 viral coreceptor. Our results suggest that compound 1 is a specific CCR5 antagonist, because it binds to the CCR5 receptor without triggering cell signaling or receptor internalization, and inhibits RANTES (regulated on activation normal T cell expressed and secreted)-mediated CCR5 internalization, intracellular calcium mobilization, and cell chemotaxis. Furthermore, compound 1 appeared not to interact with β-chemokine receptors CCR1, CCR2b, CCR3, or CCR4. Thereby, the 30-oxo-calenduladiol-associated anti-HIV-1 activity against R5-tropic virus appears to rely on the selective occupancy of the CCR5 receptor to inhibit CCR5-mediated HIV-1 infection. Therefore, it is plausible that the chemical structure of 30-oxo-calenduladiol or other related dihydroxylated lupane-type triterpenes could represent a good model to develop more potent anti-HIV-1 molecules to inhibit viral infection by interfering with early fusion and entry steps in the HIV life cycle. The existence of drug-resistant human immunodeficiency virus (HIV) viruses in patients receiving antiretroviral treatment urgently requires the characterization and development of new antiretroviral drugs designed to inhibit resistant viruses and to complement the existing antiretroviral strategies against AIDS. We assayed several natural or semi-synthetic lupane-type pentacyclic triterpenes in their ability to inhibit HIV-1 infection in permissive cells. We observed that the 30-oxo-calenduladiol triterpene, compound 1, specifically impaired R5-tropic HIV-1 envelope-mediated viral infection and cell fusion in permissive cells, without affecting X4-tropic virus. This lupane derivative competed for the binding of a specific anti-CCR5 monoclonal antibody or the natural CCL5 chemokine to the CCR5 viral coreceptor with high affinity. 30-Oxo-calenduladiol seems not to interact with the CD4 antigen, the main HIV receptor, or the CXCR4 viral coreceptor. Our results suggest that compound 1 is a specific CCR5 antagonist, because it binds to the CCR5 receptor without triggering cell signaling or receptor internalization, and inhibits RANTES (regulated on activation normal T cell expressed and secreted)-mediated CCR5 internalization, intracellular calcium mobilization, and cell chemotaxis. Furthermore, compound 1 appeared not to interact with β-chemokine receptors CCR1, CCR2b, CCR3, or CCR4. Thereby, the 30-oxo-calenduladiol-associated anti-HIV-1 activity against R5-tropic virus appears to rely on the selective occupancy of the CCR5 receptor to inhibit CCR5-mediated HIV-1 infection. Therefore, it is plausible that the chemical structure of 30-oxo-calenduladiol or other related dihydroxylated lupane-type triterpenes could represent a good model to develop more potent anti-HIV-1 molecules to inhibit viral infection by interfering with early fusion and entry steps in the HIV life cycle. The human immunodeficiency virus (HIV) 7The abbreviations used are: HIV-1human immunodeficiency virus type 1VSV-Gvesicular stomatitis virus G proteinRANTESregulated on activation normal T expressed and secretedTIRFMtotal internal reflection fluorescence microscopyPEphycoerythrinMCP-1monocyte chemotactic protein 1TARCthymus- and activation-regulated chemokineEGFPenhanced green fluorescent proteinEFevanescent fieldIRinfraredmAbmonoclonal antibodyPBSphosphate-buffered salineEnvenvelope. pandemic is a medical challenge and represents the public health crisis of our time (1Inciardi J.A. Williams M.L. AIDS Care. 2005; 17: S1-S8Crossref PubMed Scopus (8) Google Scholar, 2Simon V. Ho D.D. Abdool Karim Q. Lancet. 