Novel, selective CDK9 inhibitors for the treatment of HIV infection.

Cyclin Dependent Kinases (CDKs) are important regulators of cell cycle and gene expression. Since an up-to-date review about the pharmacological inhibitors of CDK family (CDK1-10) is not available; therefore in the present paper we briefly summarize the most relevant inhibitors and point out the low number of selective inhibitors. Among CDKs, CDK9 is a validated pathological target in HIV infection, inflammation and cardiac hypertrophy; however selective CDK9 inhibitors are still not available. We present a selective inhibitor family of CDK9 based on the 4-phenylamino-6- phenylpyrimidine nucleus. We show a convenient synthetic method to prepare a useful intermediate and its derivatisation resulting in novel compounds. The CDK9 inhibitory activity of the derivatives was measured in specific kinase assay and the CDK inhibitory profile of the best ones (IC(50) < 100 nM) was determined. The most selective compounds had high selectivity over CDK1, 2, 3, 5, 6, 7 and showed at least one order of magnitude higher inhibitory activity over CDK4 inhibition. The most selective molecules were examined in cytotoxicity assays and their ability to inhibit HIV-1 replication was determined in cellular assays.

Microbe-induced epigenetic alterations in host cells: the coming era of patho-epigenetics of microbial infections. A review.

It is well documented that the double-stranded DNA (dsDNA) genomes of certain viruses and the proviral genomes of retroviruses are regularly targeted by epigenetic regulatory mechanisms (DNA methylation, histone modifications, binding of regulatory proteins) in infected cells. In parallel, proteins encoded by viral genomes may affect the activity of a set of cellular promoters by interacting with the very same epigenetic regulatory machinery. This may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes (e.g. initiation and progression of malignant neoplasms; immunodeficiency). Bacteria infecting mammals may cause diseases in a similar manner, by causing hypermethylation of key cellular promoters at CpG dinucleotides (promoter silencing, e.g. by Campylobacter rectus in the placenta or by Helicobacter pylori in gastric mucosa). I suggest that in addition to viruses and bacteria, other microparasites (protozoa) as well as macroparasites (helminths, arthropods, fungi) may induce pathological changes by epigenetic reprogramming of host cells they are interacting with. Elucidation of the epigenetic consequences of microbe-host interactions (the emerging new field of patho-epigenetics) may have important therapeutic implications because epigenetic processes can be reverted and elimination of microbes inducing patho-epigenetic changes may prevent disease development.

Epigenotypes of latent herpesvirus genomes.

Epigenotypes are modified cellular or viral genotypes which differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Restricted expression of latent, episomal herpesvirus genomes is also due to epigenetic modifications. There is no virus production (lytic viral replication, associated with the expression of all viral genes) in tight latency. In vitro experiments demonstrated that DNA methylation could influence the activity of latent (and/or crucial lytic) promoters of prototype strains belonging to the three herpesvirus subfamilies (alpha-, beta-, and gamma-herpesviruses). In vivo, however, DNA methylation is not a major regulator of herpes simplex virus type 1 (HSV-1, a human alpha-herpesvirus) latent gene expression in neurons of infected mice. In these cells, the promoter/enhancer region of latency-associated transcripts (LATs) is enriched with acetyl histone H3, suggesting that histone modifications may control HSV-1 latency in terminally differentiated, quiescent neurons. Epstein-Barr virus (EBV, a human gamma-herpesvirus) is associated with a series of neoplasms. Latent, episomal EBV genomes are subject to host cell-dependent epigenetic modifications (DNA methylation, binding of proteins and protein complexes, histone modifications). The distinct viral epigenotypes are associated with distinct EBV latency types, i.e., cell type-specific usage of latent EBV promoters controlling the expression of latent, growth transformation-associated EBV genes. The contribution of major epigenetic mechanisms to the regulation of latent EBV promoters is variable. DNA methylation contributes to silencing of Wp and Cp (alternative promoters for transcripts coding for the nuclear antigens EBNA 1-6) and LMP1p, LMP2Ap, and LMP2Bp (promoters for transcripts encoding transmembrane proteins). DNA methylation does not control, however, Qp (a promoter for EBNA1 transcripts only) in lymphoblastoid cell lines (LCLs), although in vitro methylated Qp-reporter gene constructs are silenced. The invariably unmethylated Qp is probably switched off by binding of a repressor protein in LCLs.

