scholarly journals Intracellular Metabolism of the Nucleotide Prodrug GS-9131, a Potent Anti-Human Immunodeficiency Virus Agent

2007 ◽  
Vol 52 (2) ◽  
pp. 648-654 ◽  
Author(s):  
Adrian S. Ray ◽  
Jennifer E. Vela ◽  
Constantine G. Boojamra ◽  
Lijun Zhang ◽  
Hon Hui ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Lymphoid Cells ◽  
Activity Levels ◽  
Peripheral Blood Mononuclear ◽  
Cellular Pharmacology ◽  
Efficient Delivery ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT GS-9131 is a phosphonoamidate prodrug of the novel ribose-modified phosphonate nucleotide analog GS-9148 that demonstrates potent anti-human immunodeficiency virus type 1 (HIV-1) activity and an excellent resistance profile in vitro. Prodrug moieties were optimized for the efficient delivery of GS-9148 and its active diphosphate (DP) metabolite to lymphoid cells following oral administration. To understand the intracellular pharmacology of GS-9131, incubations were performed with various types of lymphoid cells in vitro. The intracellular accumulation and antiviral activity levels of GS-9148 were limited by its lack of cellular permeation, and GS-9131 increased the delivery of GS-9148-DP by 76- to 290-fold relative to that of GS-9148. GS-9131 activation was saturable at high extracellular concentrations, potentially due to a high-affinity promoiety cleavage step. Once inside the cells, GS-9148 was efficiently phosphorylated, forming similar amounts of anabolites in primary lymphoid cells. The levels of GS-9148-DP formed in peripheral blood mononuclear cells infected with HIV-1 were similar to that in uninfected PBMCs, and approximately equivalent intracellular concentrations of GS-9148-DP and tenofovir (TVF)-DP were required to inhibit viral replication by 90%. Once it was formed, GS-9148-DP was efficiently retained in activated CD4+ cells, with a half-life of 19 h. In addition, GS-9131 showed a low potential for drug interactions with other adenine nucleoside/nucleotide reverse transcriptase inhibitors, based on the lack of competition for anabolism between suprapharmacologic concentrations of GS-9148 and TVF and the lack of activity of GS-9131 metabolites against purine nucleoside phosphorylase, an enzyme involved in the clearance of 2′,3′-dideoxyinosine. Together, these observations elucidate the cellular pharmacology of GS-9131 and illustrate its efficient loading of lymphoid cells, resulting in a prolonged intracellular exposure to the active metabolite GS-9148-DP.

scholarly journals Design and Profiling of GS-9148, a Novel Nucleotide Analog Active against Nucleoside-Resistant Variants of Human Immunodeficiency Virus Type 1, and Its Orally Bioavailable Phosphonoamidate Prodrug, GS-9131

2007 ◽  
Vol 52 (2) ◽  
pp. 655-665 ◽  
Author(s):  
Tomas Cihlar ◽  
Adrian S. Ray ◽  
Constantine G. Boojamra ◽  
Lijun Zhang ◽  
Hon Hui ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Biological Properties ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (Ki = 0.8 μM) and exhibits low inhibitory potency against host polymerases including DNA polymerase γ. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 μM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Activity In Vitro by a New Self-Stabilized Oligonucleotide with Guanosine-Thymidine Quadruplex Motifs

2002 ◽  
Vol 76 (6) ◽  
pp. 3015-3022 ◽  
Author(s):  
Jun-ichiro Suzuki ◽  
Naoko Miyano-Kurosaki ◽  
Tomoyuki Kuwasaki ◽  
Hiroaki Takeuchi ◽  
Gota Kawai ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Core Antigen ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.

scholarly journals SRR-SB3, a disulfide-containing macrolide that inhibits a late stage of the replicative cycle of human immunodeficiency virus.

1997 ◽  
Vol 41 (2) ◽  
pp. 262-268 ◽  
Author(s):  
M Witvrouw ◽  
J Balzarini ◽  
C Pannecouque ◽  
S Jhaumeer-Laulloo ◽  
J A Esté ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Late Stage ◽  
Mononuclear Cells ◽  
Antiviral Agents ◽  
Peripheral Blood Mononuclear ◽  
Disulfide Linkage ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Replicative Cycle