2006; 368: 489-504Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 3Kuehn B.M. JAMA. 2006; 296: 29-30Crossref PubMed Scopus (7) Google Scholar, 4Kallings L.O. J. Intern. Med. 2008; 263: 218-243Crossref PubMed Scopus (120) Google Scholar, 5UNAIDSReport on the Global AIDS Epidemic. UNAIDS, Joint United Nations Programme on HIV/AIDS, Geneva, Switzerland2008Google Scholar). Antiretroviral treatment achieves long-lasting viral suppression and, subsequently, reduces the morbidity and mortality of HIV-infected individuals. However, current drugs do not eradicate HIV infection and lifelong treatment might be needed (2Simon V. Ho D.D. Abdool Karim Q. Lancet. 2006; 368: 489-504Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). human immunodeficiency virus type 1 vesicular stomatitis virus G protein regulated on activation normal T expressed and secreted total internal reflection fluorescence microscopy phycoerythrin monocyte chemotactic protein 1 thymus- and activation-regulated chemokine enhanced green fluorescent protein evanescent field infrared monoclonal antibody phosphate-buffered saline envelope. Emerging drug-resistant HIV viruses, in patients receiving high active antiretroviral treatment, urgently needs the development of new antiretroviral molecules designed to inhibit resistant viruses, because many patients treated during the past decades harbor viral strains with reduced susceptibilities to many if not all available drugs (2Simon V. Ho D.D. Abdool Karim Q. Lancet. 2006; 368: 489-504Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 6Pöhlmann S. Reeves J.D. Curr. Pharm. Des. 2006; 12: 1963-1973Crossref PubMed Scopus (27) Google Scholar). In this matter, pentacyclic triterpenes represent a varied class of natural products presenting antitumor and antiviral activities (7Cichewicz R.H. Kouzi S.A. Med. Res. Rev. 2004; 24: 90-114Crossref PubMed Scopus (457) Google Scholar, 8Connolly J.D. Hill R.A. Nat. Prod. Rep. 2000; 17: 463-482Crossref PubMed Scopus (28) Google Scholar, 9Aiken C. Chen C.H. Trends Mol. Med. 2005; 11: 31-36Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). A well studied pentacyclic lupane-type triterpene is the betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid), widely distributed throughout the plant kingdom, which presents anti-inflammatory, anti-malarial, and anti-HIV-1 effects in vitro (7Cichewicz R.H. Kouzi S.A. Med. Res. Rev. 2004; 24: 90-114Crossref PubMed Scopus (457) Google Scholar, 9Aiken C. Chen C.H. Trends Mol. Med. 2005; 11: 31-36Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10Alakurtti S. Mäkelä T. Koskimies S. Yli-Kauhaluoma J. Eur. J. Pharm. Sci. 2006; 29: 1-13Crossref PubMed Scopus (537) Google Scholar). Although its mechanism of action has not been fully determined, it has been reported that some lupane-type triterpene derivatives impair HIV-1 fusion through interacting with the viral glycoprotein gp41, or disrupting the assembly and budding of emerging viral particles in infected target cells (reviewed in Ref. 9Aiken C. Chen C.H. Trends Mol. Med. 2005; 11: 31-36Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). In the present work, we aimed to test the ability of several non-acid lupane-type triterpene, natural or derivative compounds, to inhibit HIV-1 viral infection and to determine the mechanism of action. Our results indicate that the semi-synthetic 30-oxo-calenduladiol, compound 1, specifically interacts with the G protein-coupled CCR5 chemokine receptor, acting as an antagonist, inhibiting R5-tropic HIV-1 viral infection and CCL5 (regulated on activation normal T expressed and secreted (RANTES) chemokine)-mediated CCR5 internalization, cell signaling, and chemotaxis. All solvents and reagents were purified by Standard techniques, as previously described (11Perrin D.D. Amarego W.L.F. Purification of Laboratory Chemicals. 