Evaluation of selected chemotypes in coupled cellular and molecular target-based screens identifies novel HIV-1 zinc finger inhibitors

Conservation of the Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys retroviral zinc finger sequences and their absolute requirement in both the early and late phases of retroviral replication make these chemically reactive structures prime antiviral targets. We recently reported that select 2,2'-dithiobisbenzamides (DIBAs) chemically modify the zinc finger Cys residues, resulting in release of zinc from the fingers and inhibition of HIV replication. In the current study we surveyed 21 categories of disulfide-based compounds from the chemical repository of the National Cancer Institute for their capacity to act as retroviral zinc finger inhibitors. Aromatic disulfides that exerted anti-HIV activity tended to cluster in the substituted aminobenzene, benzoate, and benzenesulfonamide disulfide subclasses. Only one thiuram derivative exerted moderate anti-HIV activity, while a number of nonaromatic thiosulfones and miscellaneous disulfide congeners were moderately antiviral. Two compounds (NSC 20625 and NSC 4493) demonstrated anti-cultures. The two compounds chemically modified the p7NC zinc fingers in two separate in vitro assays, and interatomic surface molecular modeling docked the compounds efficiently but differentially into the zinc finger domains. The combined efforts of rational drug selection, cell-based screening, and molecular target-based screening led to the identification of zinc finger inhibitors that can now be optimized by medicinal chemistry for the development of biopharmaceutically useful anti-HIV agents.     

Reliability and validity of the 12-minute distance walk in patients with chronic obstructive pulmonary disease

Validity and test-retest liability of the 12-minute distance (12MD) walk, a measure of functional status, were examined in patients with chronic obstructive pulmonary disease. Four tests were administered at weekly intervals. Performance increased (p < .01) over the first three tests. Test-retest reliability was r34 = .98 (df = 46) for tests 3 and 4. The 12MD walk correlated with the Sickness Impact Profile, Physical Dimension (r = -.45); forced expiratory volume in 1 second % predicted ( r = .40); maximal inspiratory pressure (PImax) (r = .52); and exercise-related breathlessness (r = -.49). Exercise-related breathlessness and PImax accounted for 42% of the variance. The validity and reliability of the 12MD walk were supported.     

Sequence specificity in the interaction of the four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides with the supF gene

The shuttle vector pS189 was treated with each of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide, and the modified DNA was used as a template in a polymerase arrest assay examining the supF gene. Sites at which polymerase (Sequenase, version 2.0) progress along the template was blocked were presumed to be at or near sites of adduct formation. The polymerase arrest sites were compared with recently reported mutation hotspots induced by these agents in this gene (Bigger et al., Proc. Natl. Acad. Sci. USA, 89: 368-372, 1992). For 31 of 32 mutation hotspots, a polymerase arrest band was present at or 1 or 2 nucleotides 3'- to that site, indicating that adduct formation tended to be associated with mutation hotspots. However, the arrest bands near mutation hotspots were not particularly prominent in all cases, and there were many sites of substantial polymerase arrest that were not in the vicinity of mutation hotspots. Thus, factors in addition to chemical selectivity must play key roles in determining sites of mutation.     

A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.     

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.     

Anterolateral soft-tissue impingement in the ankle: diagnosis using MR imaging

OBJECTIVE: This study was performed to elucidate the MR imaging findings and pitfalls for the diagnosis of anterolateral soft-tissue impingement in the ankle, a cause of chronic ankle pain that can be relieved by arthroscopic resection. MATERIALS AND METHODS: We retrospectively reviewed MR imaging examinations of 18 patients with arthroscopically confirmed anterolateral ankle impingement. The MR images of 18 additional subjects with symptoms that could mimic anterolateral impingement, but who had a surgically confirmed alternate diagnosis (instability, peroneal tendon injury, osteochondral defect, normal arthroscopy) and no evidence of impingement at arthroscopy, served as controls. RESULTS: On the MR imaging studies, nine patients had an ankle effusion, eight of whom showed an abnormal soft-tissue structure in the anterolateral gutter, 2-15 mm in maximal diameter. No soft-tissue mass was seen in the patients without joint fluid. Four control subjects with instability had a similar soft-tissue structure in the anterolateral gutter, but in the control subjects the finding represented a portion of the torn anterior talofibular ligament. CONCLUSION: Anterolateral soft-tissue impingement of the ankle can be suggested by MR imaging when fluid in the lateral gutter outlines an abnormal soft-tissue structure separate from the anterior talofibular ligament.