Evaluation of Combinations of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine with Clinically Used Antiretroviral Drugs

Drug combination studies of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) with FDA-approved drugs were evaluated by two different methods, MacSynergy II and CalcuSyn. Most of the combinations, including the combination of the two adenosine analogs EFdA and tenofovir, were essentially additive, without substantial antagonism or synergism. The combination of EFdA and rilpivirine showed apparent synergism. These studies provide information that may be useful for the design of EFdA combination regimens for initial and salvage therapy assessment.     

Pharmacokinetics of 4′-cyano-2′-deoxyguanosine,a novel nucleoside analog inhibitor of the resistant hepatitis B virus,in a rat model of chronic kidney disease

IntroductionThe novel nucleoside analog, 4′-cyano-2′-deoxyguanosine (CdG), possesses inhibitory activity against both the wild-type and resistant hepatitis B virus. Since the dosage of the currently available nucleoside analog preparations needs to be adjusted, depending on renal function, we investigated the effect of renal dysfunction on the pharmacokinetics of CdG in a rat model of chronic kidney disease (CKD).MethodsCKD model rats were either intravenously or orally administered CdG at a dose of 1 mg/kg. The concentration of CdG in plasma, organs (liver and kidney) and urine samples were determined by means of a UPLC system interfaced with a TOF-MS system.ResultsFollowing intravenous administration, the plasma retention of CdG was prolonged in CKD model rats compared to healthy rats. In addition, the clearance of CdG was well correlated with plasma creatinine levels in CKD model rats. Similar to the results for intravenous administration, the plasma concentration profiles of CdG after oral administration were also found to be much higher in CKD model rats than in healthy rats. However, the results for the organ distribution and urinary excretion of CdG, the profiles of which were similar to that of healthy rats, indicated that CdG did not accumulate to a significant extent in the body.ConclusionThe extent of renal dysfunction has a direct influence on the pharmacokinetics (plasma retention) of CdG without a significant accumulation, indicating that the dosage of CdG will be dependent on the extent of renal function. .     

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β-l-2′,3′-Didehydro-2′,3′-Dideoxy-N4-Hydroxycytidine: Relevance for Activation in HepG2 Cells

ß-l-2′,3′-Didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM; 50% cytotoxic dose [CD₅₀] = 2,500 μM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[³H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5′-mono-, 5′-di-, and 5′-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ß-d-N⁴-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5′-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 μM l-[³H]Hyd4C for 24 h, the 5′-triphosphate accumulated to 19.4 ± 2.7 pmol/10⁶ cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/10⁶ cells. The intracellular half-life of the 5′-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5′-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5′-diphosphate during a drug-free incubation. The high 5′-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.A large number of nucleoside analogues have been described as inhibitors of hepatitis B virus (HBV) and HIV replication. Recently l-nucleoside analogues in particular have gained increasing interest. They are characterized by an opposite configuration from that of the natural d-nucleoside analogues and represent one of the most attractive groups of antiretroviral compounds, including ß-l-2′,3′-dideoxy-3-thiacytidine (3TC) and its 5-fluoro derivative (FTC), ß-l-2′,3′-didehydro-2′,3′-dideoxy-cytidine (l-d4C) and its 5-fluoro derivative (l-d4FC), ß-l-thymidine, ß-l-fluoroarabinosylyluracil (l-FMAU), and ß-l-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-cytidine (l-2′Fd4C) (3, 5, 22).Some of them not only have been found to be more potent than their corresponding d-nucleosides but seem to exhibit lower cytotoxicity and have been proved to be effective and selective agents for the treatment of chronic hepatitis B virus infections (4). However, only long-term therapy with a single nucleoside for several years was shown to be able to completely suppress HBV DNA in serum of patients and to reverse the progression of the disease. The disadvantage connected with such therapy regimens is the development of drug-resistant HBV strains (22). Therefore, the challenge will be to develop more-efficient drugs for shorter treatment regimens and to combine them to reach synergistic or at least additive drug action. This approach has been described not only as being highly efficient for the treatment of HIV infections but also as preventing the development of resistant mutants. Therefore, AIDS therapy is considered a model for future therapy of chronic HBV infections (17).Recently we described a series of new ß-l-N⁴-hydroxydeoxycytidine and ß-l-5-methyl-deoxycytidine derivatives as inhibitors of HBV replication. Between them, ß-l-2′,3′-didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) (Fig. ​(Fig.1)1) emerged as the most effective in suppression of virus production in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM), displaying an extremely low cytotoxicity (50% cytotoxic dose [CD₅₀] for HepG2 cells = 2,500 μM) (12).Open in a separate windowFIG. 1.Structure of l-Hyd4C and possible metabolites formed by reduction (l-d4C) or by deamination (l-d4U).These encouraging features have prompted us to investigate the cellular pharmacology of l-Hyd4C in a hepatic cell line. This included the activation of this unnatural l-deoxycytidine nucleoside to its 5′-mono-, 5′-di-, and 5′-triphosphate, the search for other metabolites, and the estimation of the intracellular half-lives (t_1/2) of the 5′-di- and 5′-triphosphate of l-Hyd4C.(This work was presented in part at BIT''s 5th Anniversary Congress of International Drug Discovery Science and Technology, 7 to 13 November 2007, Xi''an and Beijing, China.)     