Reversal of HIV drug resistance and novel strategies to curb HIV infection: the viral infectivity factor Vif as a target and tool of therapy.

Due to the high genetic variability of human immunodeficiency virus (HIV), treatment of AIDS (acquired immunodeficiency syndrome) patients with inhibitors of reverse trancriptase (RT) and drugs blocking the viral protease regularly results in the accumulation of drug resistant HIV variants and treatment failure. The sensitivity of clinically derived resistant HIV-1 strains to nucleotide RT inhibitors could be restored, however, in several laboratories by pharmacological depletion of the appropriate endogenous deoxynucleotide triphosphate (dNTP), and such a manipulation (induction of dCTP pool imbalance during reverse transcription in the presence of a non-nucleoside RT inhibitor) altered the mutation spectrum of the HIV-1 genome, resulting in a lower level of HIV resistance to certain drugs. The cytoplasmic single-stranded DNA cytidine deaminases APOBEC3G and APOBEC3F block HIV replication by introducing premature stop codons into the viral genome. We suggest that the resulting crippled, defective HIV (dHIV) variants could interfere with replication of "wild type" viruses and curbe disese progression in long term non-progressor individuals. Vif, an accessory protein encoded by HIV, counteracts APOBEC3G/F action. We speculate that small molecule inhibitors of Vif could permit lethal or sublethal mutagenesis of HIV genomes. We suggest that an artificial dHIV construct carrying a mutated vif gene (coding for a Vif protein unable to block APOBEC3G/F) could have a therapeutic effect as well in HIV infected individuals and AIDS patients.

Nef: a pleiotropic modulator of primate lentivirus infectivity and pathogenesis.

The lentiviral protein Nef recruits cellular signalling proteins to lipid rafts at the cell membrane and acts thereby as a master regulator affecting the transcription of a series of cellular genes. By activating resting T cells, Nef creates an optimal environment for lentivirus replication. In human immunodeficiency virus (HIV) infected macrophages and microglial cells Nef activates the production of T-cell attracting chemokines and contributes to the development HIV infection associated brain damage. Nef also functions as an adaptor or connector protein downregulating CD4 and CCR5, the key receptor and one of the coreceptors for HIV. It also downregulates cell surface expression of a subset of class I MHC molecules which contributes to viral immune evasion. Extracellular, soluble Nef may facilitate the spread of T-cell-tropic HIV variants and mediate a switch in dominant replicating HIV strains (from macrophage-tropic to T-cell-tropic viruses) in AIDS (acquired immunodeficiency syndrome) patients. Virion-bound Nef enhances infectivity. Nef is a potential target of antiretroviral therapy and nef-deleted (attenuated) retroviruses have been considered as candidate vaccines against HIV. We suggest that nef-deleted or highly mutated defective HIV (dHIV) genomes interfere with replication of "wild type" HIV in certain long-term non-progressor individuals. This implies that introduction of artificially constructed dHIV genomes (by infusion of leukocytes carrying dHIV proviruses) into HIV infected individuals could slow disease progression and could be considered as a therapeutic possibility.

Increased incidence of genetic human prion disease in Hungary.

The authors performed analysis of the prion protein gene (PRNP) in 27 out of 109 confirmed prion disease patients between 1994 and 2004. E200K mutation was found in 17 cases. Another 10 patients, lacking PRNP analysis, showed positive family history. The mean annual incidence (0.27/million) and proportion (25.6%) of genetic prion disease is unusually high in Hungary and might be related to the migration of ancestors from the Slovakian focus.

