Inhibition of ClC-2 chloride channels by a peptide component or components of scorpion venom.

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.

Adefovir and tenofovir susceptibilities of HIV-1 after 24 to 48 weeks of adefovir dipivoxil therapy: genotypic and phenotypic analyses of study GS-96-408.

OBJECTIVE: To determine whether genotypic changes in HIV-1 (HIV) reverse transcriptase (RT) occur during adefovir dipivoxil (ADV) therapy that may alter the susceptibility of HIV to adefovir or the related nucleotide inhibitor, tenofovir. DESIGN AND METHODS: GS-96-408 was a 1:1 randomized, double-blind, phase III clinical trial assessing the safety and efficacy of 120-mg daily ADV compared with placebo for the treatment of HIV when added to stable background antiretroviral therapy (ART). Of 442 patients enrolled, 142 were prospectively selected for a virology substudy. Baseline and posttreatment (weeks 24-48) plasma samples were genotypically analyzed in HIV RT. HIV from ADV-treated patients who developed RT mutations at week 24 were also phenotypically analyzed. RESULTS: Nucleoside-associated RT mutations arose with similar frequency among the ADV-and placebo-treated patients, 32% (n = 23) and 28% (n = 20), respectively, during the 24-week blinded treatment phase. RT mutations previously selected by adefovir in vitro (K70E or K65R) did not develop in any patient. Most mutations were typical zidovudine (ZDV)-resistance mutations (e.g., M41L, D67N, K70R, T215Y) in patients taking ZDV or stavudine (d4T) concomitantly, demonstrating directly in the placebo arm that d4T is able to select for these mutations. There appeared to be more patients developing D67N and K70R mutations in the ADV arm versus more T215Y mutations in the placebo arm. Between weeks 24 and 48, 19 of 50 patients (38%) in the ADV arm developed similar RT mutations. The mean HIV RNA responses at weeks 24 and 48 among the ADV-treated patients developing RT mutations were -0.68 log(10) copies/ml (n = 23) and -0.52 log(10) copies/ml (n = 19), respectively, similar to the overall week-24 and week-48 responses (-0.53 and 0.48 log(10) copies/ml, respectively). Patient-derived HIV expressing the observed RT mutations showed insignificant decreases in adefovir susceptibility compared with wild-type in 12 of 16 cases (< threefold). HIV from 1 patient showed significantly reduced susceptibility to tenofovir, which was in association with a double insertion mutation after codon 69 that was also present at baseline. CONCLUSIONS: HIV RT changes that arose during ADV therapy appear attributable to the patient's background ART. ADV therapy may have influenced the pattern of ZDV-associated resistance mutations that developed, but this did not result in an observed loss of viral load suppression. There was a trend toward decreased phenotypic susceptibility to adefovir in ADV-treated patients, with 4 of 16 analyzed patients showing mild, but significantly decreased susceptibility associated with the additional ZDV-associated mutations. Decreased susceptibility to the related nucleotide analog, tenofovir, was not observed to develop in ADV-treated patients.

The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1.

Nephrotoxicity is the dose-limiting clinical adverse effect of cidofovir and adefovir, two potent antiviral therapeutics. Because renal uptake likely plays a role in the etiology of cidofovir- and adefovir-associated nephrotoxicity, we attempted to identify a renal transporter capable of interacting with these therapeutics. A cDNA clone was isolated from a human renal library and designated human organic anion transporter 1 (hOAT1). Northern analysis detected a specific 2.5-kilobase pair hOAT1 transcript only in human kidney. However, reverse transcription-polymerase chain reaction revealed hOAT1 expression in human brain and skeletal muscle, as well. Immunoblot analysis of human kidney cortex demonstrated that hOAT1 is an 80- to 90-kilodalton heterogeneous protein modified by abundant N-glycosylation. Xenopus laevis oocytes expressing hOAT1 supported probenecid-sensitive uptake of [(3)H]p-aminohippurate (K(m) = 4 microM), which was trans-stimulated in oocytes preloaded with glutarate. Importantly, both hOAT1 and rat renal organic anion transporter 1 (rROAT1) mediated saturable, probenecid-sensitive uptake of cidofovir, adefovir, and other nucleoside phosphonate antivirals. The affinity of hOAT1 toward cidofovir and adefovir (K(m) = 46 and 30 microM, respectively) was 5- to 9-fold higher compared with rROAT1 (K(m) = 238 and 270 microM, respectively). These data indicate that hOAT1 may significantly contribute to the accumulation of cidofovir and adefovir in renal proximal tubules and, thus, play an active role in the mechanism of nephrotoxicity associated with these antiviral therapeutics.