From a series of macrocyclic diamides possessing the disulfide linkage, only SRR-SB3, a compound that complexes with zinc, was found to inhibit human immunodeficiency virus type 1 (HIV-1; strain IIIB) replication at a concentration of 1.8 to 6.5 micrograms/ml in MT-4, CEM, and peripheral blood mononuclear cells. SRR-SB3 was toxic to MT-4 cells at a concentration of 15.9 micrograms/ml, resulting in a selectivity index of 9 in these cells. This macrolide was also effective against various other HIV-1 strains, including clinical isolates and HIV-1 strains resistant to protease inhibitors and nucleoside and nonnucleoside reverse transcriptase inhibitors. It was also active against various HIV-2 strains, simian immunodeficiency virus (strain MAC251), and Moloney murine sarcoma virus, but not against viruses other than retroviruses. In addition, the compound was found to inhibit chronic HIV-1 infections in vitro. The compound in combination with other antiviral agents, such as zidovudine, zalcitabine, and stavudine, showed an effect that was between additive and synergistic. Time-of-addition experiments indicated that SRR-SB3 acts at a late stage of the HIV-1 replicative cycle.

scholarly journals Integration of Human Immunodeficiency Virus Type 1 in Untreated Infection Occurs Preferentially within Genes

2006 ◽  
Vol 80 (15) ◽  
pp. 7765-7768 ◽  
Author(s):  
Hongbing Liu ◽  
Eugene C. Dow ◽  
Reetakshi Arora ◽  
Jason T. Kimata ◽  
Lara M. Bull ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Hiv 1

ABSTRACT Previous analyses of human immunodeficiency virus type 1 (HIV-1) integration sites generated in infections in vitro or in patients in whom viral replication was repressed by antiviral therapy have demonstrated a preference for integration within protein-coding genes. We analyzed integration sites in peripheral blood mononuclear cells (PBMCs), spleen, lymph node, and cerebral cortex from patients with untreated HIV-1 infections. The great majority of integration sites in each tissue were within genes. Statistical analyses of the frequencies of integration in genes in PBMCs and lymph tissue demonstrated a strong preference for integration within genes. Although the sample size for brain tissue was too small to demonstrate a clear statistical preference for integration in genes, four of the five integration sites identified in brain were within genes. Taken together, our data indicate that HIV-1 preferentially integrates within genes during untreated infection.

scholarly journals Shift of Clinical Human Immunodeficiency Virus Type 1 Isolates from X4 to R5 and Prevention of Emergence of the Syncytium-Inducing Phenotype by Blockade of CXCR4

1999 ◽  
Vol 73 (7) ◽  
pp. 5577-5585 ◽  
Author(s):  
José A. Esté ◽  
Cecilia Cabrera ◽  
Julià Blanco ◽  
Arantxa Gutierrez ◽  
Gary Bridger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Lymphoid Cells ◽  
Complete Suppression ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of X4 human immunodeficiency virus type 1 (HIV-1) strains in HIV-1-infected individuals has been associated with CD4+ T-cell depletion, HIV-mediated CD8+ cell apoptosis, and an impaired humoral response. The bicyclam AMD3100, a selective antagonist of CXCR4, selected for the outgrowth of R5 virus after cultivation of mixtures of the laboratory-adapted R5 (BaL) and X4 (NL4-3) HIV strains in the presence of the compound. The addition of AMD3100 to peripheral blood mononuclear cells infected with X4 or R5X4 clinical HIV isolates displaying the syncytium-inducing phenotype resulted in a complete suppression of X4 variants and a concomitant genotypic change in the V2 and V3 loops of the envelope gp120 glycoprotein. The recovered viruses corresponded genotypically and phenotypically to R5 variants in that they could no longer use CXCR4 as coreceptor or induce syncytium formation in MT-2 cells. Furthermore, the phenotype and genotype of a cloned R5 HIV-1 virus converted to those of the R5X4 virus after prolonged culture in lymphoid cells. However, these changes did not occur when the infected cells were cultured in the presence of AMD3100, despite low levels of virus replication. Our findings indicate that selective blockade of the CXCR4 receptor prevents the switch from the less pathogenic R5 HIV to the more pathogenic X4 HIV strains, a process that heralds the onset of AIDS. In this article, we show that it could be possible to redirect the evolution of HIV so as to impede the emergence of X4 strains or to change the phenotype of already-existing X4 isolates to R5.

scholarly journals Inhibition of Human Immunodeficiency Virus Replication by a Dual CCR5/CXCR4 Antagonist