A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca²⁺. This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3′,4′-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.     

Metabolism in Human Cells of the d and l Enantiomers of the Carbocyclic Analog of 2′-Deoxyguanosine: Substrate Activity with Deoxycytidine Kinase,Mitochondrial Deoxyguanosine Kinase,and 5′-Nucleotidase

The carbocyclic analog of 2′-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the l enantiomer rather than, as expected, with the d enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5′-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the l enantiomer was the better substrate. Even though the d enantiomer was the preferred substrate with 5′-nucleotidase, the rate of phosphorylation of the l enantiomer was substantial. The phosphorylation of d-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of d-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5′-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. d-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of d-CdG and related analogs and in the design of new biologically active analogs.d-CdG is an analog of CdG that has broad-spectrum antiviral activity (27–29). Until recently, the biological activity of a nucleoside analog was assumed to be due only to the “natural” β-d form. However, there are now numerous observations of antiviral activity associated with nucleosides in l configurations (for reviews, see references 10 and 26) and one report of antitumor activity associated with a β-l enantiomer (11). Even though l-CdG is much less active against HSV (4) and HCMV (unpublished results), it is essential that the metabolism of both enantiomers be examined to thoroughly understand the mechanism of action of a nucleoside analog. We have reported earlier that both enantiomers of CdG are extensively phosphorylated in cells infected with HSV-1 and that the enantiomers had equal activities as substrates for the virus-encoded kinase (4, 5). It was also observed that d-CdG was converted to its triphosphate (but to a much lesser extent) in noninfected cells and that more than one enzyme appeared to be involved in the initial phosphorylation. Since cellular enzymes presumably are responsible for the activation of CdG in cells infected with HBV (which is not known to code for a kinase) and may also be important for the activation of CdG in cells infected with HCMV (which induces cellular kinases [6, 8, 21, 23, 32, 44], in addition to encoding a ganciclovir-phosphorylating enzyme [22, 35]), we have evaluated the enantiomers of CdG as substrates for the three cellular enzymes known to catalyze phosphorylation of deoxyguanosine (2): dCyd kinase, mitochondrial dGuo kinase, and 5′-nucleotidase. We also report here observations on the metabolism of d-CdG in cells infected with HCMV and in duck hepatocytes infected with DHBV.(Some of these results have been presented elsewhere in preliminary form [1].)     

PSI-7851, a Pronucleotide of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine Monophosphate,Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine-5′-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ± 0.050 μM (mean ± standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 μM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.Hepatitis C virus (HCV) currently affects more than 170 million people worldwide. Approximately 70% of infected individuals develop chronic hepatitis, among whom about 20% will develop liver cirrhosis and fibrosis and up to 5% will progress to hepatocellular carcinoma (2). The current standard of care (SOC), which combines pegylated alpha interferon (PegIFN-α) and ribavirin (RBV), has limited efficacy in providing a sustained virological response (SVR), especially in individuals with HCV genotype 1 (∼50%), the most prevalent genotype in Western countries (8, 11, 35). The impact of genetic diversity of HCV in patients receiving SOC therapy has been reviewed (26): SVR rates are higher in patients infected with genotype 2 or 3 (∼80%), patients infected with genotype 4 appear to have a slightly better SVR rate (∼60%) than patients infected with genotype 1, and patients infected with genotypes 5 and 6 may achieve an SVR at a level between those of genotypes 1 and 2/3. In addition to the variability in efficacy, the lengthy treatment (24 to 48 weeks) with SOC is frequently associated with undesirable side effects that may include anemia, fatigue, and depression (7). There is an urgent medical need to develop anti-HCV therapies that are safer and more effective. Direct-acting antivirals (DAAs) are compounds that target a specific viral protein. Currently, four major classes of DAAs are being investigated in phase II or III clinical trials: NS3 protease inhibitors, NS5A inhibitors, allosteric nonnucleoside NS5B polymerase inhibitors, and nucleoside/-tide NS5B polymerase inhibitors (21, 27, 46). Challenges for these DAAs include safety, pan-genotypic activity, and/or emergence of resistant viruses. An effective antiviral therapy against hepatitis C should encompass a broad spectrum of activity against all HCV genotypes, shorten treatment duration, have minimal side effects, and have a high barrier to resistance.The HCV NS5B RNA-dependent RNA polymerase (Pol) is a critical component of the replicase complex and is responsible for initiating and catalyzing viral RNA synthesis (16, 32, 58). There is no human homolog of this protein, and it is absolutely required for viral infectivity (19). As a result, the HCV NS5B is an attractive target for the development of antiviral compounds. There are two major classes of NS5B inhibitors: nucleoside analogs, which are anabolized to their active triphosphates and act as alternative substrates for the polymerase, and nonnucleoside inhibitors (NNIs), which bind to allosteric regions on the protein. Two major drawbacks associated with NNIs are that the activity appears to vary significantly among different HCV genotypes and even subtypes (15, 33) and that there is a relatively low barrier for resistance as evidenced by the numerous naturally occurring resistant variants reported in the literature (18). In contrast, nucleoside analogs are similarly active across HCV genotypes (13, 15, 33) and have a higher barrier of resistance compared to the NNIs and NS3 protease inhibitors (36). To date only two amino acid changes within the NS5B polymerase that confer resistance to nucleoside inhibitors have been identified: S96T and S282T (1, 29). The S96T mutation confers resistance to 4′-azidocytidine (R1479), while the S282T mutation is resistant to a number of 2′-C-methyl-modified nucleoside inhibitors (1, 29, 38, 43).In order for nucleoside analogs to be active as alternative substrates, they must first be phosphorylated by cellular kinases to their corresponding 5′-triphosphates, which are active alternative substrate inhibitors for the NS5B polymerase. The efficiency of these metabolic steps, the stability of the triphosphates, and the affinity of the triphosphates for the NS5B polymerase are all important factors in determining the antiviral activities of nucleoside inhibitors. PSI-6130, 2′-F-2′-C-methylcytidine, was previously shown to be a specific inhibitor of HCV RNA replication in the replicon assay system (52). However, when the uridine analog, 2′-F-2′-C-methyluridine (referred to as PSI-6206), was tested in the replicon assay, it failed to inhibit HCV RNA synthesis due to the inability of cellular enzymes to metabolize PSI-6206 to its triphosphate, PSI-7409 (5, 34, 42). Biochemical studies with PSI-7409 showed that this compound was able to inhibit RNA synthesis mediated by the HCV replicase complex and by purified recombinant HCV NS5B polymerase (34, 42). Furthermore, in vitro stability studies using primary human hepatocytes demonstrated that PSI-7409 has a significantly longer half-life (t_1/2, 38 h) than PSI-6130-TP (t_1/2, 4.7 h), which could be a desirable pharmacologic benefit (34).In order to bypass the initial nonproductive phosphorylation step of PSI-6206, the phosphoramidate prodrug methodology was explored as an approach to deliver 2′-F-2′-C-methyluridine monophosphate (47, 48). An extensive series of phosphoramidate prodrugs were synthesized, and PSI-7851 demonstrated the desired characteristics with regard to activity and in vitro toxicity. Herein we present the results of in vitro studies characterizing PSI-7851, a potent and specific anti-HCV compound with pan-genotype activity.     

