Aviva Lapidot

Metabolic pathways leading to liver glycogen repletion in vivo, studied by GC-MS and NMR

A quantitative analysis of the pathways leading to glycogen repletion in rats was conducted. [U‐13C]Glucose was administered intra‐intestinally into awake fasted animals. The distribution of glucose isotopomers derived from liver glycogen, liver extracts and plasma was performed by GC‐MS and 13C NMR. The potential gluconeogenic precursors for liver glycogen, lactate, alanine, glutamate and glutamine, were also analyzed. The amount of glycogen that is synthesized by the direct pathway was found to be 35%. The 13C enrichment of liver lactate, alanine and glucose is similar, indicating that they are the major precursors for liver glycogen synthesis via the indirect pathway. Our results demonstrate that after 24 h fasting, when glucose is supplied, gluconeogenesis from endogenous sources is not shut off.

Arginine‐aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat‐transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of l‐arginine with aminoglycosides. Using a gel‐shift assay and affinity chromatography on an l‐arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with K d values in the range of 20–400 nM, which is comparable to the K d of native Tat bound to the transactivation responsive element (10–12 nM). Confocal microscopy studies demonstrated that fluorescein‐labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates

The potential of RNAs and RNA–protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa‐arginine derivative of neomycin (NeoR) and a tri‐arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans‐activator protein (Tat) and the Tat‐responsive RNA (trans‐activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.

Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system.

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage λ DNA by EcoRI endonuclease at 0.4 mm and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mm. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mm. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mm THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.

Anti-Human Immunodeficiency Virus Activity of Novel Aminoglycoside-Arginine Conjugates at Early Stages of Infection

Conjugates of L-arginine with aminoglycosides have already been described as potent in vitro inhibitors of the HIV-1 Tat-trans-activation responsive element interaction. The polycationic nature of these agents leads us to suggest that they may be active against HIV-1 replication by inhibiting earlier stages of the virus life cycle. We have found that R4K and R3G, kanamycin A, and gentamicin C, conjugated with arginine, inhibited HIV-1 NL4-3 replication at EC50 values of 15 and 30 microM for R3G and R4K, respectively, without a detectable tonic effect on MT-4 cells at concentrations higher than 4000 and about 1000 microM, respectively. Both compounds inhibited the binding of a monoclonal antibody (12G5) directed to CXCR4 as well as the intracellular Ca2+ signal induced by the chemokine SDF-1alpha on CXCR4+ cells, suggesting that aminoglycoside-arginine conjugates interact with CXCR4, the coreceptor used by T-tropic, X4 strains of HIV-1. On the other hand, CB4K, a conjugate of kanamycin A with gamma-guanidinobutyric acid, structurally similar to R4K, failed to display any anti-HIV activity of CXCR4 antagonist activity. An HIV-1 strain that was made resistant to the known CXCR4 antagonist AMD3100 was cross-resistant to both R4K and R3G. However, unlike SDF-1alpha and R4K, R3G inhibited the binding of HIV-1 to MT-4 cells. Aminoglycoside-arginine conjugates inhibit HIV replication by interrupting the early phase of the virus life cycle, namely virus binding to CD4 cells and interaction with CXCR4. R3G and R4K may serve as prototypes of novel anti-HIV agents and should be further studied.

Multi-targeting the entrance door to block HIV-1.

The multistep nature of HIV-1 entry provides multisite targeting at the entrance door of HIV-1 to cells. Blocking HIV-1 entry to its host cells has clear advantages over blocking subsequent stages in the life cycle of the virus. Indeed, potent cooperative and synergistic inhibition of HIV-1 proliferation has been observed in in vitro studies with several entry inhibitor combinations, interacting with different steps of the HIV-1-cell entry cascade. Targeting a compound to several steps of the viral-cell entry and also to subsequent steps in the viral life cycle promises an even more effective therapeutic, by reducing the probability of HIV-1 to develop resistance. Using one drug that can target multiple sites and/or steps in the viral life cycle will have obvious advantages in clinical use. In this article we review the multistep process of HIV-1 cell entry and the current repertoire of inhibitors of this critical stage in the viral life cycle, and introduce an example of multisite HIV-1 targeting of the cell entry and subsequent critical steps in the viral life cycle.

Structure-activity relationships of aminoglycoside-arginine conjugates that bind HIV-1 RNAs as determined by fluorescence and NMR spectroscopy.

