Karine Alvarez

The flavivirus polymerase as a target for drug discovery

Flaviviruses are emerging pathogens of increasingly important public health concern in the world. For most flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) neither vaccine nor antiviral treatment is available. The viral RNA-dependent RNA polymerase (RdRp) non-structural protein 5 (NS5) has no equivalent in the host cell and is essential for viral replication. Here, we give an overview of the current knowledge regarding Flavivirus RdRp function and structure as it represents an attractive target for drug design. Flavivirus RdRp exhibits primer-independent activity, thus initiating RNA synthesis de novo. Following initiation, a conformational change must occur to allow the elongation process. Structure-function studies of Flavivirus RdRp are now facilitated by the crystal structures of DENV (serotype 3) and WNV RdRp domains. Both adopt a classic viral RdRp fold and present a closed pre-initiation conformation. The so-called priming loop is thought to provide the initiation platform stabilizing the de novo initiation complex. A zinc-ion binding site at the hinge between two subdomains might be involved in opening up the RdRp structure towards a conformation for elongation. Using two different programs we predicted common potential allosteric inhibitor binding sites on both structures. We also review ongoing approaches of in vitro and cell-based screening programs aiming at the discovery of nucleosidic and non-nucleosidic inhibitors targeting Flavivirus RdRps.

Coronavirus Nonstructural Protein 16 Is a Cap-0 Binding Enzyme Possessing (Nucleoside-2′O)-Methyltransferase Activity

ABSTRACT The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-l-methionine (AdoMet)-dependent RNA (nucleoside-2′O)-methyltransferase (2′O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap (7MeGpppAC3-6) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2′O position of the first transcribed nucleotide, thus converting 7MeGpppAC3-6 into 7MeGpppA2′OMeC3-6. The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2′O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2′O methylation follows an order of events in which 2′O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.

The Y181C Substitution in 3′-Azido-3′-deoxythymidine-resistant Human Immunodeficiency Virus, Type 1, Reverse Transcriptase Suppresses the ATP-mediated Repair of the 3′-Azido-3′-deoxythymidine 5′-Monophosphate-terminated Primer

Resistance to zidovudine (3′-azido-3′-deoxythymidine, AZT) by the human immunodeficiency virus, type 1, requires multiple amino acid substitutions such as D67N/K70R/T215F/K219Q in the viral reverse transcriptase (RT). In this background of AZT resistance, additional “suppressive” substitutions such as Y181C restore sensitivity to AZT. In order to characterize the mechanism of this AZT resistance suppression, the Y181C substitution was introduced into both wild-type and AZT-resistant reverse transcriptase. The introduction of the Y181C substitution suppresses the increased repair (or unblocking) of the AZTMP-terminated primer provided by the AZT resistance substitutions in RT using either DNA or RNA templates, independently from the RT RNase H activity. Contrary to wild-type RT, the low level of unblocking activity is not due to inhibition by the next correct nucleotide binding to the RT/AZTMP-terminated primer complex. When Y181C is added to the AZT resistance substitutions, ATP binds with less affinity to the AZTMP-terminated primer-RT binary complex. These results provide an insight into one possible molecular mechanism of re-sensitization of AZT-resistant viruses by suppressive substitutions.

High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, 7MeG5′-ppp5′-A2′OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and 7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppACn and 7MeGpppACn (1 ≤ n ≤ 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 ≤ n ≤ 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

Dengue virus replicons: production of an interserotypic chimera and cell lines from different species, and establishment of a cell-based fluorescent assay to screen inhibitors, validated by the evaluation of ribavirin's activity.

