Switching to Tenofovir Alafenamide Fumarate in Chronic Hepatitis B Patients Who Had Detectable HBV DNA during Treatment with Entecavir.

Nucleos(t)ide analogues (NAs) suppress hepatitis B virus (HBV) replication, but the risk of hepatocellular carcinoma still remains. The presence of detectable HBV DNA in the serum during NA therapies for chronic hepatitis B patients has been reported to be associated with the risk of hepatocellular carcinoma. In this study, we investigated the antiviral effect of switching from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in chronic hepatitis B patients who had detectable HBV DNA in the serum at least once within a year. Among a total of 77 cases in 7 hospitals that switched NAs from ETV to TAF, 23 patients with detectable HBV DNA in a year before switching were analyzed. When the detection frequencies of HBV DNA in the 1st and 2nd years after switching to TAF were analyzed, they were significantly lower than those in the year before switching (68.8% vs. 34.1% for the 1st year and 21.3% for the 2nd year, P < 0.001 for both). The HBsAg decline tended to be larger after switching than before (-2.5% vs. -3.0% for 1st year and -3.1% for 2nd year), but the difference was not significant. One patient died of a cardiovascular event 11 months after the treatment switch, but no adverse effects due to TAF including renal function were observed. In conclusion, it was suggested that switching from ETV to TAF might be effective to suppress the HBV DNA level further in patients whose HBV DNA is detectable, even if at a very low level.

Building better polymerases: Engineering the replication of expanded genetic alphabets.

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

5-Ethynyl-2'-deoxycytidine and 5-ethynyl-2'-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses.

Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.

Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs.

NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2'-fluoro-2'C-methylcytidine-5'-triphosphate and 2'-fluoro-2'C-methyluridine-5'-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2'-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.

Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR.

5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-ß Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.

Ribonucleoside-5'-diphosphates (NDPs) support RNA polymerase transcription, suggesting NDPs may have been substrates for primordial nucleic acid biosynthesis.

A better understanding of the structural basis for the preferences of RNA and DNA polymerases for nucleoside-5'-triphosphates (NTPs) could help define the catalytic mechanisms for nucleotidyl transfer during RNA and DNA synthesis and the origin of primordial nucleic acid biosynthesis. We show here that ribonucleoside-5'-diphosphates (NDPs) can be utilized as substrates by RNA polymerase (RNAP). We found that NDP incorporation is template-specific and that noncognate NDPs are not incorporated. Compared with the natural RNAP substrates, NTPs, the Km of RNAP for NDPs was increased ∼4-fold, whereas the Vmax was decreased ∼200-fold. These properties could be accounted for by molecular modeling of NTP/RNAP co-crystal structures. This finding suggested that the terminal phosphate residue in NTP (not present in NDP) is important for positioning the nucleotide for nucleolytic attack in the nucleotidyl transfer reaction. Strikingly, a mutational substitution of the active-center ßR1106 side chain involved in NTP positioning also strongly inhibited NDP-directed synthesis, even though this residue does not contact NDP. Substitutions in the structurally analogous side chain in RB69 DNA polymerase (Arg-482) and HIV reverse transcriptase (Lys-65) were previously observed to inhibit dNDP incorporation. The unexpected involvement of these residues suggests that they affect a step in catalysis common for nucleic acid polymerases. The substrate activity of NDPs with RNAP along with those reported for DNA polymerases reinforces the hypothesis that NDPs may have been used for nucleic acid biosynthesis by primordial enzymes, whose evolution then led to the use of the more complex triphosphate derivatives.

The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans.

The yeast Candida albicans is the most prevalent opportunistic fungal pathogen in humans. Drug resistance among C. albicans isolates poses a common challenge, and overcoming this resistance represents an unmet need in managing this common pathogen. Here, we investigated CDC8, encoding thymidylate kinase (TMPK), as a potential drug target for the management of C. albicans infections. We found that the region spanning amino acids 106-123, namely the Ca-loop of C. albicans TMPK (CaTMPK), contributes to the hyperactivity of this enzyme compared with the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, expression of CaTMPK, but not of hTMPK, produced dUTP/5-FdUTP-mediated DNA toxicity in budding yeast (Saccharomyces cerevisiae). CRISPR-mediated deletion of this Ca-loop in C. albicans revealed that the Ca-loop is critical for fungal growth and susceptibility to 5-fluorouridine (5-FUrd). Of note, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd, and we also found that CaTMPK is essential for the growth of C. albicans In conclusion, these findings not only identified a target site for the development of CaTMPK-selective drugs, but also revealed that 5-FUrd may have potential utility as drug for managing C. albicans infections.

2'-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase.

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

A computational approach for predicting off-target toxicity of antiviral ribonucleoside analogues to mitochondrial RNA polymerase.

