13 research outputs found
Oral Pre-Exposure Prophylaxis by Anti-Retrovirals Raltegravir and Maraviroc Protects against HIV-1 Vaginal Transmission in a Humanized Mouse Model
Sexual HIV-1 transmission by vaginal route is the most predominant mode of viral transmission, resulting in millions of new infections every year. In the absence of an effective vaccine, there is an urgent need to develop other alternative methods of pre-exposure prophylaxis (PrEP). Many novel drugs that are currently approved for clinical use also show great potential to prevent viral sexual transmission when administered systemically. A small animal model that permits rapid preclinical evaluation of potential candidates for their systemic PrEP efficacy will greatly enhance progress in this area of investigation. We have previously shown that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and displays CD4 T cell loss typical to that seen in the human. Thus far systemic PrEP studies have been primarily limited to RT inhibitors exemplified by tenofovir and emtricitabine. In these proof-of-concept studies we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically administered chemo-prophylactics. Our results showed that oral administration of either of these drugs fully protects against vaginal HIV-1 challenge in the RAG-hu mouse model. Based on these results both these drugs show great promise for further development as orally administered PrEPs
Viral Decay Kinetics in the Highly Active Antiretroviral Therapy-Treated Rhesus Macaque Model of AIDS
To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2–58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans
Identification and characterization of nucleoside and nucleotide kinases : Pharmacological activation of anti-cancer and anti-viral nucleoside analogs
Cells require deoxyribonucleotides to synthesize DNA. Structurally
modified precursors of deoxyribonucleotides, i.e. nucleoside analogs, are
used as anti-viral and anti-cancer compounds. The deoxyribonucleosides
and the nucleoside analogs are phosphorylated to the triphosphate form in
three subsequent phosphorylation steps, that are catalyzed by nucleoside
kinases, nucleoside monophosphate kinases and nucleoside diphosphate
kinases. Five of these enzymes have been cloned and characterized in this
thesis.
Deoxyribonucleoside kinases catalyze the phosphorylation of
deoxyribonucleosides to deoxyribonucleoside monophosphates. We have
cloned and expressed the cDNA of the Drosophila melanogaster
deoxyribonucleoside kinase. This enzyme showed a broader substrate
specificity and higher catalytic rate compared to the human enzymes and
the herpes simplex virus type-1 thymidine kinase. The enzyme
phosphorylated all four natural deoxyribonucleosides as well as several
nucleoside analogs. The broad substrate specificity and high catalytic
rate of Dm-dNK makes this enzyme an interesting candidate for suicide
gene therapy.
Two human uridine-cytidine kinases (UCK) were cloned and shown to
catalyze the phosphorylation of uridine and cytidine to their
monophosphate forms. UCK1 mRNA was ubiquitously expressed in twelve
different human tissues, whereas human UCK2 mRNA was detected only in
placenta. The genes encoding UCK1 and UCK2 were mapped to chromosome
9q34.2-9q34.3 and lq22-lq23.2, respectively. We tested 28 cytidine and
uridine nucleoside analogs and several of these were shown to be
substrates of the enzymes.
UCK1 and UCK2 were overexpressed in a Chinese hamster ovary cancer cell
line to determine the importance of UCK mediated nucleoside analog
phosphorylation. The transfected cells showed a nuclear and cytosolic
location for UCK1 and UCK2, respectively. The UCK1 and UCK2 overexpressed
cell line exhibited increased sensitivity to several analogs. Our data
imply that UCK1 and UCK2 activity are ratelimiting for the efficacy of
some ribonucleoside analogs in the cell line investigated.
Nucleoside monophosphate kinases catalyze the second phosphorylation
step, from the monophosphate to the diphosphate form. A novel human
adenylate kinase, AK5, was cloned and shown to be predominantly expressed
in brain. The enzyme phosphorylated AMP and dAMP with ATP as phosphate
donor. The gene that encoded this cytosolic enzyme was localized to
chromosome 1p3 1. The cytosolic human UMPCMP kinase was cloned and
characterized. The UMP-CMP kinase phosphorylated CMP, dCMP, UMP and dUMP
and also the nucleoside analogs dFdC-MP, AraC-MP and ddC-MP. UMP-CMP
kinase mRNA was ubiquitously present in human tissues. The human UMP-CMP
kinase gene was localized to chromosome 1p34 1-1p33.
The cloning, recombinant expression and characterization of these five
nucleoside and nucleotide kinases will allow the pathways of nucleoside
and nucleoside analog metabolism to be outlined in more detail. Knowledge
of the metabolic pathways will be important for the development of novel
ribonucleoside- and deoxyribonucleoside analogs and for the
characterization of their pharmacological activation in different
tissues
Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases. Mol Pharmacol
ABSTRACT Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer. We have cloned the cDNA of two human UCKs. The Ϸ30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected as two isoforms of Ϸ1.8 and Ϸ2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of Ϸ1.8 kb was present in skeletal muscle, heart, liver, and kidney. The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N 4 -acetylcytidine, N 4 -benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N 4 -anisoylcytidine. The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation
Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo
Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I)
We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85–95% and a between-laboratory-reproducibility of 90%
Microarray analyses in dendritic cells reveal potential biomarkers for chemical-induced skin sensitization
Prolonged tenofovir treatment of macaques infected with K65R reverse transcriptase mutants of SIV results in the development of antiviral immune responses that control virus replication after drug withdrawal
BACKGROUND: We reported previously that while prolonged tenofovir monotherapy of macaques infected with virulent simian immunodeficiency virus (SIV) resulted invariably in the emergence of viral mutants with reduced in vitro drug susceptibility and a K65R mutation in reverse transcriptase, some animals controlled virus replication for years. Transient CD8+ cell depletion or short-term tenofovir interruption within 1 to 5 years of treatment demonstrated that a combination of CD8+ cell-mediated immune responses and continued tenofovir therapy was required for sustained suppression of viremia. We report here follow-up data on 5 such animals that received tenofovir for 8 to 14 years. RESULTS: Although one animal had a gradual increase in viremia from 3 years onwards, the other 4 tenofovir-treated animals maintained undetectable viremia with occasional viral blips (≤ 300 RNA copies/ml plasma). When tenofovir was withdrawn after 8 to 10 years from three animals with undetectable viremia, the pattern of occasional episodes of low viremia (≤ 3600 RNA/ml plasma) continued throughout the 10-month follow-up period. These animals had low virus levels in lymphoid tissues, and evidence of multiple SIV-specific immune responses. CONCLUSION: Under certain conditions (i.e., prolonged antiviral therapy initiated early after infection; viral mutants with reduced drug susceptibility) a virus-host balance characterized by strong immunologic control of virus replication can be achieved. Although further research is needed to translate these findings into clinical applications, these observations provide hope for a functional cure of HIV infection via immunotherapeutic strategies that boost antiviral immunity and reduce the need for continuous antiretroviral therapy
The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT
The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 μM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 μM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC)·oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses