In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Increased Resistance to ABT-378, a Novel Protease Inhibitor

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 μM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P₁′ residue Leu to Phe) and p7/p1 (P₂ residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 μM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 μM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763–3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662–6670, 1997).     

Grafting an RGD motif onto an epidermal growth factor-like module: chemical synthesis and functional characterization of the chimeric molecule.

A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.     

Synthesis and SAR of novel 1,1-dialkyl-2(1H)-naphthalenones as potent HCV polymerase inhibitors

A series of gem-dialkyl naphthalenone derivatives with varied alkyl substitutions were synthesized and evaluated according to their structure-activity relationship. This investigation led to the discovery of potent inhibitors of the hepatitis C virus at low nanomolar concentrations in both enzymatic and cell-based HCV genotype 1a assays.     

Survivin, the starlet of the passenger-protein complex : check-up for its tenth anniversary

Ten year after its discovery Survivin has gained a strategic place within the chromosomal passenger complex. Whereas INCENP, Borealin and Aurora B are fully immobile in the complex, Survivin is mobile on centromere. Its mobility is regulated both by phosphorylation and ubiquitination. Survivin is a dynamic messenger that senses the central spindle tension and participates to the control of the mitotic chekpoint. In this review, we have detailed the multiple mitotic activities of Survivin and discussed them in light of the recent reported crystallographic data.     

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.     

Optimization of human d-amino acid oxidase expression in Escherichia coli

Human d-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAAO are indeed required for the investigation of its structure–function relationships and for the screening of new inhibitors to be used in the treatment of schizophrenia. A recombinant hDAAO has been over-expressed in BL21(DE3)Star E. coli cells. By alternating screenings of medium components at flask level and investigating physiological parameters in 2 L controlled batch fermentations, an improved, robust and scalable microbial process was set up giving almost a 40- and 4-fold improvement in volumetric productivity and specific activity, respectively. Under these conditions 770 U/L culture hDAAO with a specific activity of 0.4 U/mg protein and a specific productivity of 24.9 U/g biomass were produced. Optimization of medium ingredients, of the time and the amount of inducer’s addition, pH control at the moment of induction and harvest, low mechanical shear stress regime during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is higher than any previously described production of hDAAOs. A yield of 100 mg of pure hDAAO/L culture thus became available in comparison to the 1–10 mg/L previously reported.     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Redox potentials and their pH dependence of D-amino-acid oxidase of Rhodotorula gracilis and Trigonopsis variabilis.

The redox potentials and pH characteristics of D-amino-acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5-8.5 at 15 degrees C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two single-electron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single-electron transfer potentials. The first electron potential is pH-independent, while the second electron transfer is pH-dependent exhibiting a approximately -60 mV/pH unit slope, consistent with a one-electron/one-proton transfer. In the presence of the substrate analogue benzoate, the two-electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of Em vs. pH (-30 mV/pH unit) corresponds to an overall two-electron/one-proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO-benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

New biotech applications from evolved D-amino acid oxidases

D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxidative deamination of amino acid D-isomers with absolute stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Recently, the extraordinary functional plasticity of DAAO has become evident; in turn, boosting research on this flavoprotein. Protein engineering has allowed for a redesign of DAAO substrate specificity, oxygen affinity, cofactor binding, stability, and oligomeric state. We review recent developments in utilizing DAAO, including as a biocatalyst for resolving racemic amino acid mixtures, as a tool for biosensing, and as a new mechanism of herbicide resistance. Perspectives for future biotechnological applications of this oxidative biocatalyst are also outlined.