Phosphonoacetate Research Articles

Combined In Situ and In Silico Studies of Guest Intercalation into the Layered Double Hydroxide [LiAl2(OH)6]X·yH2O

Phosphonoacetate (PAA), diethyl phosphonoacetate (DPA), and sulfoacetate (SAA) anions have been intercalated into the galleries of the layered double hydroxide (LDH) [LiAl2(OH)6·X]·yH2O (LiAl-X; X = Cl, NO3). X-ray diffraction (XRD), Fourier transform infrared spectroscopy, and elemental microanalysis confirmed the successful intercalation of the guest ions into the LDH. The guests could also be de-intercalated and recovered from the host intact. In situ XRD was used to probe the mechanisms of the reactions, and the intercalation of PAA proceeded via clear intermediate phases. In contrast, the SAA and DPA reactions did not show any intermediates, but the organic intercalates exhibited changes in their interlayer spacing as the reaction progressed. Molecular dynamics (MD) simulations were used to investigate the interlayer structure of the intercalation compounds. It was found that the intermediates observed in situ correspond to local energy minima in the MD simulations. MD can thus predict the course of ...

Synthetic tools for studying the chemical biology of InsP8.

To synthesise stabilised mimics of InsP8, the most phosphorylated inositol phosphate signalling molecule in Nature, we replaced its two diphosphate (PP) groups with either phosphonoacetate (PA) or methylenebisphosphonate (PCP) groups. Utility of the PA and PCP analogues was verified by structural and biochemical analyses of their interactions with enzymes of InsP8 metabolism.

Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2′-O-methyl oligoribonucleotides

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

Asymmetric synthesis of the highly potent anti-metastatic prostacyclin analogue cicaprost and its isomer isocicaprost.

An asymmetric synthesis of the anti-metastatic prostacyclin analogue cicaprost and a formal one of its isomer isocicaprost by a new route are described. A key step of these syntheses is the coupling of a chiral bicyclic C6-C14 ethynyl building block with a chiral C15-C21 omega-side chain amide building block with formation of the C14-C15 bond of the target molecules. A highly stereoselective reduction of the thereby obtained C6-C21 intermediate carrying a carbonyl group at C15 of the side chain was accomplished by the chiral oxazaborolidine method. The chiral phosphono acetate method was used for the highly stereoselective attachment of the alpha-side chain to the bicyclic C6-C21 intermediate carrying a carbonyl group at C6. Asymmetric syntheses of the bicyclic C6-C14 ethynyl building blocks were carried out starting from achiral bicyclic C6-C12 ketones by using the chiral lithium amide method. In the course of these syntheses, a new method for the introduction of an ethynyl group at the alpha-position of the carbonyl group of a ketone with formation of the corresponding homopropargylic alcohol was devised. Its key steps are an aldol reaction of the corresponding silyl enol ether with chloral and the elimination of a trichlorocarbinol derivative with formation of the ethynyl group. In addition, a new aldehyde to terminal alkyne transformation has been realized. Its key steps are the conversion of an aldehyde to the corresponding 1-alkenyl dimethylaminosulfoxonium salt and the elimination of the latter with a strong base. Two basically different routes have been followed for the synthesis of the enantiomerically pure C15-C21 omega-side chain amide building block. The first is based on the chiral oxazolidinone method and features a highly stereoselective alkylation of (4R)-N-acetyl-4-benzyloxazolidin-2-one, and the second encompasses a malonate synthesis of the racemic amide and its efficient preparative scale resolution by HPLC on a chiral stationary phase containing column.

Expression of human cytomegalovirus DNA polymerase in insect cells using baculovirus expression system: Purification and biochemical characterizations

Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr II-EcoRI DNA fragment with intact HCMV pol gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pol gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pol gene. Infection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 108 cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3′–5′ and 5′–3′ exonuclease activities. This enzyme is also sensitive to phosphono acetate.

α-Cl-α-Br-phosphonoacetic acid is a potent and selective inhibitor of Na +/Pi cotransport across renal cortical brush border membrane

We found that alpha-Cl-alpha-Br-phosphonoacetate (ClBrPAA) is a competitive, solute-specific inhibitor of Na+/Pi cotransport across renal cortical brush border membrane. Inhibition by ClBrPAA (Ki = 62 microM) is more than three times more effective than inhibition by phosphonoformate (PFA), the most potent Na+/Pi cotransport inhibitor known to date, and 26 times more effective than the parent compound, phosphonoacetate (PAA). These observations indicate that substitution of bromine and chlorine atoms at the alpha-carbon of PAA greatly enhances its efficacy as a competitive inhibitor of Na+/Pi cotransport. As ClBrPAA is much less inhibitory than PAA and PFA towards viral DNA polymerases and did not inhibit human alpha-DNA polymerase (ref. 10), the results also demonstrate that Na+/Pi cotransport inhibition can be dissociated from inhibition of DNA polymerases by phosphonocarboxylate compounds.

The glycoprotein C gene of herpes simplex virus 1 resident in clonal L cell lines manifests two regulatory domains conferring a dominant B and a subordinate γ2 regulation

Earlier studies have shown that late, γ 2 genes of herpes simplex virus 1 stably incorporated into the environment of the cell are regulated as β genes. For example, the induction of the intact gene specifying glycoprotein C (gC) resident in a clonal L cell line superinfected with a gC- virus was enhanced in the presence of inhibitory concentrations of phosphonoacetate (PAA), a viral DNA synthesis inhibitor. Moreover, the gene was induced by superinfection at the nonpermissive temperature with tsHA1, a temperature-sensitive (ts) DNA − virus mutant. In the viral genome, the gC gene is not expressed by the tsHA1 mutant at the nonpermissive temperature or by the mutant or wild-type virus at the permissive temperature in the presence of inhibitory concentrations of PAA. We report that expression of a truncated gC gene introduced in the same fashion and resident in a clonal L cell line was at least partially sensitive to PAA and was not induced by tsHA1 at the nonpermissive temperature. The gene was transcribed from a prokaryotic transcription initiation site in the plasmid. Induction of gene expression by superinfecting virus did not result in appreciable amplification of the gene. We conclude that gC, a γ 2 gene, contains two regulatory domains. The gene domain upstream from nucleotide +22 confers β-like regulation, whereas the gene domain downstream from +22 confers γ 2 regulation. In the gene resident in cells in culture the upstream regulatory domain is dominant, whereas the converse is true for the gene resident in the viral genome.

Synergistic inhibition of anatid herpesvirus replication by acyclovir and phosphonocompounds.

Plaque reduction assays were used to evaluate the inhibitory effects of acyclovir (ACV), phosphonoacetate (PAA), and phosphonoformate (PFA) with a plaque-purified isolate of anatid herpesvirus (AHV-ppc3). A plaque assay employing a liquid overlay medium was developed to facilitate the drug inhibition studies. From dose-response curves, the ID50 for PAA, PFA, and ACV were 20, 12, and 0.14 micrograms/ml, respectively. From data obtained from combination dose-response curves, dose isobolograms were prepared and used to determine the types of interactions between drug pairs. Whereas the interaction between PFA and PAA was additive, synergism occurred with ACV and either PAA or PFA. Drug-resistant mutants of AHV-ppc3 resistant to 8.0 micrograms/ml ACV, 250 micrograms/ml PAA, 180 micrograms/ml PFA or 6.0 micrograms/ml AHV, and 220 micrograms/ml PAA were isolated.

Genetic analysis of the susceptibility of mouse cytomegalovirus to acyclovir.

Eight independently derived mouse cytomegalovirus (MCMV) mutants resistant to acyclovir (ACV) were obtained by the sequential plating of wild-type virus in increasing concentrations of ACV. Results of complementation studies among these eight mutants suggest that all had mutations within the same or closely associated genes. A ninth MCMV mutant resistant to phosphonoacetate (PAA) derived by plating wild-type virus in the presence of 100 micrograms of PAA per ml displayed coresistance to ACV and was unable to complement any of the ACV-derived mutants. Recombination experiments among all combinations of the nine MCMV mutants were performed and supported the complementation data in that no recombination could be detected. Seven of the eight ACV-resistant mutants demonstrated cross-resistance to PAA and hypersensitivity to aphidicolin. The one mutant not coresistant to PAA was more susceptible to PAA than was the parent virus. Only a few mutants demonstrated coresistance when the mutants were tested against 9-beta-D-arabinofuranosyladenine (ara-A). The ACV mutant that demonstrated increased susceptibility to PAA was 30-fold more susceptible to ara-A but remained unchanged in susceptibility to aphidicolin. Two of the parent-mutant combinations were selected for DNA synthesis analysis in the presence of ACV (5 microM). A significant decrease in DNA synthesis was demonstrated for both parent viruses, and there was little effect on mutant virus DNA synthesis at the same drug concentration. These results suggest that susceptibility of MCMV to ACV is confined to a product of a single gene and that a mutation of this gene can lead to an altered phenotype when compared with parent virus in susceptibility of DNA synthesis to PAA, ara-A, and aphidicolin, drugs that are known to inhibit DNA polymerase activity.

HSV1-specific thymidylate kinase activity in infected cells.