A 30 kb region of the Epstein-Barr virus genome is colinear with the rearranged human immunoglobulin gene loci: implications for a "ping-pong evolution" model for persisting viruses and their hosts. A review.

The left part of the Epstein-Barr virus (EBV) genome exhibits a strong colinearity of structural and functional elements with the immunoglobulin (Ig) gene loci which is only partially reflected in nucleotide sequence homologies. We propose that this colinearity may be the result of an inter-dependent co-evolution of the immunoglobulin loci together with EBV. Our observation could help elucidating the mechanisms of somatic hypermutation, explaining the ability of EBV to accidentally cause tumors, and shedding more light on the general mechanisms of viral and organismal evolution. We suggest that persisting viruses served as a complement for the organismal germline like in a ping-pong game and outline The Ping-Pong Evolution Hypothesis.

Protein-DNA interaction and CpG methylation at rep*/vIL-10p of latent Epstein-Barr virus genomes in lymphoid cell lines.

The viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the Epstein-Barr virus (EBV) genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes.

Epigenetics of latent Epstein-Barr virus genomes: high resolution methylation analysis of the bidirectional promoter region of latent membrane protein 1 and 2B genes.

We analysed the methylation patterns of CpG dinucleotides in a bidirectional promoter region (LRS, LMP 1 regulatory sequences) of latent Epstein-Barr virus (EBV) genomes using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. Transcripts for two latent membrane proteins, LMP 1 (a transforming protein) and LMP 2B, are initiated in this region in opposite directions. We found that B cell lines and a clone expressing LMP 1 carried EBV genomes with unmethylated or hypomethylated LRS, while highly methylated CpG dinucleotides were present at each position or at discrete sites and within hypermethylated regions in LMP 1 negative cells. Comparison of high resolution methylation maps suggests that CpG methylation-mediated direct interference with binding of nuclear factors LBF 2, 3, 7, AML1/LBF1, LBF5 and LBF6 or methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing of the LMP 1 promoter. Although a role for CpG methylation within binding sites of Sp1 and 3, ATF/CRE and a sis-inducible factor (SIF) cannot be excluded, hypermethylation of LRS or regions within LRS in LMP 1 negative cells suggests a role for an indirect mechanism, via methylcytosine binding proteins, in silencing of the LMP 1 promoter.

Protein-DNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp, and LMP1p of Epstein-Barr virus.

Epstein-Barr viral (EBV) latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five representative cell lines and correlated the results with the known in vitro binding data and activities of these promoters from previous transfection experiments. Promoter activity inversely correlated with the methylation state of promoters, although Qp was a remarkable exception. Novel protein binding data were obtained for all promoters. For Cp, binding correlated well with promoter activity; for LMP1p and Qp, binding patterns looked similar regardless of promoter activity.

Effect of simulated microgravity on human lymphocytes.

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).

Relative deficiency in CpG dinucleotides is a widespread but not unique feature of Gammaherpesvirinae genomes.

Since methylcytosine is relatively unstable, a deficiency of CpG dinucleotides and accumulation of mutations that manifest as TpG (and its complement CpA) is a diagnostic feature of higher eukaryotic DNA sequences subjected to methylation by DNA (cytosine-5) methyltransferases. Latent viral genomes may also be affected by DNA methylation in their host cells. We calculated, therefore, frequencies of dinucleotides in 20 completely sequenced herpesvirus genomes. We found a relative deficiency of CpG dinucleotides and a surplus of TpG + CpA dinucleotides in all lymphotropic gammaherpesvirus genomes except for two strains of rhesus rhadinovirus. DNAs of two strains of human herpesvirus 7, a betaherpesvirus targeting helper T cells, and equine herpesvirus 4, an alphaherpesvirus residing in the lymphoreticular system, also had a moderate CpG deficiency and TpG + CpA surplus. In contrast, most members of Alpha-, and Betaherpesvirinae subfamilies contained a relative surplus of CpG dinucleotides in their DNAs. Our data are consistent with the idea that methylated latent genomes are involved, after reactivation and productive replication, in the natural transmission cycle of most members of Gammaherpesvirinae and certain lymphotropic members of Alpha- and Betaherpesvirinae.

