Silvio Spadari

Effect of aphidicolin on viral and human DNA polymerases

Abstract DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity in vivo . Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both in vitro and in vivo and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either in vitro on in vivo suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase δ assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N′-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

Replication protein A as a "fidelity clamp" for DNA polymerase alpha.

The current view of DNA replication in eukaryotes predicts that DNA polymerase α (pol α)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol α elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol α displays a low processivity and, because of the lack of an intrinsic or associated 3′→ 5′ exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol α-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol α playing a dual role: (i) it stabilizes the pol α/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol α. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a “fidelity clamp” for pol α.

Do DNA polymerases δ and α act coordinately as leading and lagging strand replicases

The activity ratio of DNA polymerases δ and α in calf thymus was found to be invariably 1:1, irrespective of extraction procedure (8 types) and subcellular localization (cytoplasm, nucleus and microsomes). This was established by separation of the two forms by hydroxyapatite chromatography and by their response to specific inhibitors and monoclonal antibodies. This finding supports the dimeric DNA polymerase model [(1980) J. Biol. Chem. 255, 4290–4303], which proposes that DNA polymerases δ and α act coordinately as leading and lagging strand enzymes, respectively, at the replication fork.

Synchronization of nuclear DNA synthesis in cultured Daucus carota L. cells by aphidicolin

By inhibiting the α‐like DNA polymerase, and therefore nuclear DNA synthesis, aphidicolin induces accumulation of suspension cultured carrot cells at the G1/S boundary of the cell cycle. After a 24‐h treatment with the drug the accumulation is complete, affecting all the cycling cells (95% of the population). Upon removal of the inhibitor, all cycling cells immediately resume nuclear DNA synthesis and move synchronously throughout the S‐phase.

Slow-, Tight-Binding HIV-1 Reverse Transcriptase Non-Nucleoside Inhibitors Highly Active against Drug-Resistant Mutants

The potent combination therapy in use for the treatment of infection with human immunodeficiency virus type 1 (HIV-1), known as highly active antiretroviral therapy (HAART), has produced sustained reductions in plasma HIV-1 RNA to levels below the limits of detection and has resulted in dramatic reduction in disease progression and mortality in the developed world. However, to maximize the benefits of treatment, suppression of virus replication must be continuous. Failure to completely suppress virus replication, inevitably leads to the selection of drug resistant variants that limits the long term success of antiretroviral therapy. Actually, three classes of antiretroviral drugs are available: protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTIs), and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Among them, the NNRTIs comprise a very large number of chemically different compounds that bind to a hydrophobic pocket on the HIV-1 RT. These compounds are attractive because of their high potency, low toxicity, and favorACHTUNGTRENNUNGable pharmacokinetic properties. Unfortunately, they rapidly select for the outgrowth of HIV-1 strains with mutations that alter the shape of the hydrophobic NNRTI-binding pocket. Resistance to one NNRTI is usually associated with cross-resistance to many other NNRTIs. For this reason the necessity to improve and expand the therapeutic arsenal is still pursued. Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs) are a potent class of NNRTIs developed in the last decade. The structure– activity relationship, supported by molecular modeling, suggested that the C2 substituent is a structural determinant necessary for the antiviral activity of these derivatives. The various DABOs synthesized to date are characterized by different C2side chains (S-DABOs and NH-DABOs, Figure 1).

Design and Synthesis of Phosphonoacetic Acid (PPA) Ester and Amide Bioisosters of Ribofuranosylnucleoside Diphosphates as Potential Ribonucleotide Reductase Inhibitors and Evaluation of Their Enzyme Inhibitory, Cytostatic and Antiviral Activity

Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonu-cleotides to deoxyribonucleotides, we have now prepared and evaluated 5′-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 μM for the UDP analogue 5′-deoxy-5′-N-(phosphon-acetyl)uridine sodium salt (amide) to 600 μM for the CDP analogue 5′-O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100–500 μM concentrations, whereas ADP analogue 5′-N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5′-deoxy-5′-N-(phos-phon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 μM) against HSV-2 and a modest inhibitory activity (EC50: 110 μM) against HIV-1, respectively.

Sensitivity of Monkey B Virus (Cercopithecine herpesvirus 1) to Antiviral Drugs: Role of Thymidine Kinase in Antiviral Activities of Substrate Analogs and Acyclonucleosides

ABSTRACT Herpes B virus (B virus [BV]) is a macaque herpesvirus that is occasionally transmitted to humans where it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to “activate” nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease.

Drug Resistance Mutations in the Nucleotide Binding Pocket of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Differentially Affect the Phosphorolysis-Dependent Primer Unblocking Activity in the Presence of Stavudine and Zidovudine and Its Inhibition by Efavirenz

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) derivatives with D113E, Y115F, F116Y, Q151E/N, and M184V mutations were studied for their phosphorolysis-mediated resistance to the nucleoside RT inhibitors (NRTIs) zidovudine and stavudine and for their inhibition by the nonnucleoside analogs (NNRTIs) efavirenz and nevirapine. The results presented here indicate that these single amino acid substitutions within the nucleotide binding pocket of the viral RT can independently affect different enzymatic properties, such as catalytic efficiency, drug binding, and phosphorolytic activity. Moreover, small local alterations of the physicochemical properties of the microenvironment around the active site can have profound effects on some NRTIs while hardly affecting other ones. In conclusion, even though different mutations within the nucleotide binding pocket of HIV-1 RT can result in a common phenotype (i.e., drug resistance), the molecular mechanisms underlying this phenotype can be very different. Moreover, the same mutation can give rise to different phenotypes depending on the nature of the substrates and/or inhibitors.

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin.Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.