Several 5-methoxymethyldeoxyuridine (MMdU)-resistant mutants of herpes simplex virus type 1 (HSV1) were classified by measuring their sensitivities to the deoxythymidine kinase (dTK)-dependent antiviral drugs 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV), 1-beta-D-arabinofuranosylthymine (araT), and E-(2)-5-bromovinyldeoxyuridine (BVdU) and to the dTK-independent antiviral drug phosphonoacetate (PAA). Compared to wild-type (WT) virus, all five of the dTK- mutants were highly resistant (greater than or equal to 500-fold) to BVdU and MMdU, moderately resistant to ACV (50- to 100-fold) and araT (10- to 20-fold), but not resistant to PAA. The dTK of the mutant MMdUr-20 (dTK+) appeared to phosphorylate dTMP less well than that of the WT virus, while its affinity for deoxythymidine was not altered. Two other drug-resistant HSV mutants-S1 (isolated against ACV) and B3 (isolated against BVdU)--also showed reduced phosphorylation of dTMP. This suggests that alterations in both dTK and thymidylate kinase activities may determine sensitivity to antiviral drugs.

Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.

In vitro antiviral effect of 9-(2-hydroxyethoxymethyl) guanine on the fish herpesvirus, Oncorhynchus masou virus (OMV)

The antiviral activity of 9-(2-hydroxyethoxymethyl) guanine (Acyclovir, ACV) on the salmon herpesvirus, Oncorhynchus masou virus (OMV), was studied in vitro. ACV showed high efficacy against the fish herpesvirus OMV, Herpesvirus salmonis and channel catfish virus (CCV). Cytopathic effect (CPE) induced by 100 TCID 50/ml of OMV in rainbow trout gonad (RTG-2) cells was inhibited by 2.5 μg/ml of ACV. ACV was more effective than other compounds such as 9-β- d-arabinofuranosyladenine (Ara-A), 5-iodo-2′-deoxyuridine (IUdR) and phosphonoacetate (PA). Growth of RTG-2 cells was considerably inhibited by ACV at 25 μg/ml, but no morphological changes were observed in the cells. Replication of OMV in RTG-2 cells inoculated with 100 TCID 50/ml was completely suppressed by 2.5 μg/ml of ACV. Addition of ACV within 4 days post infection was effective in reducing OMV replication. In order to be effective, ACV had to be present continuously.

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.

Effect of acyclovir combined with other antiherpetic agents on varicella zoster virus in vitro

The sensitivity of varicella zoster virus (VZV) strain Ellen to acyclovir in combination with other antiherpetic agents in vitro has been examined by the plaque-reduction and infectious center assay methods. (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU), trifluorothymidine (TFT), or 5-iodo-2′-deoxyuridine (IdUrd) produced, in general, additive antiviral activity when examined by either assay method. Acyclovir in combination with phosphonoacetate (PAA) resulted in additive effects as measured by the plaque-reduction assay. The combination of acyclovir with 9-β-D-arabinofuranosyladenine (ara-A) gave generally additive or occassionally synergistic antiviral activity when tested in MRC-5 or WI-38 cells respectively, regardless of assay method.

5-Methoxymethyldeoxyuridine-resistant mutants of herpes simplex virus type 1.

Abstract 5-Methoxymethyldeoxyuridine (MMdU)-resistant (MMdU r ) mutants of herpes simplex virus type 1 (HSV-1) were isolated by a single passage of the virus in MMdU at 100 μg/ml (3.7 × 10 −4 M ). The isolates were cloned and passed in the presence of MMdU at 5 μg/ml (0.18 × 10 −4 M ). All MMdU r mutants showed considerable cross-resistance to the nucleoside analogs acycloguanosine (ACG), (E)-5-bromovinyldeoxyuridine (BVdU), and arabinosylthymine (araT), but were as sensitive as the parental strain to phosphonoacetate (PAA). One mutant, MMdU r -20, induced significant deoxythymidine kinase (dTK) activity. Because it was only 30 times more resistant to BVdU than the parental wild-type virus, while mutants MMdU r -2 and -12-3 were about 10,000 times more resistant, it was suspected that the mutation was in the dTK locus. All three mutants, however, showed a similar pattern of resistance to the other nucleoside analogs ACG, MMdU, and araT. These results suggest differences in the active sites for PAA and nucleoside analogs (with respect to the viral DNA polymerase), and also among the nucleoside analogs (with respect to the viral dTK).

Identification of target antigen for antibody-dependent cellular cytotoxicity on cells carrying Epstein-Barr virus genome.