3rd Ed. Pergamon Press, New York1988Google Scholar). All reactions were monitored by thin layer chromatography (TLC) (on silica gel POLYGRAM® SIL G/UV254 foils). Pre-coated SIL G-100 UV254 (Machery-Nagel, Düren, Germany) TLC plates were used for preparative TLC purification. 1H nuclear magnetic resonance spectra were recorded in CDCl3 or C6D6 at 300 and 400 MHz, using Bruker AMX300 and AMX400 instruments. For 1H spectra, chemical shifts are given in parts per million (ppm) and are referenced to the residual solvent peak. The following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Proton assignments and stereochemistry were supported by 1H-1H COSY and ROESY where necessary. Data are reported in the following manner with chemical shift (integration, multiplicity, and coupling constant, if appropriate). Coupling constants (J) are given in Hertz (Hz) to the nearest 0.5 Hz. 13C NMR spectra were recorded at 75 and 100 MHz using Bruker AMX300 and AMX400 instruments. Carbon spectra assignments were supported by DEPT-135 spectra, 13C-1H (HMQC), and 13C-1H (HMBC) correlations where necessary. Chemical shifts are quoted in ppm and are referenced to the appropriate residual solvent peak. MS and HRMS were recorded at VG Micromass ZAB-2F. IR spectra were taken on a Bruker IFS28/55 spectrophotometer. Six natural or derivative lupanes were assayed (1–6). Compounds 1–3 and 5 were semi-synthesized as described below, whereas natural compounds 4 and 6 were isolated from Maytenus apurimacensis, using a previously described method (12Mesa-Siverio D. Chavez H. Estevez-Braun A. Ravelo A.G. Tetrahedron. 2005; 61: 429-436Crossref Scopus (15) Google Scholar, 13Mesa-Siverio D. Estevez-Braun A. Ravelo A.G. Murguia J.R. Rodriguez-Afonso A. Eur. J. Org. Chem. 2003; : 4243-4247Crossref Scopus (27) Google Scholar, 14Gutiérrez F. Estévez-Braun A. Ravelo A.G. Astudillo L. Zarate R. J. Nat. Prod. 2007; 70: 1049-1052Crossref PubMed Scopus (27) Google Scholar, 15Delgado-Méndez P. Herrera N. Chávez H. Estévez-Braun A. Ravelo A.G. Cortes F. Castanys S. Gamarro F. Bioorg. Med. Chem. 2008; 16: 1425-1430Crossref PubMed Scopus (28) Google Scholar, 16Neukirch H. D'Ambrosio M. Sosa S. Altinier G. Della Loggia R. Guerriero A. Chem. Biodivers. 2005; 2: 657-671Crossref PubMed Scopus (36) Google Scholar). The degree of purity of the tested compounds was estimated higher than 99% by NMR spectroscopy. 10 mg (0.023 mmol) of calenduladiol in 2 ml of EtOH were treated with 2.6 mg (1 eq) of SeO2. The reaction mixture was heated under reflux for 10 h. Then, the reaction mixture was cooled and EtOH was removed under reduced pressure. The crude was treated with water and extracted three times with CH2Cl2. The organic layer was dried and concentrated under reduced pressure. The residue was purified by preparative TLC with hexanes/EtOAc (7:3) as solvent to obtain 3.6 mg (30.4%) of 1 as amorphous pale yellow solid. Compound 1 showed identical spectroscopic data to those previously reported (16Neukirch H. D'Ambrosio M. Sosa S. Altinier G. Della Loggia R. Guerriero A. Chem. Biodivers. 2005; 2: 657-671Crossref PubMed Scopus (36) Google Scholar). 