Inhibition of Herpesvirus Replication by 5-Substituted 4′-Thiopyrimidine Nucleosides

A series of 4′-thionucleosides were synthesized and evaluated for activities against orthopoxviruses and herpesviruses. We reported previously that one analog, 5-iodo-4′-thio-2′-deoxyuridine (4′-thioIDU), exhibits good activity both in vitro and in vivo against two orthopoxviruses. This compound also has good activity in cell culture against many of the herpesviruses. It inhibited the replication of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus with 50% effective concentrations (EC₅₀s) of 0.1, 0.5, and 2 μM, respectively. It also inhibited the replication of human cytomegalovirus (HCMV) with an EC₅₀ of 5.9 μM but did not selectively inhibit Epstein-Barr virus, human herpesvirus 6, or human herpesvirus 8. While acyclovir-resistant strains of HSV-1 and HSV-2 were comparatively resistant to 4′-thioIDU, it retained modest activity (EC₅₀s of 4 to 12 μM) against these strains. Some ganciclovir-resistant strains of HCMV also exhibited reduced susceptibilities to the compound, which appeared to be related to the specific mutations in the DNA polymerase, consistent with the observed incorporation of the compound into viral DNA. The activity of 4′-thioIDU was also evaluated using mice infected intranasally with the MS strain of HSV-2. Although there was no decrease in final mortality rates, the mean length of survival after inoculation increased significantly (P < 0.05) for all animals receiving 4′-thioIDU. The findings from the studies presented here suggest that 4′-thioIDU is a good inhibitor of some herpesviruses, as well as orthopoxviruses, and this class of compounds warrants further study as a therapy for infections with these viruses.Iterative medicinal chemistry studies can optimize molecules for the inhibition of viral enzymes and yield molecules that are highly effective and remarkably specific. This approach, as well as others, has led to the development of highly selective therapies for the treatment of viral infections and has been reviewed previously (5, 9, 20). While these therapeutic agents have excellent activities against the target enzymes from specific viruses, they can have reduced or poor efficacy against other closely related viruses. Thus, it is important to assess the activities of candidate molecules against an array of viruses to determine their spectra of activity. The development of compounds with activities against many viruses is challenging, yet essential, given the vast array of viral pathogens that cause human disease. A broad spectrum of activity is particularly important in the development of therapies for infections caused by emerging pathogens.Nucleoside phosphonate antiviral agents have been approved for the treatment of infections with hepatitis B virus and human immunodeficiency virus and are under development for infections caused by hepatitis C virus and DNA viruses (6). Cidofovir (CDV) is one such compound that has activity against many viruses, and recently, alkoxyalkyl ester prodrug forms of CDV have been reported to be active at concentrations up to 3 orders of magnitude lower than the active concentrations of the parent compound (36). The compound CMX001 (hexadecyloxypropyl CDV) has excellent activity against adenoviruses, orthopoxviruses, and polyomaviruses, as well as the herpesviruses (13, 18, 32, 36). Equally important is the increased oral bioavailability conferred by the alkoxyalkyl substituent (1, 3, 17, 30), which appears to reduce drug exposure in the mouse kidney and may reduce the potential of the drug for renal toxicity (4). While the discovery of truly broad spectrum antiviral drugs will be difficult, the development of CMX001 represents a significant advance.Previously, the efficacies of a series of nucleoside analogs against vaccinia and cowpox viruses were evaluated and a number of thymidine analogs that have good activities against these viruses were identified (8, 26). This result was intriguing since the orthopoxviruses encode thymidine kinase (TK) homologs that are closely related to the human cytosolic TK (TK1) (15). The TK homologs encoded by the orthopoxviruses and the human cytosolic TK are both type II TK homologs, which are characterized by a number of features including their homotetrameric structures and their comparatively narrow substrate specificities (2). Some herpesviruses encode distinct type I TK enzymes, which are active as dimers and have broader substrate specificities that include thymidine, cytidine, and even some purine analogs such as acyclovir (ACV) (16). Results from genetic studies with orthopoxvirus and herpesvirus TK mutants suggested that these enzymes could confer sensitivity to a few thymidine analogs, presumably through selective phosphorylation to the level of the monophosphate (26, 29). This result was confirmed subsequently by the findings of enzymatic studies, which showed that the orthopoxvirus and herpesvirus TK enzymes share the ability to catalyze the phosphorylation of certain thymidine analogs, some of which are not substrates for the cellular cytosolic TK (25). This group of analogs includes a few 2′-deoxyuridine analogs with large substituents in the 5 position (25). The carbocyclic analog N-methanocarbathymidine has also been reported previously to exhibit good activity against both the orthopoxvirus and herpesvirus families (26) and is selectively phosphorylated by each of the TK homologs encoded by these viruses (data not presented). The unexpected finding that orthopoxvirus TK homologs possess broader substrate specificities than the human cytosolic TK homolog was interesting and suggested a potential avenue for the development of therapies for infections with these viruses. Importantly, the overlapping specificities of vaccinia virus TK and herpes simplex virus (HSV) TK also suggested that it might be possible to identify additional thymidine analogs with spectra of activity that included both the orthopoxviruses and some of the human herpesviruses (HHVs). Compounds with this property would be desirable since therapies for herpesvirus infections are more viable economically than those for orthopoxvirus infections, which may help drive the continued development of these compounds. Ideally, they should also retain activity against drug-resistant strains of these viruses.A series of 2′-deoxy-4′-thiopyrimidine nucleosides were synthesized previously and reported to be active against human cytomegalovirus (HCMV) (33). Other related analogs identified by Rahim and colleagues were also reported to have good activities against varicella-zoster virus (VZV), as well as HSV (31). One analog in this series, 5-iodo-4′-thio-2′-deoxyuridine (4′-thioIDU), also exhibited activity against HCMV. Recently, we described the antiviral activities of this and related molecules against vaccinia virus and cowpox virus (19). The most active analog was 4′-thioIDU, which inhibited viral replication in vitro at submicromolar concentrations. This compound also significantly reduced the mortality of mice infected with cowpox virus when administered orally at concentrations of 5 mg/kg of body weight and when therapy was initiated as late as 4 days after infection. The activity of the compound was shown to be largely dependent on the orthopoxvirus TK homologs, so we examined the activities of this and related compounds against HSV-2 and confirmed that 4′-thioIDU had the highest activity. The spectrum of activity of this compound also included HSV-1, VZV, and cytomegalovirus, but selective activity against the other HHVs was not observed. Immunofluorescence studies showed that the compound was specifically phosphorylated in infected cells, incorporated into viral DNA, and dependent on viral TK activity. However, in contrast to previous results that indicated good in vivo efficacy against two orthopoxviruses, the findings of the present study revealed that the compound did not significantly reduce the mortality of mice infected intranasally with HSV-2.     

