RESUMEN
Some anti-inflammatory molecules are also known to possess anti-human immunodeficiency virus (HIV) activity. We found that o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS), a recently synthesized non-steroidal anti-inflammatory molecule can inhibit HIV-1 replication. The aim of this study was to clarify the mechanism of action of APHS. When administered during the first steps of the infection, APHS was capable of inhibiting the replication of several HIV-1 strains (macrophage-tropic and/or lymphocytotropic) in a dose-dependent manner in both peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages and peripheral blood lymphocytes with 50% inhibitory concentration values of approximately 10 microM. The 50% toxic concentration of APHS varied between 100 and 200 microM in the different primary cells tested. APHS did not affect HIV-1 replication once the provirus was already inserted into the cellular genome. APHS also did not inhibit HIV-1 entry into the host cells as determined by quantification of gag RNA inside PBMC 2h after infection. However, APHS did inhibit gag DNA synthesis during reverse transcription in primary cells, which indicates that APHS may target the reverse transcription process.
Asunto(s)
Acetileno/análogos & derivados , Acetileno/farmacología , Antiinflamatorios no Esteroideos/farmacología , VIH-1/efectos de los fármacos , Sulfuros/farmacología , Replicación Viral/efectos de los fármacos , Acetileno/química , Alquinos , Antiinflamatorios no Esteroideos/química , Aspirina/química , Aspirina/farmacología , Línea Celular , Células Cultivadas , ADN Viral/análisis , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/virología , Linfocitos/virología , Macrófagos/virología , Monocitos/virología , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Sulfuros/química , Polimerasa Taq/metabolismo , Transcripción GenéticaRESUMEN
OBJECTIVES: The use of multiple drug combinations in current anti-human immunodeficiency virus (HIV) therapy allows lower dosages of individual drugs and results in enhancement of the therapeutic effect due to synergic interactions between different drugs. We have shown that o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently developed non-steroidal anti-inflammatory drug, shows anti-HIV activity in a dose-dependent manner. The first aim of this study was to investigate whether APHS can act synergically with the clinically available reverse transcriptase and protease inhibitors (RTIs and PIs, respectively) in vitro. Because of the increasing prevalence of RTI- and PI-resistant HIV-1 strains, the second aim of this study was to assess the antiviral activity of APHS against drug-resistant HIV-1 strains in vitro. MATERIALS AND METHODS: HIV-infected peripheral blood mononuclear cells (PBMC) were treated for 7 days with different combinations of APHS and RTIs or PIs. The MT-2 cell line was infected with different HIV-1 strains and treated with APHS for 5 days. RESULTS: APHS showed synergic interactions with the RTIs zidovudine, lamivudine and efavirenz and with the PIs indinavir and ritonavir. The 50% inhibitory concentration (IC50) of APHS in this assay dropped from 13 microM when used alone, to 5 micro M after combination with an RTI or PI. In combination with APHS the IC50 of the RTI and PI drugs tested also dropped. APHS inhibits the replication of HIV-1 strains resistant to zidovudine, lamivudine, stavudine, didanosine, zalcitabine and ritonavir. CONCLUSIONS: These results indicate that APHS can be combined with RTIs and PIs and can inhibit several NRTI and PI-resistant HIV-1 strains.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Heptanos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Sulfuros/farmacología , Alquinos , Farmacorresistencia Viral , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Técnicas In Vitro , Neutrófilos/efectos de los fármacos , Neutrófilos/virologíaRESUMEN
Herpes simplex virus type 1 and 2 (HSV-1 and -2) glycoproteins D (gD-1 and gD-2) play a role in the entry of the virus into the host cell. Availability of substantial amounts of these proteins, or large fragments thereof, will be needed to allow studies at the molecular level. We studied the potency of the Pichia pastoris yeast expression system to produce soluble forms of gD. The DNA sequences encoding the extracellular domains of gD [amino acids 1-314 (gD-1(1-314)) and amino acids 1-254 (gD-1(1-254)) of gD-1 and amino acids 1-314 of gD-2 (gD-2(1-314))] were cloned into the P. pastoris yeast expression vector pPIC9. Two truncated forms of gD-1 were fitted with a His tail (designated as gD-1(1-314His) and gD-1(1-254His)) to facilitate their purification. Large amounts of gD-1(1-314) and gD-1(1-314His) (280-300mg/L induction medium) were produced. The yields of recombinant gD-1(1-254) and gD-1(1-254His) were lower: 20-36mg/L, and the yield of the gD-2(1-314) fragment was much lower: 6mg/L. SDS-PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78kDa. The smaller gD-1(1-254) fragment appeared as two bands with molecular weights of 33 and 31kDa. All recombinant proteins produced by P. pastoris were recognized, as expected, by a panel of MAbs (A16, DL6, A18, DL11, HD1, ABDI, and AP7). In addition, we showed that gD-1(1-314), gD-2(1-314), and gD-1(1-254His) were able to interfere with binding of HSV to susceptible cells. These results indicate that the conformations of the recombinant proteins closely resemble those of native gD.