15 mg (0.035 mmol) of resinone (17Núñez M.J. Reyes C.P. Jiménez I.A. Moujir L. Bazzocchi I.L. J. Nat. Prod. 2005; 68: 1018-1021Crossref PubMed Scopus (50) Google Scholar) in 2 ml of EtOH were treated with 7.3 mg (3 eq) of hydroxylamine hydrochloride and a solution of 5.75 mg (2 eq) of sodium acetate in 0.5 ml of H2O. The reaction mixture was heated under reflux for 15 h. Then, the reaction mixture was cooled and the solvent was removed under reduced pressure. The crude was treated with water and extracted three times with CH2Cl2. The organic layer was dried and concentrated under reduced pressure. The residue was purified by preparative TLC with hexanes/EtOAc (7:3) as solvent to obtain 13.0 mg (80%) of resinone oxime as amorphous pale yellow solid. Then 6 mg (0.013) of resinone oxime in 2 ml of CH2Cl2 were treated with pyridine, the catalytic amount of 4-dimethylaminopyridine, and an excess of Ac2O (10 μl). The reaction mixture was stirred at room temperature for 10 h. Then the solvent was removed under reduced pressure and the residue was purified by TLC preparative with hexanes/EtOAc (4:1) as solvent to afford 2.6 mg (38.5%) of compound 2 as an amorphous solid: [α]D20, +4.6 (c 0.26, CHCl3); UV (EtOH) λmax (log ϵ) nm, 340 (2.42); 273 (2.72); IR (CHCl3) νmax, 2925, 2854, 1735, 1459, 1370, 1023, 755 cm−1; 1H NMR(CDCl3) δ, 4.89 (1H, dd, J = 8.5; 3.9 Hz, H-16), 4.72 (1H, s, H-29b), 4.62 (1H, s, H-29a), 2.20 (3H, s, OCOCH3), 2.04 (3H, s, OCOCH3), 1.69 (3H, s, H-30), 1.14 (3H, s, H-26), 1.08 (H, s, H-23), 1.05 (3H, s, H-27), 0.94 (3H, s, H-24), 0.87 (6H, s, H-25, H-28); 13C NMR (CDCl3) δ, 170.0 (2xs, OCOCH3), 167.0 (s, C-3), 149.5 (s, C-20), 109.7 (t, C-29), 78.7 (d, C-16), 54.9 (d, C-5), 49.3 (d, C-9), 47.3 (d, C-18), 47.1 (d, C-19), 47.0 (s, C-17), 43.9 (s, C-14), 40.9 (s, C-4), 40.7 (s, C-8), 38.9 (t, C-1), 37.4 (t, C-22), 37.3 (d, C-13), 36.9 (s, C-10), 33.6 (t, C-15), 33.3 (t, C-7), 31.6 (t, C-2), 29.1 (t, C-21), 27.2 (c, C-23), 24.3 (t, C-12), 22.4 (2xc, C-24, C-11), 21.1 (2x c, OCOCH3), 20.8 (t, C-6), 19.8 (c, C-30), 15.8 (c, C-25), 15.6 (c, C-26), 13.8 (c, C-27), and 12.4 (c, C-28); EIMS m/z (%), 481 [M+-C2H5ON] (40Fernandez E.J. Lolis E. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 469-499Crossref PubMed Scopus (511) Google Scholar); 466 (6Pöhlmann S. Reeves J.D. Curr. Pharm. Des. 2006; 12: 1963-1973Crossref PubMed Scopus (27) Google Scholar); 452 (1Inciardi J.A. Williams M.L. AIDS Care. 2005; 17: S1-S8Crossref PubMed Scopus (8) Google Scholar); 422 (5UNAIDSReport on the Global AIDS Epidemic. UNAIDS, Joint United Nations Programme on HIV/AIDS, Geneva, Switzerland2008Google Scholar); HREIMS m/z (%), 481.3688 (calculated for C32H49O3 481.3682), 452.3545 (M+-C4H8O2) (calculated for C30H46O2N, 452.3529). 5.8 mg (0.01 mmol) of 3β,16β-dihydroxylup-12-ene were acetylated with 2.5 eq of Ac2O (2.5 μl), following the same procedure described for compound 2. The residue was purified by TLC preparative with hexane/EtOAc (4:1) as solvent to yield 6.8 mg (86%) of compound 3 as an amorphous white solid: [α]D20, +36.4 (c, 0.6, CHCl3); UV (EtOH) λmax (log ϵ) nm, 258 (3.09); IR (CHCl3) νmax, 2926, 2855, 1737, 1456, 1368, 1243, and 1025 cm−1; 1H NMR (CDCl3) δ, 5.45 (1H, dd, J = 11.3; 5.4 Hz, H-16), 5.19 (1H, t, J = 3.4 Hz, H-12), 4.50 (1H, dd, J = 5.5;9.0 Hz, H-3), 2.04 (3H, s, OCOCH3), 2.02 (3H, s, OCOCH3), 1.18 (3H, s, H-27), 1.02 (3H, s, H-26), 0.97 (3H, s, H-23), 0.92 (3H, s, H-25), 0.87 (3H, s, H-24), 0.86 (3H, d, J = 7.7 Hz, H-29), 0.85 (3H, s, H-28), 0.79 (3H, d, J = 6.2 Hz, H-30); 13C NMR (CDCl3) δ: 170.7 (s, OCOCH3), 170.5 (s, OCOCH3), 137.3 (s, C-13), 124.9 (d, C-12), 80.6 (d, C-3), 70.5 (d, C-16), 60.5 (d, C-18), 54.9 (d, C-5), 46.5 (d, C-9), 43.6 (s, C-14), 39.8 (s, C-8), 39.2 (2xd, C-19, C-20), 38.1 (t, C-1), 37.4 (s, C-17), 37.3 (s, C-4), 36.4 (s, C-10), 35.1 (t, C-15), 32.5 (t, C-7), 32.0 (t, C-22), 30.3 (t, C-21), 27.8 (c, C-23), 24.8 (c, C-27), 23.3 (t, C-2), 23.1 (t, C-11), 22.4 (c, C-28), 21.0 (C-30), 20.9 (2x c, OCOCH3), 17.9 (t, C-6), 17.3 (c, C-25), 16.6 (c, C-26), 16.4 (t, C-29), 15.5 (c, C-24); EIMS m/z (%), 526 [M+] (1Inciardi J.A. Williams M.L. AIDS Care. 2005; 17: S1-S8Crossref PubMed Scopus (8) Google Scholar), 466 (16Neukirch H. D'Ambrosio M. Sosa S. Altinier G. Della Loggia R. Guerriero A. Chem. Biodivers. 