CD8 cis-targeted IL-2 drives potent antiviral activity against hepatitis B virus.

CD8+ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8+ T cell dysfunction showed that interleukin-2 (IL-2)-based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2's effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (Treg) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8+ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8+ T cells in the liver without substantially altering Treg or NK cell counts. These expanded CD8+ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen-positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8+ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8+ T cells without affecting NK or Treg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.

SAR studies on dihydropyrimidinone antibiotics.

There is an urgent need for the development of novel antimicrobial agents that offer effective treatment against MRSA. Using a new class of dipeptide antibiotic TAN-1057A/B as lead, we designed, synthesized and evaluated analogs of TAN-1057A/B. Several novel dihydropyrimidinone antibiotics demonstrating comparable antibiotic efficacy while possessing favorable selectivity were identified.

Phenotypic susceptibility to bevirimat in isolates from HIV-1-infected patients without prior exposure to bevirimat.

Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir.

Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 A of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT-DNA complex at a resolution of 3.1 A with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.

Biochemical characterization of rhinovirus RNA-dependent RNA polymerase.

Human rhinoviruses (HRV) represent the single most important causative agent of the common cold. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome. We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus. The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E. coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity. The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined. The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism. A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme. In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity. However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template. Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation. The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical. The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold.

Trends in HIV-1 reverse transcriptase resistance-associated mutations and antiretroviral prescription data from 2003-2010.

BACKGROUND: The selection of antiretroviral (ARV) drugs for treatment of HIV-1 infection is based on several factors including potency, toxicity, resistance and ease of administration. Emtricitabine (FTC) or lamivudine (3TC), components of recommended initial ARV regimens, are structurally related and share the same resistance mutation (M184V/I). However they differ with respect to potency and incidence of M184V/I. METHODS: Resistance-associated mutation (RAM) prevalence data were obtained from genotype test results performed in a large reference laboratory from 2003-2010; subsets of data were defined by mutation pattern to resemble those following failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based combination therapy. Mutational trend data were compared to contemporaneous ARV prescription information. RESULTS: In the unfiltered data set (n=107,231), the prevalence in 2010 decreased compared to 2003 for all nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs, such as M184V/I (44.0% to 17.9%), T215Y (22.7% to 4.1%), and K65R (4.3% to 2.1%). Among samples resembling those typical of first-line NNRTI-based failures, prevalence of K103N increased slightly, but prevalence of M184V/I decreased (49.8% to 36.8%), as did other NRTI RAMs. These decreases were coincident with a shift in ARV prescriptions away from zidovudine and 3TC towards tenofovir and FTC, and an increase in use of fixed-dose combinations. CONCLUSIONS: RAM prevalence decreased substantially since 2003 among samples submitted for resistance testing in the US. The causes of this decrease are multifactorial, but our results suggest a possible role of increased use of potent ARVs that are available as fixed-dose combinations or as single-tablet regimens.

Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131.

GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4'-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-2'-fluoro-1'-furanyladenine, respectively} are novel alkylalaninyl phenyl ester prodrugs of tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV) and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2'-fluoro-2', 3'-dideoxydidehydroadenosine), respectively. Both prodrugs exhibit potent antiretroviral activity against both wild-type and drug-resistant human immunodeficiency virus type 1 strains and excellent in vivo pharmacokinetic properties. In this study, the main enzymatic activity responsible for the initial step in the intracellular activation of GS-7340 and GS-9131 was isolated from human peripheral blood mononuclear cells and identified as lysosomal carboxypeptidase A (cathepsin A [CatA]; EC 3.4.16.5). Biochemical properties of the purified hydrolase (native complex and catalytic subunit molecular masses of 100 and 29 kDa, respectively; isoelectric point [pI] of 5.5) matched those of CatA. Recombinant CatA and the isolated prodrug hydrolase displayed identical susceptibilities to inhibitors and identical substrate preferences towards a panel of tenofovir phosphonoamidate prodrugs. Incubation of both enzymes with 14C-labeled GS-7340 or [3H]difluorophosphonate resulted in the covalent labeling of identical 29-kDa catalytic subunits. Finally, following a 4-h incubation with GS-7340 and GS-9131, the intracellular concentrations of prodrug metabolites detected in CatA-negative fibroblasts were approximately 7.5- and 3-fold lower, respectively, than those detected in normal control fibroblasts. Collectively, these data demonstrate the key role of CatA in the intracellular activation of nucleotide phosphonoamidate prodrugs and open new possibilities for further improvement of this important class of antiviral prodrugs.

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin.

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery.

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.

Combinations of adefovir with nucleoside analogs produce additive antiviral effects against hepatitis B virus in vitro.