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3′-end primer region on real-time PCR using the 5′-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A–C, C–A, T–G, G–T) to severe impact (>7.0 cycle threshold, eg, A–A, G–A, A–G, C–C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.During the past decade, real-time polymerase chain reaction (PCR) has established itself as an essential technique for reliable detection and quantification of nucleic acids.^1,2,3 The result is a widespread application of real-time PCR assays in both research^1,2,3,4 and diagnostic^4,5,6 laboratories. Vital to the specificity, sensitivity, and efficiency of real-time PCR are the primers. The most important primer characteristics contributing to a successful amplification are primer-template association and dissociation kinetics, possible secondary structures, and primer-template complementarity (Watson-Crick base-pairing).^7,8 Full complementarity between primer and template sequences is generally considered crucial for the specific amplification of a nucleic acid sequence, but can be difficult to achieve, in particular for applications depending on highly heterogenic nucleic acid input for amplification (eg, diagnostic assays for influenza virus and human immunodeficiency virus). Conserved regions are often too small to accommodate a typical real-time PCR assay (50 to 150 bp), exhibit inferior G-C contents or are prone to the formation of secondary structures. Primer-template mismatches can therefore be unavoidable.Unfortunately, mismatches between primers and template are known to affect both the stability of the primer-template duplex and the efficiency with which the polymerase extends the primer,^{7,8,9,10,11,12,13} potentially leading to biased results or even PCR failure.^14,15 Even apparently small effects on nucleic acid quantification (0.5 to 1.0 log underestimation of initial copy number) can have serious consequences, as illustrated by studies on the relation between viral load and disease prognosis in HIV-1.¹⁶The detrimental effects of primer-template mismatches can however also prove beneficial. They provide a discriminative force that can be used for PCR assays opting to distinguish between different nucleic acids (eg, single nucleotide polymorphism detection, allele-specific PCR), which have become important tools for modern molecular diagnostics.⁴Every mismatch, irrespective of its location within the primer sequence, will result in a decreased thermal stability of the primer-template duplex, thus potentially affecting PCR specificity. However, mismatches located in the 3′ end region (defined as the last 5 nucleotides of the 3′ end region) of a primer have significantly larger effects on priming efficiency than more 5′ located mismatches,^{9,11,13,14,15} since 3′ end mismatches can disrupt the nearby polymerase active site.^17,18Strategies to alter mismatch impact, eg, degenerate/modified bases or extensive adaptation of PCR conditions, can prove helpful in specific situations, but these strategies often require a lot of time-consuming optimization and can result in unwanted side effects (eg, increased primer-dimer formation). Quantitative data on the effects of 3′ end mismatches is necessary to improve knowledge and reliable prediction of mismatch behavior, which is beneficial for the development and optimization of real-time PCR assays involving mismatches.Several studies on the effects of 3′ end primer-template mismatches have been published.^{9,10,19,20,21,22} However, only few systematically examined the behavior of 3′ end primer-template mismatches (including the relationship between these effects and the position of the mismatch) using modern quantitative methods. In this study, we comprehensively investigate the effects of 3′ end primer-template mismatches using different commercially available 5′-nuclease assay master mixes. Diagnostic laboratories often employ such optimized pre-mixed reagents, which are generally directly used with few adaptations. Our approach therefore provides a relevant system for quantification of mismatch impact on diagnostic real-time PCR assays. Our experiments resulted in a large quantitative dataset from which different aspects of mismatch effects on PCR amplification were further analyzed, ultimately leading to the formulation of a set of general guidelines for improved prediction of primer-template mismatch impact.     