2005; 2: 657-671Crossref PubMed Scopus (36) Google Scholar), 451 (7Cichewicz R.H. Kouzi S.A. Med. Res. Rev. 2004; 24: 90-114Crossref PubMed Scopus (457) Google Scholar); HREIMS, 526.4029 (calculated for C34H54O4, 526.4022), 466.3778 (M+-C2H4O2) (calculated for C32H50O2, 466.3811). 5.9 mg (84.6%) of compound 5 as amorphous solid were obtained from 6.0 mg (0.013 mmol) of 3α,16β-dihydroxylup-12-ene under identical reaction and purification conditions as those used for compound 3: [α]D20, +11.2 (c 0.6, CHCl3); UV (EtOH) λmax (log ϵ), 202 (3.79) nm; IR (CHCl3) νmax, 2926, 2856, 1737, 1457, 1372, 1244, 1024, and 756 cm−1; 1H NMR (CDCl3) δ, 5.47 (1H, dd, J = 11.4; 5.4 Hz, H-16), 5.20 (1H, t, J = 3.4, H-12), 4.63 (1H, bs, H-3), 2.08 (3H, s, OCOCH3), 2.03 (3H, s, OCOCH3), 1.24 (3H, s, H-27), 1.22 (3H, d, J = 7.1 Hz, H-29), 1.03 (3H, s, H-26), 0.97 (3H, s, H-25), 0.89 (3H, s, H-23), 0.87 (6H, s, H-28, H-24), and 0.80 (3H, d, J = 6.3 Hz, H-30); 13C NMR (CDCl3) δ, 170.6 (2xs, OCOCH3), 137.2 (s, C-13), 125.1 (d, C-12), 77.8 (d, C-3), 70.5 (d, C-16), 60.5 (d, C-18), 49.7 (d, C-5), 46.4 (d, C-9), 43.6 (s, C-14), 40.0 (s, C-8), 39.2 (2xd, C-19, C-20), 37.3 (s, C-17), 36.5 (s, C-4), 36.2 (s, C-10), 35.1 (t, C-7), 33.6 (t, C-1), 32.4 (t, C-15), 32.1 (t, C-22), 30.3 (t, C-21), 27.5 (c, C-23), 24.1 (c, C-27), 23.0 (t, C-2), 22.9 (c, C-24), 22.4 (t, C-11), 21.7 (c, C-28), 21.1 (c, OCOCH3), 21.0 (c, OCOCH3), 20.9 (c, C-29), 17.8 (t, C-6), 17.3 (c, C-30), 16.6 (c, C-26), and 15.2 (t, C-25); EIMS m/z (%), 526 [M+] (1Inciardi J.A. Williams M.L. AIDS Care. 2005; 17: S1-S8Crossref PubMed Scopus (8) Google Scholar), 466 (14Gutiérrez F. Estévez-Braun A. Ravelo A.G. Astudillo L. Zarate R. J. Nat. Prod. 2007; 70: 1049-1052Crossref PubMed Scopus (27) Google Scholar), 451 (6Pöhlmann S. Reeves J.D. Curr. Pharm. Des. 2006; 12: 1963-1973Crossref PubMed Scopus (27) Google Scholar); HREIMS: 526.4038 (calculated for C34H54O4, 526.4022), 467.3917 (M+-OCOCH3) (calculated for C32H51O2, 467.3889). The monoclonal antibodies (mAbs) CD184 (clone 12G5) and CD195 (clone 2D7/CCR5), used as phycoerythrin (PE) conjugates (BD Bioscience/BD Pharmingen, San Jose, CA), are directed against the second extracellular loop of CXCR4 and CCR5, respectively. The PE-labeled mAb RPT-4 is a neutralizing antibody against CD4 (eBioscience, San Diego, CA). PE-conjugated mAbs against human CCR1 (FAB145P), CCR2b (FAB151P), CCR3 (FAB155P), and CCR4 (FAB1567P) chemokine receptors were from R&D Systems (Minneapolis, MN). The Cremophor® EL emulsifying agent that is used in aqueous preparations of hydrophobic substances was from Sigma. In all experiments, 30-oxo-calenduladiol and the other assayed triterpenes were dissolved in the following working buffer solution: Cremophor EL/dimethyl sulfoxide/culture medium at a ratio of 1:1:8 (v/v/v). The Fura 2-AM probe was from Invitrogen. The FluorokineTM human biotinylated RANTES (CCL5), monocyte chemotactic protein 1 (MCP-1), Eotaxin, or thymus- and activation-regulated chemokine (TARC) kits, and the human recombinant RANTES were from R&D Systems. The human CEM.NKR-CCR5 permissive cell line (catalog number 4376, NIH AIDS