Combination therapies may be required for long-term management of some patients chronically infected with hepatitis B virus (HBV). Adefovir is a nucleotide analog that has similar activity against wild-type and lamivudine-resistant HBV. In contrast to lamivudine, clinical resistance to the prodrug adefovir dipivoxil emerges infrequently. Based on its clinical efficacy and low frequency of resistance, adefovir dipivoxil may form an important component of combination regimens. We therefore investigated the in vitro antiviral efficacy of combinations of adefovir with other nucleoside analogs (lamivudine, entecavir, emtricitabine [FTC],and telbivudine [L-dT]) and the nucleotide analog tenofovir. Using a novel stable cell line that expresses high levels of wild-type HBV, we assayed the antiviral activity of each drug alone and in combination with adefovir. All two-drug combinations resulted in greater antiviral effects than treatments with single agents and could be characterized as additive by the Bliss independence model. Analysis using the Loewe additivity model indicated that adefovir exerted additive antiviral effects when combined with lamivudine, FTC, or L-dT and moderately synergistic effects when combined with entecavir or tenofovir. There was no evidence of cytotoxicity with any of the drugs when used alone or in combination at the tested doses.

Characterization of a novel antibacterial agent that inhibits bacterial translation.

Bacterial protein synthesis is the target for several classes of established antibiotics. This report describes the characterization of a novel translation inhibitor produced by the soil bacterium Flexibacter. The dipeptide antibiotic TAN1057 A/B was synthesized and designated GS7128. As reported previously, TAN1057 inhibits protein synthesis in both Escherichia coli and Staphylococcus aureus, leaving transcription unaffected. Cell-free translation systems from E. coli were used to further dissect the mechanism of translational inhibition. Binding of mRNA to ribosomes was unaffected by the drug, whereas the initiation reaction was reduced. Elongation of translation was completely inhibited by GS7128. Detailed analysis showed that the peptidyl transferase reaction was strongly inhibited, whereas tRNA binding to both A- and P-site was unaffected. Selection and analysis of drug-resistant mutants of S. aureus suggests that drug uptake may be mediated by a dipeptide transport mechanism.

Inhibitory effect of adefovir on viral DNA synthesis and covalently closed circular DNA formation in duck hepatitis B virus-infected hepatocytes in vivo and in vitro.

The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model.

BACKGROUND/AIMS: Combination of antiviral drugs with immunotherapeutic approaches may be a promising approach for the treatment of chronic hepatitis B. We used the duck HBV (DHBV) infection model to evaluate the efficacy of the combination of adefovir with DNA-immunization by comparison with the respective monotherapies. METHODS: Pekin ducks chronically infected with DHBV received adefovir treatment alone or in association with intramuscular immunization with a plasmid (pCI-preS/S) expressing the DHBV large envelope protein. Ducks immunized with pCI-preS/S plasmid alone and two control groups receiving empty plasmid injections or no treatment were followed in parallel. RESULTS: All animals treated with adefovir showed a marked drop in viremia titers during drug administration, followed by a rebound of viral replication after drug withdrawal. Eight weeks after the third DNA boost, the median of viremia within the duck group receiving the combination therapy tended to be lower compared to that of the other groups. In addition, our results suggest a trend to an additive effect of adefovir and DNA vaccine since a 51% decrease in DHBV DNA was observed in autopsy liver samples from combination therapy group, whereas pCI-preS/S or adefovir monotherapies decreased intrahepatic viral DNA by 38 and 14%, respectively. This effect was sustained since it was observed 12 weeks after the end of therapy. CONCLUSIONS: Our results suggest that combination of adefovir with DNA-vaccine may be able to induce a sustained antiviral effect in vivo.

Hepatitis B virus genotypes and virologic response in 694 patients in phase III studies of adefovir dipivoxil1.

BACKGROUND & AIMS: Hepatitis B virus (HBV) genotype may influence disease progression and antiviral response. We therefore analyzed the frequency and distribution of genotypes in patients from 2 multinational phase III studies of adefovir dipivoxil. Antiviral efficacy of adefovir dipivoxil 10-mg therapy was examined with respect to HBV genotype, hepatitis B e antigen (HBeAg) serostatus, and race. METHODS: HBV genotypes were assigned by phylogenetic analyses of DNA sequences amplified from baseline serum samples (n = 694). RESULTS: Patients from Asia/Oceania were infected predominantly with genotypes B and C, whereas patients from Western European countries were infected predominantly with genotypes A and D. In Mediterranean countries, genotype D was dominant. The most common genotype in North America was C, followed by A, B, and D. Regardless of location, Asian patients were infected predominantly with genotypes B or C, whereas Caucasian patients were infected predominantly with A or D. There were significant differences in the baseline serum HBV-DNA levels of patients infected with different HBV genotypes regardless of HBeAg serostatus. Forty-eight weeks of adefovir dipivoxil 10-mg therapy resulted in potent reductions in serum HBV DNA with no significant differences based on genotype, HBeAg status, or race; similarly, there was no statistical difference in HBeAg seroconversion rates between genotypes in these patients. CONCLUSIONS: HBV genotypes were distributed asymmetrically with respect to race, geography, and HBeAg status. Forty-eight weeks of adefovir dipivoxil therapy resulted in significant decreases in serum HBV-DNA levels in patients regardless of HBV genotype, HBeAg status, or race.