In the development of antiviral drugs that target viral RNA-dependent RNA polymerases, off-target toxicity caused by the inhibition of the human mitochondrial RNA polymerase (POLRMT) is a major liability. Therefore, it is essential that all new ribonucleoside analogue drugs be accurately screened for POLRMT inhibition. A computational tool that can accurately predict NTP binding to POLRMT could assist in evaluating any potential toxicity and in designing possible salvaging strategies. Using the available crystal structure of POLRMT bound to an RNA transcript, here we created a model of POLRMT with an NTP molecule bound in the active site. Furthermore, we implemented a computational screening procedure that determines the relative binding free energy of an NTP analogue to POLRMT by free energy perturbation (FEP), i.e. a simulation in which the natural NTP molecule is slowly transformed into the analogue and back. In each direction, the transformation was performed over 40 ns of simulation on our IBM Blue Gene Q supercomputer. This procedure was validated across a panel of drugs for which experimental dissociation constants were available, showing that NTP relative binding free energies could be predicted to within 0.97 kcal/mol of the experimental values on average. These results demonstrate for the first time that free-energy simulation can be a useful tool for predicting binding affinities of NTP analogues to a polymerase. We expect that our model, together with similar models of viral polymerases, will be very useful in the screening and future design of NTP inhibitors of viral polymerases that have no mitochondrial toxicity.

Amino acid residues in HIV-2 reverse transcriptase that restrict the development of nucleoside analogue resistance through the excision pathway.

Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) are the backbone of current antiretroviral treatments. However, the emergence of viral resistance against NRTIs is a major threat to their therapeutic effectiveness. In HIV-1, NRTI resistance-associated mutations either reduce RT-mediated incorporation of NRTI triphosphates (discrimination mechanism) or confer an ATP-mediated nucleotide excision activity that removes the inhibitor from the 3' terminus of DNA primers, enabling further primer elongation (excision mechanism). In HIV-2, resistance to zidovudine (3'-azido-3'-deoxythymidine (AZT)) and other NRTIs is conferred by mutations affecting nucleotide discrimination. Mutations of the excision pathway such as M41L, D67N, K70R, or S215Y (known as thymidine-analogue resistance mutations (TAMs)) are rare in the virus from HIV-2-infected individuals. Here, we demonstrate that mutant M41L/D67N/K70R/S215Y HIV-2 RT lacks ATP-dependent excision activity, and recombinant virus containing this RT remains susceptible to AZT inhibition. Mutant HIV-2 RTs were tested for their ability to unblock and extend DNA primers terminated with AZT and other NRTIs, when complexed with RNA or DNA templates. Our results show that Met73 and, to a lesser extent, Ile75 suppress excision activity when TAMs are present in the HIV-2 RT. Interestingly, recombinant HIV-2 carrying a mutant D67N/K70R/M73K RT showed 10-fold decreased AZT susceptibility and increased rescue efficiency on AZT- or tenofovir-terminated primers, as compared with the double-mutant D67N/K70R. Molecular dynamics simulations reveal that Met73influences ß3-ß4 hairpin loop conformation, whereas its substitution affects hydrogen bond interactions at position 70, required for NRTI excision. Our work highlights critical HIV-2 RT residues impeding the development of excision-mediated NRTI resistance.

Concentration of 2'C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase.

Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virus Leishmania RNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2'-C-methyladenosine (2CMA) potently inhibited LRV1 in Leishmania guyanensis (Lgy) and Leishmania braziliensis, leading to viral eradication at concentrations above 10 µm Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation, and inhibition of the viral RNA-dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMA-TP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50 values ranging from 150 to 910 µm, depending on the CsCl density of the virion (empty, ssRNA-, and dsRNA-containing). Lgy parasites incubated in vitro with 10 µm 2CMA accumulated 2CMA-TP to 410 µm, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

Is HBV viral load at admission associated with development of acute-on-chronic liver failure in patients with acute decompensation of chronic hepatitis B related cirrhosis?

BACKGROUND: Hepatitis B virus (HBV) reactivation is one of the most common precipitating events associated with acute decompensation (AD) or acute-on-chronic liver failure (ACLF) in chronic hepatitis B (CHB)-related cirrhotic patients. However, whether their serum HBV deoxyribonucleic acid (DNA) levels are associated with ACLF incidence and short-term mortality rate is still ambiguous. METHODS: The ACLF incidences, 28-day and 90-day liver transplantation (LT)-free mortality rates, previous nucleoside/nucleotide analogues (NUCs) treatments and serum HBV DNA levels at admission (ad-levels) of 111 hospitalized patients with AD of CHB-related cirrhosis were analyzed. RESULTS: 43 (38.7%) patients developed ACLF. The 28-day and 90-day LT-free mortality rates of the ACLF cases were 15.4 and 40.9%, respectively. Though NUCs inhibited HBV replication effectively, there were no differences in the ACLF incidence between antiviral treatment-naïve patients and NUCs treatment-experienced patients with or without interruptions (37.5, 41.7 and 45.5%, respectively, P>0.05). The serum HBV DNA ad-level was similar between the patients with and without ACLF development (logarithms: 4.50 ± 1.96 vs 4.32 ± 1.99; ≥2000 IU/ml: 67.4% vs 67.6%; both P>0.05), so was between the ACLF patients died or survived in 28 or 90 days (logarithms: 4.31 ± 1.91 vs 5.54 ± 2.53, 4.81 ± 1.76 vs 4.84 ± 2.40, respectively, both P>0.05). CONCLUSION: Serum HBV DNA ad-level and previous NUCs treatment are not associated with incidence of ACLF and short-term mortality rate in the hospitalized patients with AD of CHB-related cirrhosis.

Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3.

Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.

Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase.

Eukaryotic class I ribonucleotide reductases (RRs) generate deoxyribonucleotides for DNA synthesis. Binding of dNTP effectors is coupled to the formation of active dimers and induces conformational changes in a short loop (loop 2) to regulate RR specificity among its nucleoside diphosphate substrates. Moreover, ATP and dATP bind at an additional allosteric site 40 Å away from loop 2 and thereby drive formation of activated or inactive hexamers, respectively. To better understand how dNTP binding influences specificity, activity, and oligomerization of human RR, we aligned >300 eukaryotic RR sequences to examine natural sequence variation in loop 2. We found that most amino acids in eukaryotic loop 2 were nearly invariant in this sample; however, two positions co-varied as nonconservative substitutions (N291G and P294K; human numbering). We also found that the individual N291G and P294K substitutions in human RR additively affect substrate specificity. The P294K substitution significantly impaired effector-induced oligomerization required for enzyme activity, and oligomerization was rescued in the N291G/P294K enzyme. None of the other mutants exhibited altered ATP-mediated hexamerization; however, certain combinations of loop 2 mutations and dNTP effectors perturbed ATP's role as an allosteric activator. Our results demonstrate that the observed compensatory covariation of amino acids in eukaryotic loop 2 is essential for its role in dNTP-induced dimerization. In contrast, defects in substrate specificity are not rescued in the double mutant, implying that functional sequence variation elsewhere in the protein is necessary. These findings yield insight into loop 2's roles in regulating RR specificity, allostery, and oligomerization.

Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine: IMPLICATIONS FOR THE PHARMACOLOGY OF ADENOSINE ANTIMETABOLITES.

Trypanosoma brucei causes African sleeping sickness for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mm deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP knockdown cells. Recombinant TbMTAP exhibited higher turnover number (kcat) and Km values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. For deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors.

A metal-containing nucleoside that possesses both therapeutic and diagnostic activity against cancer.

Nucleoside transport is an essential process that helps maintain the hyperproliferative state of most cancer cells. As such, it represents an important target for developing diagnostic and therapeutic agents that can effectively detect and treat cancer, respectively. This report describes the development of a metal-containing nucleoside designated Ir(III)-PPY nucleoside that displays both therapeutic and diagnostic properties against the human epidermal carcinoma cell line KB3-1. The cytotoxic effects of Ir(III)-PPY nucleoside are both time- and dose-dependent. Flow cytometry analyses validate that the nucleoside analog causes apoptosis by blocking cell cycle progression at G2/M. Fluorescent microscopy studies show rapid accumulation in the cytoplasm within 4 h. However, more significant accumulation is observed in the nucleus and mitochondria after 24 h. This localization is consistent with the ability of the metal-containing nucleoside to influence cell cycle progression at G2/M. Mitochondrial depletion is also observed after longer incubations (Δt ∼48 h), and this effect may produce additional cytotoxic effects. siRNA knockdown experiments demonstrate that the nucleoside transporter, hENT1, plays a key role in the cellular entry of Ir(III)-PPY nucleoside. Collectively, these data provide evidence for the development of a metal-containing nucleoside that functions as a combined therapeutic and diagnostic agent against cancer.

Evaluation of anti-HIV-1 mutagenic nucleoside analogues.

Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.

The crystal structure of the PB2 cap-binding domain of influenza B virus reveals a novel cap recognition mechanism.

The influenza RNA-dependent RNA polymerase is a core enzyme required for both transcription and replication of the virus RNA genome, making it a potential drug target for the influenza virus. To detect the feature of cap-dependent transcription of influenza B virus (FluB) polymerase, we determined the crystal structures of the wild-type FluB polymerase PB2 subunit cap-binding domain (PB2cap) with bound GDP and the mutant FluB Q325F PB2cap with bound m(7)GDP or GDP. These structures revealed that, distinct from influenza A virus (FluA) PB2cap, the guanine and ribose moieties of substrates invert in FluB PB2caps. Moreover, we characterized the substrate specificity and affinity of the PB2caps using isothermal titration calorimetry. FluB PB2cap has a weaker affinity for m(7)GDP than FluA PB2cap. Unlike FluA PB2cap that has a preference for m(7)GDP in comparison with GDP, FluB PB2cap shows an analogous affinity for both substrates. Replacement of FluB PB2 Glu(325) by Phe, the corresponding residue of FluA PB2, increased the binding affinity of FluB PB2cap for m(7)GDP to a level approximate to that of FluA PB2cap and caused a significant higher affinity to GDP. This study indicated that FluB PB2cap has a unique cap recognition mechanism compared with FluA PB2cap, providing molecular insight into inhibitor design targeting FluB PB2cap.

The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.