Research and Reference Reagent Program) was grown at 37 °C in a humidified atmosphere with 5% CO2 in RPMI 1640 medium (Lonza, Verviers, Belgium) supplemented with 10% fetal calf serum (Lonza), 1% l-glutamine, and 1% penicillin-streptomycin antibiotics. Cells were regularly passaged every 3 days. The 293T cell line was similarly cultured, in supplemented Dulbecco's modified Eagle's medium (Lonza), and were regularly passaged every 2–3 days. 24 h before cell transfection with viral or human DNA constructs, cells were harvested and resuspended at a density of 50–70% in fresh supplemented Dulbecco's modified Eagle's medium. The HeLa-P5 cells, stably transfected with human CD4 and C-terminal enhanced green fluorescent protein (EGFP)-tagged CCR5 cDNAs and with an HIV-long terminal repeat-driven β-galactosidase reporter gene (18Pleskoff O. Tréboute C. Brelot A. Heveker N. Seman M. Alizon M. Science. 1997; 276: 1874-1878Crossref PubMed Scopus (286) Google Scholar), as well as, HeLa-243 and HeLa-ADA cells, co-expressing the Tat and X4- and R5-tropic HIV-1-Env proteins, respectively, were provided by Dr. M. Alizon (Hôpital Cochin, Paris, France) (18Pleskoff O. Tréboute C. Brelot A. Heveker N. Seman M. Alizon M. Science. 1997; 276: 1874-1878Crossref PubMed Scopus (286) Google Scholar, 19Valenzuela-Fernández A. Alvarez S. Gordon-Alonso M. Barrero M. Ursa A. Cabrero J.R. Fernández G. Naranjo-Suárez S. Yáñez-Mo M. Serrador J.M. Muñoz-Fernández M.A. Sánchez-Madrid F. Mol. Biol. Cell. 2005; 16: 5445-5454Crossref PubMed Scopus (103) Google Scholar). Human astroglia U87 cell line, stably expressing human CD4 and CCR3 receptors (U87.CD4.CCR3), was kindly provided by Dr. Guido Poli (San Raffaele Scientific Institute, Milano, Italy) and Dr. Dan R. Littman (Skirball Institute of Biomolecular Medicine, New York). Human cDNAs of the β-chemokine CCR1, CCR2b, CCR3, and CCR4 receptors were from OriGene (Origene Technologies, Inc., Rockville, MD). The pNL4-3.Luc.R-E- provirus (catalog number 6070013), the HXB2-env (catalog number 5040154), and pCAGGS-SF162-gp160-env (catalog number 3041817) glycoprotein vectors, and the pHEF-VSV-G vector (catalog number 4693), encoding the vesicular stomatitis virus G (VSV-G) protein, were obtained through the NIH AIDS Research and Reference Reagent Program. A β-galactosidase cell fusion assay was performed as previously described (19Valenzuela-Fernández A. Alvarez S. Gordon-Alonso M. Barrero M. Ursa A. Cabrero J.R. Fernández G. Naranjo-Suárez S. Yáñez-Mo M. Serrador J.M. Muñoz-Fernández M.A. Sánchez-Madrid F. Mol. Biol. Cell. 2005; 16: 5445-5454Crossref PubMed Scopus (103) Google Scholar, 20Valenzuela-Fernández A. Palanche T. Amara A. Magerus A. Altmeyer R. Delaunay T. Virelizier J.L. Baleux F. Galzi J.L. Arenzana-Seisdedos F. J. Biol. Chem. 2001; 276: 26550-26558Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, HeLa-243 or HeLa ADA cells were mixed with HeLa-P5 cells, in 96-well plates, in a 1:1 ratio (20,000 total cells), in the absence or presence of 5 μm of the different molecules assayed. These co-cultures were kept at fusion for 16 h at 37 °C. The fused cells were washed with Hanks' balanced salt solution, lysed, and the enzymatic activity was evaluated by chemiluminescence (β-galactosidase reporter gene assay; Roche Diagnostics, Germany). Anti-CD4 neutralizing mAb (5 μg/ml was preincubated in HeLa-P5 cells for 30 min at 37 °C before co-culture with Env+ HeLa cells) was used as a control for the blockage of cell fusion. X4- or R5-tropic HIV-1 viral particles were produced by co-transfecting 293T cells (70% of confluence) in 75-cm2 flasks with pNL4-3.Luc.R-E- (20 μg) and CXCR4-tropic (HXB2-env) or CCR5-tropic (pCAGGS SF162 gp160) env glycoprotein (10 μg) vector, as previously described (21Barrero-Villar M. Barroso-González J. Cabrero J.R. Gordón-Alonso M. Alvarez-Losada S. Muñoz-Fernández M.A. Sánchez-Madrid F. Valenzuela-Fernández A. J. Immunol. 