Activities of Certain 5-Substituted 4′-Thiopyrimidine Nucleosides against Orthopoxvirus Infections

As part of a program to identify new compounds that have activity against orthopoxviruses, a number of 4′-thionucleosides were synthesized and evaluated for their efficacies against vaccinia and cowpox viruses. Seven compounds that were active at about 1 μM against both viruses in human cells but that did not have significant toxicity were identified. The 5-iodo analog, 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-5-iodouracil (4′-thioIDU), was selected as a representative molecule; and this compound also inhibited viral DNA synthesis at less than 1 μM but only partially inhibited the replication of a recombinant vaccinia virus that lacked a thymidine kinase. This compound retained complete activity against cidofovir- and ST-246-resistant mutants. To determine if this analog had activity in an animal model, mice were infected intranasally with vaccinia or cowpox virus and treatment with 4′-thioIDU was given intraperitoneally or orally twice daily at 50, 15, 5, or 1.5 mg/kg of body weight beginning at 24 to 120 h postinfection and was continued for 5 days. Almost complete protection (87%) was observed when treatment with 1.5 mg/kg was begun at 72 h postinfection, and significant protection (73%) was still obtained when treatment with 5 mg/kg was initiated at 96 h. Virus titers in the liver, spleen, and kidney were reduced by about 4 log₁₀ units and about 2 log₁₀ units in mice infected with vaccinia virus and cowpox virus, respectively. These results indicate that 4′-thioIDU is a potent, nontoxic inhibitor of orthopoxvirus replication in cell culture and experimental animal infections and suggest that it may have potential for use in the treatment of orthopoxvirus infections in animals and humans.The potential use of orthopoxviruses as weapons of bioterrorism and the fact that one member of this group, monkeypox virus, is indigenous in West and Central Africa and has also been introduced through zoonotic spread into the United States have prompted the search for effective and nontoxic antiviral agents for the treatment of orthopoxvirus infections in both animals and humans. Since there is perceived to be little financial reward for the development of a new agent for the treatment of these infections, initial investigations focused on the development of agents that were already approved for use for another indication, such as herpes, AIDS, or hepatitis. One of these agents, cidofovir (CDV), which is approved for use for the treatment of cytomegalovirus retinitis in human immunodeficiency virus-infected patients, is very active in tissue culture cells against all of the orthopoxviruses, including variola virus (2, 7, 8, 9, 13, 15), and has been shown to be highly effective for the treatment of animals experimentally infected with vaccinia, cowpox, ectromelia, and monkeypox viruses (4, 5, 10, 19, 26, 27, 33, 34). Although CDV is approved for use for the emergency treatment of smallpox and complications of vaccination under an experimental Investigational New Drug application, its lack of oral bioavailability somewhat limits its use in a large orthopoxvirus outbreak.In order to obtain a compound that retains the activity of CDV but that can be administered orally, a series of analogs was synthesized by attaching a long-chain alkoxyalkanol to CDV. One of these compounds, hexadecyloxypropyl CDV (CMX001), was about 100-fold more active than CDV against vaccinia virus and cowpox virus (15, 16). A similar level of enhanced activity of CMX001 against variola and monkeypox viruses has been reported (12, 15). In mice infected with vaccinia, cowpox, or ectromelia virus, CMX001 given orally as either a single dose or multiple doses was at least as effective as CDV, if not more so, in preventing mortality and reducing viral replication in target organs (6, 21, 28). This compound is currently in phase I/II clinical studies for the treatment of orthopoxvirus and herpesvirus infections.A second compound