Week 48 resistance surveillance in two phase 3 clinical studies of adefovir dipivoxil for chronic hepatitis B.

Seven hundred nucleoside treatment-naive patients were enrolled in two phase 3 trials of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B. To monitor for the emergence of potential adefovir resistance mutations over the first 48 weeks, all intent-to-treat patients (467 ADV-treated and 228 placebo patients) were included in a prospectively defined, treatment-blinded, virology substudy. The study protocol mandated genotypic analysis for all patients with detectable hepatitis B virus (HBV) DNA by Roche Amplicor polymerase chain reaction (PCR) at baseline and week 48, and in vitro phenotypic analyses for patients with conserved site substitutions in HBV polymerase or 1.0 log(10) or greater increase in HBV DNA from nadir. Paired sequences of the entire HBV reverse transcriptase were obtained for 271 ADV-treated and 227 placebo patients by using a sequencing method that detects down to 30% of minor species present within mixtures. Four substitutions (rtS119A, rtH133L, rtV214A, and rtH234Q) developed once each at conserved sites in HBV polymerase in 4 ADV-treated patients. Seven conserved site substitutions developed in 6 placebo patients. HBV mutants encoding the 4 substitutions that emerged in ADV-treated patients remained fully susceptible to adefovir in vitro. Furthermore, these 4 ADV-treated patients had HBV-DNA reductions of 3.3 to 5.9 log(10) copies/mL by week 48 with no rebound. All other substitutions occurred at very low frequencies (<1.6%) at polymorphic sites and were not associated with HBV-DNA increases in patients or adefovir resistance in vitro. In conclusion, no adefovir resistance mutations were identified in a large group of chronic hepatitis B patients treated with ADV for 48 weeks.

Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient.

BACKGROUND/AIMS: In contrast to lamivudine, adefovir dipivoxil (ADV) therapy is associated with delayed and infrequent selection of drug resistant hepatitis B virus (HBV). METHODS: A 52 year-old man was treated with lamivudine for an HBV recurrence on his liver graft. A viral breakthrough was observed and the patient received ADV. Serum HBV DNA decreased rapidly and lamivudine was discontinued while ADV monotherapy was maintained. Serum HBV DNA levels remained suppressed until a second breakthrough was observed. Lamivudine was then reintroduced together with ADV, and serum HBV DNA became undetectable by polymerase chain reaction. RESULTS: Sequence analyses of the HBV polymerase gene revealed a sequential selection of lamivudine resistance mutations L180M+M204V, followed by a reversion to wild-type, and subsequently the selection of a novel adefovir resistance mutation N236T. Phenotypic analyses in cell culture assays demonstrated that the HBV isolates at the time of ADV breakthrough had reduced susceptibility to ADV. This mutant remained sensitive to lamivudine, entecavir and emtricitabine in vitro. CONCLUSIONS: We describe the first case of sequential selection of lamivudine and adefovir resistant strains of HBV in a liver transplantation patient. The selection of the N236T polymerase mutant was associated with resistance to ADV but remained sensitive to lamivudine in vitro and in vivo.

The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro.

Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase [rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.

Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection.

Edema factor (EF), a key virulence factor in anthrax pathogenesis, has calmodulin (CaM)-activated adenylyl cyclase activity. We have found that adefovir dipivoxil, a drug approved to treat chronic infection of hepatitis B virus, effectively inhibits EF-induced cAMP accumulation and changes in cytokine production in mouse primary macrophages. Adefovir diphosphate (PMEApp), the active cellular metabolite of adefovir dipivoxil, inhibits the adenylyl cyclase activity of EF in vitro with high affinity (K(i) = 27 nM). A crystal structure of EF-CaM-PMEApp reveals that the catalytic site of EF forms better van der Waals contacts and more hydrogen bonds with PMEApp than with its endogenous substrate, ATP, providing an explanation for the approximately 10,000-fold higher affinity EF-CaM has for PMEApp versus ATP. Adefovir dipivoxil is a clinically approved drug that can block the action of an anthrax toxin. It can be used to address the role of EF in anthrax pathogenesis.

Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase.

BACKGROUND & AIMS: Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment. METHODS: HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant. RESULTS: Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log(10) decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir. CONCLUSIONS: The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.