In vitro antiviral susceptibilities of isolates from cytomegalovirus retinitis patients receiving first- or second-line cidofovir therapy: relationship to clinical outcome.

Blood culture isolates from patients receiving first- (peripheral retinitis) or second-line (relapsing retinitis) therapy with intravenous cidofovir were obtained from three clinical trials for in vitro antiviral susceptibility analyses. Isolates from 6 patients obtained after 14.3 weeks (mean) of first-line cidofovir therapy showed complete susceptibility to cidofovir, ganciclovir, and foscarnet. Isolates from 20 patients were obtained after 17.3 weeks (mean) of second-line cidofovir therapy. Ten showed complete susceptibility to all inhibitors, 3 showed low-level ganciclovir resistance (<6-fold) but were sensitive to cidofovir and foscarnet, and 7 showed moderately reduced susceptibility (<8-fold) to cidofovir and high-level resistance (8- to 23-fold) to ganciclovir in vitro. Four of these 7 isolates showed reduced susceptibility (4-fold) to foscarnet. Notably, there was no difference in time to retinitis progression in patients that were on cidofovir therapy when sensitive isolates were compared with those showing reduced susceptibility to cidofovir in vitro.

Human immunodeficiency virus type 1 reverse transcriptase expressing the K70E mutation exhibits a decrease in specific activity and processivity.

Adefovir dipivoxil [9-(2-(bispivaloyloxymethyl)phosphonylmethoxyethyl)adenine (bis-POM PMEA)], an oral prodrug of adefovir (PMEA), is currently in phase III clinical testing for the treatment of human immunodeficiency virus-1 (HIV-1) infection. Previous in vitro experiments have shown that HIV-1 recombinant viruses expressing either a K65R or a K70E mutation in reverse transcriptase (RT) have reduced sensitivity to PMEA and that the K70E mutant also has impaired replication capacity in vitro. Genotypic analyses of samples from patients enrolled in a phase I/II clinical trial of adefovir dipivoxil demonstrated that the K70E RT mutation developed in two of 29 patients during extended therapy. To further investigate the molecular mechanisms involved in the resistance to PMEA, we cloned, expressed, and purified HIV-1 RT enzymes carrying either the K65R or K70E and, for comparison, the M184V mutation. The Km values of dNTPs for these mutant enzymes were not significantly altered from wild-type RT. The Ki values for the K65R mutant were increased from wild-type by 2-5-fold against a variety of inhibitors, whereas the Ki values for the M184V mutant were increased 12-fold specifically for 2', 3'-dideoxy-3'-thiacytidine (3TC) triphosphate. The Ki values for the K70E mutant were increased for PMEA diphosphate and 3TC triphosphate by 2-3-fold. These results are in agreement with antiviral drug susceptibility assay results. The three recombinant enzymes were also evaluated for their specific activities and processivities. All mutants were reduced in specific activity with respect to wild-type RT. In single-cycle processivity studies, the M184V mutant was, as expected, notably impaired. The K70E mutant was also slightly impaired, whereas the K65R mutant was slightly more processive than wild-type. These results with recombinant K70E RT are consistent with the reduced in vitro replication capacity of the K70E RT mutant of HIV-1 and further demonstrate that the K70E mutation confers minor PMEA and 3TC resistance to HIV-1.

A point mutation in the human cytomegalovirus DNA polymerase gene selected in vitro by cidofovir confers a slow replication phenotype in cell culture.