Genetic subtypes of HIV type 1 in Hungary.

We examined the diversity of HIV-1 subtypes in 11 adults from Hungary, using the heteroduplex mobility assay (HMA) and DNA sequencing. HMA results showed that HIV-1 gp120 sequences from 10 patients were of subtype B, whereas 1 patient, infected in Africa, carried a subtype C strain. DNA sequencing confirmed the HMA results and revealed a high intrasubtype diversity in the C2V3 region of env in different clade B isolates, which suggests multiple introduction of subtype B to Hungary. This study shows that subtype B is the predominant HIV-1 clade in Hungary.

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes.

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.

Strong correlation between the complement-mediated antibody-dependent enhancement of HIV-1 infection and plasma viral load.

OBJECTIVE: We have previously demonstrated that complement-mediated antibody-dependent enhancement (C-ADE) of HIV-1 infection correlates with accelerated immunosuppression and disease progression in HIV-1-infected individuals. In the present work the relationship between C-ADE and plasma HIV-1 RNA concentrations was studied to determine the effect of C-ADE on viral replication. METHODS: Three studies were performed: (a) C-ADE and HIV-1 RNA concentrations were determined in the serum and plasma aliquots taken at the same time from 98 HIV patients, mostly in the advanced stage of the disease; (b) the above two parameters as well as HIV enzyme-linked immunosorbent assay (ELISA)-reactive antibodies (Abbott HIV 1/2 test), and p24 antigen levels (Abbott antigen test; Abbott, Delkenheim, Germany) were determined in four seroconversion panels purchased from the Boston Biomedica firm; (c) changes of HIV-1 RNA concentration and C-ADE during a 17 month follow-up period were determined in 18 HIV-infected patients. C-ADE was measured by the method previously established in our laboratories. The results were expressed by an enhancement/neutralization index (E/NI). HIV-1 RNA levels were determined with the Amplicor monitor kit (Roche, Basel, Switzerland), and in some experiments with the nucleic acid sequence based amplification (Organon Teknika, Turnhout, Belgium) kits. RESULTS: (a) We found a highly significant (P<0.0001) positive correlation between E/NI values reflecting the extent of HIV-1 infection enhancement and plasma HIV-1 RNA levels. Both E/NI and HIV-1 RNA levels negatively correlated to the CD4 cell counts. (b) C-ADE was first detected just before, or concomitantly with, seroconversion in 4/4 seroconversion panels. (c) Both E/NI values and HIV-1 RNA levels significantly (P<0.001) increased during a 17 month observation period in 18 HIV-infected patients. CONCLUSION: We found strong association between the extent of the complement-mediated antibody-dependent enhancement of HIV-1 infection and the plasma viral load in HIV patients. On the basis of these findings, C-ADE correlates with HIV replication in vivo, and potentially contributes to the progression of HIV disease.

The effect of simulated microgravity conditions on the TNF-alpha production by human PBMCS.

Our earlier space experiments demonstrated that the interferon production of human lymphocytes in microgravity is 4-8 times higher than those of the synchronous ground controls in vitro (Talas et al. 1983). These data suggested that the microgravity has a significant effect on cells. Since the possibilities to perform space-experiments are very limited and our study raised many interesting questions, we wished to simulate microgravity conditions in our laboratory. For this reason we purchased a Rotary Cell Culture System (RCCS) equipment to study different cell lines and human peripheral blood mononuclear cells (PBMCs) in experimental microgravity conditions. RCCS is a horizontally rotated bubble free culture vessel with membrane diffusion gas exchange. We report here an analysis of TNF-alpha (tumor necrosis factor-alpha) production by human PBMCs (control cultures exposed to simulated microgravity in RCCS). The cells were incubated in the presence or absence of either NDV (Newcastle Disease Virus) or one of the different forms (PHA-M or -P) of Phytohaemagglutinin.