We present here a new set of aminoglycoside‐arginine conjugates (AACs) that are either site‐specific or per‐arginine conjugates of paromomycin, neamine, and neomycin B as well as their structure–activity relationships. Their binding constants (K D) for TAR and RRE RNAs, measured by fluorescence anisotropy, revealed dependence on the number and location of arginines in the different aminoglycoside conjugates. The binding affinity of the per‐arginine aminoglycosides to TAR is higher than to RRE, and hexa‐arginine neomycin B is the most potent binder (K D=5 and 23 nM, respectively). The 2D TOCSY NMR spectrum of the TAR monoarginine‐neomycin complex reveals binding at the bulge region of TAR.

Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine-containing peptide, to HIV TAR RNA in vitro

The ability of a small molecule, 2‐methyl,4‐carboxy,5‐hydroxy‐3,4,5,6‐tetrahydropyrimidine (THP(A)), which accumulates intracellularly in various streptomyces, to inhibit the interaction of Tat peptide (R52) with TAR RNA is presented. Using gel‐shift assay, we found that the inhibition constant K i of THP(A) is 50–100 nM, which is in the range of the binding constants of Tat peptide and protein. THP(A) is ∼ 106 times more tightly bound than the free l‐arginine. The high binding affinity may be attributed to the special delocalized positive charge on the NCN group and the hydroxyl group at the 5 position of this molecule. A model for THP(A)‐TAR interaction, analogous to the arginine guanidinum group‐TAR interaction, is presented. The relatively high uptake of THP(A) by mammalian cells warrants in vivo Tat/TAR inhibition studies.

Regulation of pool sizes and turnover rates of amino acids in humans: 15N-glycine and 15N-alanine single-dose experiments using gas chromatography—Mass spectrometry analysis☆

Gas chromatography--mass spectrometry (GCMS) of plasma amino acid derivatives has been used to determine directly the 15N-enrichment of plasma glycine and alanine in ten volunteers at various metabolic states. Isotope-enrichment time-decay curves of plasma glycine and alanine, following a single intravenous dose of 15N-glycine or 15N-L alanine were obtained and provide an estimate of the extracellular compartment. Relatively narrow ranges were obtained for the glycine pool (7.7--11.8 micromole/100 g body wt), rate constants of transport (3.7--4.2 hr-1) and flux (28--43 micromole hr-1/100 g body wt) in the postabsorptive statcine pool (7.7--11.8 mumole/100 g body wt), rate constants of transport (3.7--4.2 hr-1) and flux (28--43 mumole hr-1/100 g body wt) in the postabsorptive state. In postprandial humans, pool sizes showed only a modest variation whereas the rate constants of transport of glycine and alanine were significantly lower. The plasma 15N-glycine and 15N-alanine isotope-enrichment time-decay curves over the first hour following a single i.v. dose of 15-amino acid represent mostly the hepatic uptake of glycine and alanine from the extracellular pool. The results presented in this study establish the stable isotope GCMS method as a more accurate, more convenient, and safe alternative to the use of radioactive labeled amino acids in studies of amino acid metabolism in human subjects.

The Tat antagonist neomycin B hexa-arginine conjugate inhibits gp-120-induced death of human neuroblastoma cells.

Several patients with acquired immunodeficiency syndrome (AIDS) develop neurological complications, which are referred to as human immunodeficiency virus (HIV)‐associated dementia (HAD). The HIV‐1 coat glycoprotein gp‐120 has been proposed as the major etiologic agent for neuronal loss reported postmortem in the brain of AIDS patients. Chemokine receptors may play a role in gp‐120‐triggered neurotoxicity, both in vitro and in vivo, thus being an intriguing target for developing therapeutic strategies aimed to prevent or reduce neuronal damage occurring during HIV infection. We have previously shown that human CHP100 neuroblastoma cells express CXCR4 and CCR5 chemokine receptors and that interaction between gp‐120 and these receptors contributes to cytotoxicity elicited by the protein. Here, we examined the neuroprotective potential of neomycin B hexa‐arginine conjugate (NeoR), a recently synthesized compound with anti‐HIV activity. We found that gp‐120‐triggered death is significantly reduced by NeoR, and this protective effect seems related to the ability of NeoR to interact with CXCR4 receptors. The ability of NeoR to cross the blood–brain barrier, as demonstrated in mice by systemic administration of the fluorescein conjugate drug, makes this compound a powerful and attractive therapeutic agent.