In cell culture, cidofovir (CDV) was used to select a human cytomegalovirus (HCMV) strain with decreased drug susceptibility. The genotypic characterization of this virus revealed a single base substitution resulting in a K513N amino acid alteration in the viral DNA polymerase (UL54). Performed in parallel, the selection of HCMV for replication in the presence of ganciclovir (GCV) selected an M460V substitution in the phosphotransferase (UL97), as well as a K513N/V812L double substitution in DNA polymerase. Neither of the two DNA polymerase mutations has been previously identified in HCMV drug-resistant strains. To precisely elucidate their role in drug resistance, corresponding recombinant mutant viruses were generated by recombination of nine overlapping viral DNA fragments. The K513N recombinant virus showed 13- and 6.5-fold decreased susceptibility to CDV and GCV in vitro, respectively, compared with the wild-type recombinant virus. Mutation V812L was associated with a moderate (2-3-fold) decrease in susceptibility to CDV, GCV, foscarnet, and adefovir. A multiplicative interaction of the K513N and V812L mutations with regard to the profile and level of drug resistance was demonstrated in recombinant virus expressing both mutations. In vitro replication kinetic experiments revealed that the K513N mutation significantly decreased HCMV replication capacity. Consistent with this finding, the K513N mutant DNA polymerase exhibited reduced specific activity in comparison with the wild-type enzyme and was severely impaired in its 3'-5' exonuclease function. Unexpectedly, the K513N mutant enzyme showed no decrease in susceptibility to CDV-diphosphate or GCV-triphosphate. However, the K513N mutation decreased the susceptibility to CDV and GCV of the oriLyt plasmid replication in the transient transfection/infection assay, suggesting that the DNA replication of the K513N mutant virus is less sensitive to the corresponding inhibitors.

Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments.

A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and delta-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.

High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes.

Mutations in both the viral phosphotransferase gene, UL97, and the DNA polymerase gene, UL54, have been shown to confer ganciclovir resistance to cytomegalovirus (CMV). Moreover, UL54 alterations have been associated with in vitro cross-resistance of CMV to cidofovir. To investigate the relative significance of UL97 versus UL54 alterations in conferring antiviral resistance, phenotypic and genotypic characterization of 28 ganciclovir-resistant clinical CMV isolates was undertaken. Isolates were either low-level ganciclovir-resistant, which have ganciclovir ID50 values > or =8 microM and <30 microM and sensitivity to cidofovir, or high-level ganciclovir-resistant, which have ganciclovir ID50 values > or =30 microM and cross-resistance to cidofovir. Low-level ganciclovir-resistant isolates were associated with UL97 alterations and short periods of ganciclovir treatment, while high-level ganciclovir-resistant isolates were associated with both UL97 and polymerase alterations and were cultured after extended ganciclovir therapy.

Expression of the catalytic subunit (UL54) and the accessory protein (UL44) of human cytomegalovirus DNA polymerase in a coupled in vitro transcription/translation system.

The catalytic subunit (UL54) and accessory protein (UL44) of human cytomegalovirus (HCMV) DNA polymerase have been cloned and expressed in an in vitro-coupled transcription/translation reticulocyte lysate system. The influence of the 5'-untranslated region (5'-UTR) on the efficiency of expression from the circular plasmids has been investigated. For expression of both UL54 and UL44, a truncated form of the alfalfa mosaic virus (AMV) RNA4 5'-UTR was found to be superior to the full-length AMV 5'-UTR or the original HCMV 5'-UTRs of different lengths. Protein products with Mr approximately 140 and 55 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the UL54 and UL44 in vitro expression reactions, respectively. The properties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3'-5' exonuclease activities of the expressed UL54/UL44 complex were found to be dependent on salt concentration in the same manner as the activities of the native enzyme. The in vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidofovir diphosphate, ganciclovir triphosphate, and foscarnet). This straightforward method for protein expression may also be applicable to other enzymes where reproducible generation of fully functional products is desirable.

In vitro characterization of the anti-human cytomegalovirus activity of PMEA (Adefovir).

PMEA [9-[2-(phosphonomethoxy)ethyl]adenine; adefovir] has shown anti-cytomegalovirus activity in animal models and in preliminary human trials. PMEA diphosphate (PMEApp), the active antiviral metabolite of PMEA, is a potent inhibitor of human cytomegalovirus (HCMV) DNA polymerase. PMEA is efficiently taken up and phosphorylated to PMEApp in numerous human cell lines. In vitro replication of wild type and drug resistant HCMV clinical isolates is effectively inhibited by PMEA. PMEA in combination with other anti-HCMV agents shows additive inhibition of HCMV replication.

Novel mutation (K70E) in human immunodeficiency virus type 1 reverse transcriptase confers decreased susceptibility to 9-[2-(phosphonomethoxy)ethyl]adenine in vitro.