The prevention and treatment of flavivirus infections are public health priorities. Dengue fever is the most prevalent mosquito-borne viral disease of humans, affecting more than 50 million people annually. Despite the urgent need to control dengue infections, neither specific antiviral therapies nor licensed vaccines exist and the molecular basis of dengue pathogenesis is not well understood. In this study we produced a novel dengue virus type 2 (DV2) subgenomic replicon that expresses a fusion protein comprised of Enhanced Green Fluorescent Protein (EGFP) and Puromycin N-Acetyltransferase (PAC). We successfully established BHK, COS and Huh7 cell lines that stably expressed the DV2 replicon. Using EGFP as a reporter of DV replication complex activity, we set up a new HTS assay. The assay was validated using the inhibitor ribavirin, confirmed by flow cytometry analysis and the analysis of NS5 expression by Western-blot analysis. In order to develop a system to test antivirals against the NS5 proteins of all four DV serotypes in a similar cellular environment, the replicon was further modified, to allow easy exchange of the NS5 gene between DV serotypes. As proof of principle, a chimeric replicon in which the DV2 NS5 gene was substituted with that of DV type 3 was stably expressed in BHK cells and used in ribavirin inhibition studies. The assays described in this study will greatly facilitate DV drug discovery by serving as primary or complementary screening. The approach should be applicable to the development of fluorescent cell-based HTS assays for other flaviviruses, and useful for the study of many aspects of DV, including viral replication and pathogenesis.

Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase

Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR around hit compound 1 as well as the synthesis and antiviral evaluation of 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole and 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole analogues derived from a rapid and easily accessible chemical pathway. A large number of compounds prepared by this method were shown to possess in vitro activity against the polymerase of dengue virus. The most potent inhibitors were tested against Dengue virus clinical isolates on infected cells model and exhibit submicromolar activity on the four dengue virus serotypes.

In Vitro Suppression of K65R Reverse Transcriptase-Mediated Tenofovir- and Adefovir-5′-Diphosphate Resistance Conferred by the Boranophosphonate Derivatives

ABSTRACT 9-[2-(Boranophosphonomethoxy)ethyl]adenine diphosphate (BH3-PMEApp) and (R)-9-[2-(boranophosphonomethoxy)propyl]adenine diphosphate (BH3-PMPApp), described here, represent the first nucleoside phosphonates modified on their α-phosphates that act as efficient substrates for the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). These analogues were synthesized and evaluated for their in vitro activity against wild-type (WT), K65R, and R72A RTs. BH3-PMEApp and BH3-PMPApp exhibit the same inhibition properties as their nonborane analogues on WT RT. However, K65R RT was found hypersensitive to BH3-PMEApp and as sensitive as WT RT to BH3-PMPApp. Moreover, the presence of the borane group restores incorporation of the analogue by R72A HIV RT, the latter being nearly inactive with regular nucleotides. The BH3-mediated suppression of HIV-1 RT resistance, formerly described with nucleoside 5′-(α-p-borano)-triphosphate analogues, is thus also conserved at the phosphonate level. The present results show that an α-phosphate modification is also possible and interesting for phosphonate analogues, a result that might find application in the search for a means to control HIV RT-mediated drug resistance.

Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position.

Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The α-borano or α-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the α-phosphate, resulting in substrate bad positioning.

Prooligonucleotides exhibit less serum-protein binding than phosphodiester and phosphorothioate oligonucleotides.

Abstract The protein-binding properties of dodecathymidine derivatives (prooligos) bearing either methyl- or tert-butyl-S-acylthioethyl (Me- or tBuSATE) protecting groups were evaluated. The dissociation constants (Kd) were estimated for complexes of prooligos with serum blood proteins and lactoferrin using prooligos to compete the binding of the radiolabeled, alkylating probe oligonucleotide CIRp(T)12 with the proteins. tBuSATE and MeSATE prooligos have decreased affinity of binding with serum proteins and lactoferrin compared with their parent oligos. These data suggest that prooligos should cause less side effects which combined with their stability to nucleases and enhanced permeability into cells make them potentially useful for design of therapeutics.

Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: effect of the α-phosphorus configuration on HIV-1 RT activity.

The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to determine the absolute configuration at the α-phosphorus atom in relation to the stereo-preference of studied enzymes. The RP isomer of S-PMPApp and S-PMEApp are the preferred substrate over SP for both NDPK and HIV-1 RT.