2008; 181: 6882-6888Crossref PubMed Scopus (34) Google Scholar). Co-transduction of the pNL4-3.Luc.R-E- (20 μg) vector with the pHEF-VSV-G (10 μg) vector generates non-replicative viral particles that infect with cells in a VSV-G-dependent manner. Viral plasmids were transduced in 293T cells by using linear polyethylenimine, with an average molecular mass of 25 kDa (PEI25k) (Polyscience Inc., Warrington, PA). For this purpose, viral plasmids were first dissolved in 1/10th of the final tissue culture volume of Dulbecco's modified Eagle's medium, free of serum and antibiotics. The PEI25k was prepared as a 1 mg/ml solution in water and adjusted to neutral pH. After addition of PEI25k to the viral plasmids (at a plasmids:PEI25k ratio of 1:5 (w/w)), the solution was mixed immediately, incubated for 20–30 min at room temperature and then added to 293T cells in culture. After 4 h the medium was changed to RPMI 1640, supplemented with 10% fetal calf serum and antibiotics, and the cells were cultivated to allow viral production. Viruses were harvested 40 h post-transfection. The supernatant was clarified by centrifugation at 3,000 × g for 30 min. Virions were then stored at −80 °C. Viral stocks were normalized by p24-Gag content measured with an enzyme-linked immunosorbent assay test (Innogenetics, Gent, Belgium). 1 × 106 CEM.NKR-CCR5 permissive cells were incubated in the presence of different amounts of RANTES or 30-oxo-calenduladiol, with a synchronous dose of luciferase-based X4- or R5-tropic HIV-1 or VSV-G viral inputs (500 ng of p24), in 500 μl of RPMI 1640 medium for 2 h, as described (21Barrero-Villar M. Barroso-González J. Cabrero J.R. Gordón-Alonso M. Alvarez-Losada S. Muñoz-Fernández M.A. Sánchez-Madrid F. Valenzuela-Fernández A. J. Immunol. 2008; 181: 6882-6888Crossref PubMed Scopus (34) Google Scholar). Cells were then extensively washed to remove free virions. After 32 h of infection, luciferase activity was determined by using a luciferase assay kit (Promega Corporation) with a microplate reader (GeNiosTM, Tecan Trading AG, Switzerland). Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA). CEM.NKR-CCR5 cells were incubated with PE-labeled specific antibodies against CD4, CXCR4, or CCR5 in the presence of different amounts of the 30-oxo-calenduladiol compound, for 1 h at 4 °C to avoid receptor internalization. Then, cells were washed by ice-cold PBS, fixed in PBS, 1% formaldehyde, and analyzed by flow cytometry (XL-MCL system, Beckman-Coulter Inc.). Cell surface expression analysis of CCR1, CCR2b, CCR3, or CCR4 receptors, transiently expressed in 293T cells, was similarly performed by using specific PE-conjugated mAbs, and their binding were equally assayed in the presence of 30-oxo-calenduladiol. HeLa-P5 cells on coverslips at 50% confluence were starved for 30 min at 37 °C in Dulbecco's modified Eagle's medium without serum. They were then incubated in 80 μl of Dulbecco's modified Eagle's medium (with 0.5% bovine serum albumin) with RANTES (150 nm) or 30-oxo-calenduladiol (1 μm) at 37 °C for 30 min, or for 15 min at 37 °C with 30-oxo-calenduladiol (1 μm) before adding RANTES (150 nm) for a further 30 min at 37 °C. These CCR5-EGFP+ cells were rinsed three times at the end of incubation with ice-cold PBS and fixed in PBS, 