9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), an acyclic nucleoside phosphonate analog, is active against several retroviruses and herpesviruses and has shown anti-human immunodeficiency virus (HIV) activity in clinical trials. Serial passage of HIV type 1 (strain IIIb, in MT2 cells in increasing concentrations of PMEA resulted in viruses with > 12-fold increases in their 50% inhibitory concentrations of PMEA compared with that for strain IIIb. Sequence analyses of these PMEA-selected viruses demonstrated the presence of a novel lysine-to-glutamic acid mutation at amino acid 70 (K70E) in HIV reverse transcriptase. A recombinant virus carrying the K70E mutation was constructed and showed a 10-fold increase in its 50% inhibitory concentrations of PMEA and 2',3'-dideoxy-3'-thiacytidine but showed wild-type susceptibility levels to 2',3'-dideoxycytosine, 2',3'-dideoxyinosine,2',3'-didehydro-2'3'-dideoxythymidine, 3'-azido-3'-deoxythymidine, foscarnet, and two additional phosphonates, 9-[(R)-2-(phosphonomethoxy)propyl]adenine and 9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine. Additionally, the K70E recombinant showed a minor reduction in growth kinetics compared with those of the wild-type virus in vitro.

Comparative kinetic analyses of interaction of inhibitors with Rauscher murine leukemia virus and human immunodeficiency virus reverse transcriptases.

The inhibitory effects of several nucleoside triphosphate analogs on Rauscher murine leukemia virus (RMuLV) and human immunodeficiency virus (HIV) type 1 reverse transcriptases (RTs) were studied. With RNA as the template, the apparent K(m) and apparent K(i) values of HIV RT toward its substrates and inhibitors are 12 to 500 times lower than the corresponding values for RMuLV RT. However, the k(i)/k(m) ratios (inhibition efficiencies) for HIV and RMuLV RTs'are similar for AZTTP (zidovudine triphosphate), d4TTP [3'-deoxythymidine-2'-ene-(3'-deoxy-2',3'-didehydrothymidine) triphosphate], PMEADP [9-(2-phosphonylmethoxyethyl)adenine diphosphate], FIAUTP [1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodouracil triphosphate], and HPMPCDP [(S)-1-(3-hydroxy-2-phosphylmethoxypropyl) cytosine diphosphate]. With DNA as the template, the K(m) values are similar for HIV and RMuLV RTs. However, the K(i)/K(m) values of HIV and RMuLV RTs are significantly different for ddCTP, ddATP, and 3TCTP (2',3'-dideoxy-3'-thiacytidine). The RTs of RMuLV and HIV are sufficiently different from one another that the kinetic inhibition constants for a particular antiviral compounds should be determined to indicate whether anti-RMuLV activity is likely to be predictive for the anti-HIV activity of the compound. This information, in conjunction with species-specific drug metabolism differences and tissue culture antiviral activity, is important in determining the suitability of a particular animal model.

Tetrapyrrole utilization of Bacteroides fragilis.

During growth in a defined medium containing protoheme, Bacteroides fragilis strain 2044 nd Bacteroides "l" 7CM formed protoheme-containing b-type cytochromes. The major reduced versus oxidized spectral peaks found for strain 2044 were at 562 and 428 nm whereas the peaks observed for strain 7CM were at 559 and 427 nm. When protoheme was replaced by deuteroporphyrin, mesoporphyrin, or their manganese or magnesium chelates, both organisms formed deuteroheme- or mesoheme-containing cytochromes with difference spectral peaks at 546 to 547 nm and 411 to 413 nm. Pyridine hemochromogen spectra of cells grown in the presence of tetrapyrroles containing the deuteroporphyrin or mesoporphyrin moiety confirmed the presence of deuteroheme of mesoheme in the cells. Although capable of delayed and relatively scant growth in media devoid of added tetrapyrroles or in media containing the vanadium chelates of mesoporphyrin or deuteroporphyrin, neither organism formed cytochromes under these growth conditions. The functional nature of the cytochromes containing deuteroheme or mesoheme was confirmed by their reduction by cells in the presence of 56 mM glucose and carbon dioxide compared with cells exposed to air. The use of tetrapyrroles by strains 2044 and 7CM is similar to that previously found for Bacteroides ruminicola ssp. ruminicola. Versatility in tetrapyrrole utilization for cytochrome synthesis appears to be a common characteristic of heme-requiring gastrointestinal Bacteroides species.