Analysis of methylation patterns in the regulatory region of the latent Epstein-Barr virus promoter BCR2 by automated fluorescent genomic sequencing.

We analyzed the methylation patterns of CpG dinucleotides in the regulatory region of the latent Epstein-Barrvirus (EBV) promoter BCR2 (also called C promoter, Cp) using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. BCR2 is one of the alternative promoters for transcripts encoding the growth-transformation-associated nuclear antigens EBNA 1-6 which are expressed in a host cell phenotype dependent manner. Well characterized clones isolated from the Burkitt's lymphoma (BL) line Mutu differing from each other as to their phenotype and EBV latent gene expression were used in the present study. We found that in Mutu BL III clone 99 which is actively using the BCR2 promoter the regulatory sequences are unmethylated with two exceptions (position 10702 and 10799). In contrast, there are 15 methylated cytosines in the same region in Mutu BL I clone 216 where the BCR2 promoter is silent. Cytosines which are potential targets of DNA methyltransferase in the immediate vicinity or within the attachment sites of cellular C promoter binding factors CBF1 and CBF2 remained hypomethylated in Mutu BL I clone 216. This suggests a role for a hypermethylated region (nucleotides 10666 -10865, -639 to -440 bases upstream from the beginning of the TATA box at position 11305) in silencing of the BCR2 promoter in these cells.

Plasma HIV-1 load and disease progression in HIV-infected patients in Hungary.

Nucleic Acid Sequence Based Amplification (NASBA) is a suitable method for the quantification of HIV-1 RNA in plasma and serum samples. Since determination of the viral load appears to be a valuable marker for the prediction of disease progression and for monitoring the efficiency of antiretroviral therapy, the National AIDS Committee initiated the introduction of NASBA in Hungary at the end of 1996. We obtained plasma samples from patients with ARC and AIDS of the Szt. László Hospital, Budapest. We found an increased viral burden in untreated AIDS (CDC group C) patients compared to untreated ARC (CDC group B) patients. In plasma samples of clinically stable ARC and AIDS patients treated with antiretroviral drugs we detected relatively low HIV-1 RNA copy levels while similarly treated ARC and AIDS patients with progressive disease had high HIV-1 RNA copy numbers. The CD4+ T-cell count was lower in AIDS patients compared to ARC patients, as expected. In general, there was an inverse correlation (r = -0.487, P < 0.0001) between CD4+ T-cell counts and HIV-1 RNA levels. We concluded that measurement of HIV-1 RNA plasma level has an important role in assessing prognosis and effects of antiretroviral therapy in HIV-infected patients.

S-methylthio-cysteine and cystamine are potent stimulators of thiol production and glutathione synthesis.

The effects of methylthio-cysteine disulfide (MT-Cy) and cystamine (CAM) on the thiol production and glutathione content of a human T cell line (CEM-SS) have been investigated. MT-Cy per se and CAM in the presence of cystine greatly enhanced thiol production and glutathione content of cells while cystine alone exerted no or slight influence in the first hours. The MT-Cy- or CAM-induced extracellular SH-generation was observed both in a complete nutrient medium and even more in SH-free D-PBS. The acid-soluble thiol level and glutathione content of cells elevated markedly (up to 5-6 fold in two hours) when incubating cells in complete medium. Inhibition of glutathione synthesis by DL-buthionine (S,R)-sulfoximine did not alter the MT-Cy- or CAM-induced extracellular thiol production indicating that glutathione synthesis is not involved in this effect. The results suggest that MT-Cy easily enters the cells thus accelerating the thiol cycle in SH-poor medium while CAM promotes cystine uptake into the cells. Phenylalanine and leucine inhibited both MT-Cy- and CAM-dependent thiol production in D-PBS most effectively suggesting the involvement of the L membrane transport system in these effects.