that is also in phase I/II clinical studies is a low-molecular-weight compound, ST-246, which has potent activity in vitro against all of the orthopoxviruses against which it has been tested (11, 29, 40) and which has been reported to be highly effective when it is given orally in preventing mortality or disease in mice infected with vaccinia, cowpox, or ectromelia virus (29, 40). The compound has a mechanism of action different from that of CDV, and its activity is restricted to poxviruses.Numerous nucleoside analogs have been reported to be active against vaccinia virus (7). We have evaluated most of the antiviral agents that have been licensed for other uses for their activities against orthopoxviruses (15) and found that CDV, idoxuridine (IDU), and trifluridine have significant activities in vitro without being overtly toxic. The fact that the mechanism of action of IDU against herpes simplex virus involves phosphorylation of the drug by the virus-encoded UL23 thymidine kinase (TK) was of special interest to us, as we have reported that this enzyme is involved in the selective activation of certain inhibitors of orthopoxvirus replication (23-25). In previous studies by Secrist et al. (32) and Rahim and colleagues (31), a series of 2′-deoxy-4′-thiopyrimidine nucleosides were synthesized, and a number of these analogs had significant activity predominantly against the alphaherpesviruses. We subsequently found that one of these analogs, 1-(2′-deoxy-4′-thio-β-d-ribofuranosyl)-5-iodouracil (4′-thioIDU; compound 5 in Fig. ​Fig.1),1), also has potent activity in vitro against vaccinia and cowpox viruses. Subsequently, we synthesized and evaluated 16 other analogs for their activities against orthopoxvirus infections.Open in a separate windowFIG. 1.Structures of 5-substituted 4′-thiopyrimidine nucleosides.The purpose of the studies reported in this communication was to synthesize and evaluate a number of compounds in this series for their activities against vaccinia and cowpox viruses in vitro, investigate their mechanisms of action, determine if they retain their activities against other drug-resistant mutants, and compare their activities against vaccinia and cowpox virus infections in mice to the activity of CDV.     

Characterization of MRSA transmission in an emergency medical center by sequence analysis of the 3′-end region of the coagulase gene

The distribution of methicillin-resistant Staphylococcus aureus (MRSA) isolates at the St. Marianna University affiliated emergency medical center (EMC) was studied by sequence analysis of the 3′-end region of the coagulase gene. We collected a total of 42 MRSA isolates, consisting of 20 strains from the hospital environment, 13 strains from the nostrils or fingers of medical staff, and 9 strains from inpatients in the EMC. We compared our results with those from 27 stock strains of known coagulase serotype and 2 strains reported in the literature. All 69 strains tested have four to six tandem repeats in the 3′-end region of the coagulase gene. Among the 42 MRSA isolates collected, the base sequence of the 3′-end region of the coagulase gene was identical in 28 of them (67%). The number of isolates originating from the hospital environment, medical staff, and patients, respectively, that were identical to this representative strain were 18 (90%), 6 (46%), and 4 (44%). Phylogenetic analysis using the DNA sequences of the tandem repeat region demonstrated that almost all strains from the patients formed a concordant cluster with the representative strain from the hospital ward. We also assessed the value of sequence analysis of the 3′-end region of the coagulase gene as an epidemiological marker. Our results indicate that sequence analysis of the 3′-end region of the coagulase gene of MRSA may be a potent epidemiologic typing system. Received: July 25, 2000 / Accepted: October 27, 2000     

The Extragenic Spacer Length Between the 5′ and 3′ Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors

In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.