2004 ◽  
Vol 78 (23) ◽  
pp. 12996-13006 ◽  
Author(s):  
Katrien Princen ◽  
Sigrid Hatse ◽  
Kurt Vermeire ◽  
Stefano Aquaro ◽  
Erik De Clercq ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Entry ◽  
Mononuclear Cells ◽  
Human Immunodeficiency Virus Replication ◽  
Cxcr4 Antagonist ◽  
Peripheral Blood Mononuclear ◽  
Transfected Cells ◽  
Immunodeficiency Virus ◽  
Intracellular Ca ◽  
Hiv 1

ABSTRACT Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC50] ranging from 1.2 to 26.5 μM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC50, 1.8 to 7.3 μM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca2+ signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca2+ flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca2+ signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca2+ signaling by itself at concentrations up to 400 μM. In freshly isolated monocytes, AMD3451 inhibited the Ca2+ flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.

scholarly journals Mechanism of Action and In Vitro Activity of 1′,3′-Dioxolanylpurine Nucleoside Analogues against Sensitive and Drug-Resistant Human Immunodeficiency Virus Type 1 Variants

1999 ◽  
Vol 43 (10) ◽  
pp. 2376-2382 ◽  
Author(s):  
Zhengxian Gu ◽  
Mark A. Wainberg ◽  
Nghe Nguyen-Ba ◽  
Lucille L’Heureux ◽  
Jean-Marc de Muys ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Nucleoside Analogues ◽  
Drug Resistant ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT (−)-β-d-1′,3′-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 μM when evaluated against HIV-1IIIB in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2′,3′-dideoxy-3′-thiacytidine (3TC) but 5- to 10-fold less potent than 3′-azido-2′,3′-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2′,3′-dideoxyinosine, and 2′,3′-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 μM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

scholarly journals Selection following isolation of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and herpesvirus saimiri-transformed T cells is comparable

2002 ◽  
Vol 83 (6) ◽  
pp. 1343-1352 ◽  
Author(s):  
Natalie N. Zheng ◽  
Cherelyn Vella ◽  
Philippa J. Easterbrook ◽  
Rod S. Daniels
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Herpesvirus Saimiri ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

In attempts to improve isolation rates and virus yields for human immunodeficiency virus (HIV), the use of herpesvirus saimiri-immortalized T cells (HVS T cells) has been investigated as an alternative to/improvement over peripheral blood mononuclear cells (PBMCs). Here we characterize isolates rescued, in the two cell types, from two asymptomatic, long-term non-progressing HIV-1-infected individuals. All rescued viruses replicated in PBMCs and HVS T cells only, displaying a non-syncytium inducing (NSI) phenotype, and using CCR5 as co-receptor. Furthemore, PBMC/HVS T cell virus pairs displayed similar neutralization profiles. Full-length, expression-competent env genes were rescued from all virus isolates and directly from the patient samples using proviral DNA and viral RNA as templates. Compared with the sequences retrieved directly from the patient samples, both cell types showed similar selection characteristics. Whilst the selections were distinct for individual patient samples, they shared a common characteristic in selecting for viruses with increased negative charge across the V2 domain of the viral glycoproteins. The latter was observed at the env gene sequencing level for three other patients whose HIV strains were isolated in PBMCs only. This further supports a common selection for viral sequences that display a macrophage-tropic/NSI phenotype and shows that HVS T cells are a viable alternative to PBMCs for HIV-1 isolation.

scholarly journals Human immunodeficiency virus types 1 and 2 have different replication kinetics in human primary macrophage culture

2006 ◽  
Vol 87 (2) ◽  
pp. 411-418 ◽  
Author(s):  
David Marchant ◽  
Stuart J. D. Neil ◽  
Áine McKnight
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Steady Rate ◽  
Immunodeficiency Virus ◽  
Load Characteristics ◽  
Kinetics Of ◽  
Hiv 1

This study compares the replication of primary isolates of human immunodeficiency virus type 2 (HIV-2) and type 1 (HIV-1) in monocyte-derived macrophages (MDMs). Eleven HIV-2 and five HIV-1 primary isolates that use CCR5, CXCR4 or both coreceptors to enter cells were included. Regardless of coreceptor preference, 10 of 11 HIV-2 viruses could enter, reverse transcribe and produce fully infectious virus in MDMs with efficiency equal to that in peripheral blood mononuclear cells. However, the kinetics of replication of HIV-2 compared with HIV-1 over time were distinct. HIV-2 had a burst of virus replication 2 days after infection that resolved into an apparent ‘latent state’ at day 3. HIV-1, however, continued to produce infectious virions at a lower, but steady, rate throughout the course of infection. These results may have implications for the lower pathogenesis and viral-load characteristics of HIV-2 infection.

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