2% paraformaldehyde for 3 min at room temperature. Cells were then rinsed and mounted in ProLong® Gold antifade reagent (Invitrogen) containing the 4′,6-diamidino-2-phenylindole probe to stain the nucleus of cells. The internalization of the CCR5-EGFP molecule was analyzed by fluorescence confocal microscopy (Leica DMR photomicroscope and Leica TCS-SP confocal microscope; Leica, Heidelberg, Germany). HeLa-P5 cells, stably expressing the CCR5-EGFP receptor, were imaged with an inverted microscope Zeiss 200M (Zeiss, Germany) through a 1.45 NA objective (αFluar, ×100/1.45; Zeiss) in a Krebs-Hepes buffer containing 2 mm Ca2+ at 37 °C, in the absence or presence or RANTES (150 nm), compound 1 (1 μm), or pre-incubating compound 1 (1 μm), for 15 min at 37 °C, before adding RANTES (150 nm). RANTES-induced CCR5-EGFP internalization, or its prevention by compound 1 was analyzed by TIRFM technology, as described (22Barroso-González J. Machado J.D. García-Expósito L. Valenzuela-Fernández A. J. Biol. Chem. 2009; 284: 2419-2434Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 23Steyer J.A. Almers W. Nat. Rev. Mol. Cell Biol. 2001; 2: 268-275Crossref PubMed Scopus (341) Google Scholar). Briefly, total internal reflection generates an evanescent field (EF) that declines exponentially with increasing distance from the interface, depending on the angle at which the light strikes the interface. The angle was measured using a hemicylinder, as described (24Merrifield C.J. Feldman M.E. Wan L. Almers W. Nat. Cell Biol. 2002; 4: 691-698Crossref PubMed Scopus (564) Google Scholar). The images were projected onto a back-illuminated CCD camera (AxioCam MRm, Zeiss) through a dichoric and specific band-pass filter for the EGFP fluorophor. CCR5-EGFP molecules were analyzed at 37 °C, and imagined on the cell surface of HeLa-P5 cells using Axiovision (Zeiss) with 0.5-s exposures at 10 Hz, when illuminated under EF at the indicated times for any experimental condition. CEM.NKR-CCR5 cells were incubated with 5 nm human biotinylated RANTES and competed with different amounts of unlabeled human recombinant RANTES, as indicated by the manufacturer (FluorokineTM RANTES (CCL5) kit, R&D Systems), or the 30-oxo-calenduladiol triterpene in a final volume of 300 μl for 1 h at 4 °C. Incubations were terminated by centrifugation at 4 °C. Cell pellets were resuspended in ice-cold PBS and fluorescein isothiocyanate-labeled avidin (The FluorokineTM biotinylated RANTES (CCL5) kit, R&D Systems) was added to detect bound biotinylated RANTES. The CCR5-associated biotinylated RANTES was quantified by flow cytometry. Binding assays for human CCR1, CCR2b, CCR3, or CCR4 receptor (OriGene), transiently transfected in 293T cells, were carried out in a similar way, by analyzing the ability of 30-oxo-calenduladiol to compete the specific binding of their respective biotinylated natural ligands (RANTES, MCP-1, Eotaxin, or TARC (R&D Systems), respectively). The nonspecific binding of each ligand assayed was measured in cells pre-treated with specific blocking anti-receptor Abs (1 μg/ml) (included in the respective Fluorokine kits (R&D Systems)), which entirely prevented specific ligand-receptor binding. Intracellular calcium levels were measured in a fluorescence spectrophotometer (Eclypse Variant; Melbourne, Australia) using Fura 2-AM (5 μm)-loaded CEM.NKR-CCR5 cells (5 × 106 cells/ml), as similarly described (25Valenzuela-Fernández A. Planchenault T. Baleux F. Staropoli I. Le-Barillec K. Leduc D. Delaunay T. Lazarini F. Virelizier J.L. Chignard M.

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