The target antigen for antibody-dependent-cellular cytotoxicity (ADCC) on Epstein-Barr virus-(EBV) carrying lymphoblastoid cells expressing EBV-specific membrane antigen (MA) were examined with human serum antibody and adult human peripheral lymphocytes as effector cells. These studies confirmed that anti-MA-positive but not MA-negative sera were reactive in the ADCC. The ADCC reaction was positive with cells in which the MA consisted of late (LMA) and early (EMA) components. These included 1) MA-positive cells prepared by EBV antigen-adsorption, 2) cells carrying de novo-synthesized MA without adsorbed MA, and 3) EBV-producer cells expressing MA spontaneously. In all these preparations, the target cells were lysed roughly in parallel with the frequency of MA-positive cells. Inhibition of LMA synthesis in EBV-superinfected cells by phosphonoacetate (PA) reduced ADCC sensitivity significantly and to a far greater extent than MA synthesis as measured by immunofluorescence. This suggests that a target for ADCC is the PA-sensitive LMA. No ADCC reaction occurred with the cell preparation comprised of a high percentage of MA-positive cells induced by 5-iodo-2'-deoxyuridine, which is believed to be EMA only. These results strongly suggest that the target antigen for ADCC in EBV-positive cells is a late MA but not early MA.

Inhibition of hepatitis-B-virus DNA polymerase by phosphonoformate: Studies on its mode of action

Phosphonoformate (PFA) and phosphonoacetate (PAA) were tested for their ability to inhibit the hepatitis-B-virus associated DNA polymerase. The HBV DNA polymerase was inhibited by 100 microM/liter PFA 50% while it was highly resistant to PAA. The inhibition of the Dane particle-associated DNA polymerase by PFA was not competitive to substrates and not affected by changes in the magnesium concentration. PFA was active also after initiation of the DNA polymerase reaction. Competition studies revealed that PFA had a higher affinity to a proposed pyrophosphate binding site than PAA or--alternatively--that both compounds bind to different sites.

Characteristics of herpesvirus mutants resistant to phosphonoformate and phosphonoacetate.

Mutants of herpes simplex virus type 1, resistant to phosphonoformate (PFA) and phosphonoacetate (PAA), have been selected in cell culture. The PFA-resistant mutant (HSV-PFA(r)) and the PAA-resistant mutant (HSV-PAA(r)) were also resistant to PAA and PFA, respectively. This cross-resistance indicates that PFA and PAA interact at the same site. The HSV-PFA(r) had a decreased susceptibility to vidarabine, but no difference in sensitivity to idoxuridine (5-iodo-2'-deoxyuridine) was observed when compared to the wild type. The induced deoxyribonucleic acid polymerases isolated from cells infected with HSV-PFA(r) and HSV-PAA(r) were both cross-resistant to inhibition by PFA and PAA. No increased resistance to vidarabine triphosphate could be observed, but the susceptibility to inhibition by pyrophosphate was about two times lower. None of the mutants showed any increased temperature sensitivity

Effect of Disodium Phosphonoacetate and Iododeoxyuridine on the Multiplication of African Swine Fever Virus <i>in vitro</i>

Disodium phosphonoacetate (PAA) was found to inhibit the replication of African swine fever virus (ASFV). The action of this compound has been compared with the inhibitory capacity of iododeoxyuridine (IDU) upon ASFV growing in Vero cells. The study was done by the immunofluorescence technique in order to detect formations of cytoplasmic virus antigens and inclusion bodies; both were found to be inhibited by IDU and PAA. At 100 microgram/ml, IDU blocked completely the multiplication of ASFV and with PAA, a few scattered cells showed positive fluorescence. The infectivity of the virus was reduced 1--5 log depending upon drug concentrations and time of exposure to the drugs. Inhibition of ASFV replication by PAA suggests that this virus, like other herpesviruses, involves a virus-specific DNA polymerase in its replication mechanism.

Sensitivity of the Transforming and Replicative Functions of Epstein-Barr Virus to Inhibition by Phosphonoacetate

Disodium, phosphonoacetate (PA), at concentrations of 50 to 200 microgram/ml, which still allowed continued growth of the EB virus-transformed B95-8 cell line on a routine culture regimen, was able to inhibit the production of virus capsid antigen and of virus particles by these cells down to very low but finite levels which persisted despite prolonged treatment. Further experiments measured the effects of these same drug concentrations on the EB virus-induced in vitro transformation of foetal cord blood lymphocytes and on the colony forming ability of already established EB virus-transformed foetal cell lines; in both types of culture, doses of PA up to and including 50 microgram/ml did not affect cell growth within the 8-week observation period, whereas doses of 100 microgram/ml and above were increasingly inhibitory. The cell lines established by EB virus-induced transformation in the continual presence of PA at 50 to 150 microgram/ml contained multiple copies of the virus genome per cell just as did the corresponding cell lines established in control medium. The results argue against the existence of any PA-sensitive event unique to the EB virus-induced transformation process.