Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+.

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for processing viral polyproteins which contain the enzymes and structural proteins required for the infectious virus. It was previously found that cupric chloride, in the presence of dithiothreitol or ascorbic acid, could inhibit the HIV-1 protease. It was suggested that a Cu1+ chelate was the moiety responsible for inhibition of the protease. This hypothesis has now been investigated directly by utilizing the stable Cu1+ chelate, bathocuproine disulfonic acid Cu1+ (BCDS-Cu1+). BCDS-Cu1+ inhibited the HIV-1 wild type protease as well as a mutant HIV-1 protease lacking cysteines. BCDS-Cu1+ was a competitive inhibitor of the mutant HIV-1 protease with an apparent Ki of 1 microM. Replication of HIV-1 in human lymphocytes and the cytotoxic effect of HIV-1 in CEM cells was inhibited by micromolar BCDS-Cu1+. Inhibition of the protease and of HIV replication by BCDS-Cu1+ was dependent on the presence of Cu1+ as BCDS alone was ineffective. EDTA blocked the inhibition of the protease by Cu1+ but was unable to block inhibition of the protease by BCDS-Cu1+, indicating that the Cu1+ complex was the inhibitory agent. The apparent IC50 for BCDS-Cu1+ on the inhibition of replication by primary isolates of HIV-1 was 5 microM. However, BCDS-Cu1+ did not affect polyprotein processing in an H9 cell line chronically infected with HIV-1, indicating that BCDS-Cu1+ acts by yet another mechanism to block HIV infection. Other possible targets for BCDS-Cu1+ include inhibition of viral adsorption and/or inhibition of the HIV-1 integrase.

Does the solid-state structure of endothelin-1 provide insights concerning the solution-state conformational equilibrium?

Additional NMR data (local NOE ratios and chemical shifts) for endothelin-1 supporting the existence of a relatively regular helix initiated abruptly at Lys9 (with Asp8 as an N-cap) and extending in all cases to Cys15 (and in a frayed form to Asp18 in some analogs) is presented. The recent solids-state structure [Janes et al. (1994), Nature Struct. Biol. 1, 311-319], in contrast, places the helix in the extreme C-terminal section of structure and the Lys9-Tyr13 segment is not helical. The X-ray structure does not predict the NOEs or chemical shifts observed for endothelins in aqueous media containing polar organic co-solvents. An analysis of the chemical shift data for reporter groups indicates that the helical conformational preference of endothelins is not significantly altered by the addition of acetonitrile, acetic acid, or ethylene glycol. The validity of the analytic strategy is supported by results for both more rigid and less helical analogs. We conclude that the structure observed in crystals obtained from purely aqueous media is influenced by intermolecular interactions in the solid state and is not a significant contributor to the conformational equilibrium observed for monomeric ET-1.

Conformational isomerism of endothelin in acidic aqueous media: a quantitative NOESY analysis.

The conformational features of endothelin-1 (ET-1) in mixed water/ethylene glycol media have been studied by two-dimensional 1H NMR experiments throughout the pH range 3.2-7.2. At pH less than 5 all backbone NH signals can be observed, and NOESY experiments provided a large set of dipolar cross-peaks. Cross-peak intensities from each experiment (different mixing times and H2O versus D2O) were converted to distance constraints using a novel algorithm (program DISCON) for removing spin diffusion effects and thus obtain cross-rates rather than cross-peak intensities. A set of 168 nonstereospecific distance bounds (average experimental precision, +/- 0.38 A) was used in dynamics simulated annealing refinements. Two consensus structural features were found--a reverse turn at Ser5----Asp8 and an alpha-helical stretch from Lys9 to Cys15; however, after constraint-free minimization, structures generated using XPLOR-1.5, CONGEN, and DISCOVER all violated at least 32% of the bounds by more than 0.2 A, which we ascribe to conformational isomerism. When the constraints were modified to reflect subsequent experimental data and to eliminate constraints that could not be obeyed by any single conformer structure, the relaxed structures still violated at least 15% of this more limited and looser set of constraints. Therefore, a modified procedure for constrained dynamics refinement (using XPLOR-2.1), which allows for conformational isomerism outside of the central helical core region, was developed. This "conformer search procedure" produced structures which fell into five tightly defined conformational clusters. The two most populated clusters correspond to a rotation of the 8,9-amide unit. The conformer which we propose as the major contributor at pH 3.2-5.8 was defined to a backbone rmsd of 0.51 A over residues 1----15. An alternative description of the motional averaging in segments of the endothelin structure as extensive randomization rather than rapid interconversion between a small number of discreet conformers was ruled out by an analysis of NH shift-temperature gradients and exchange rates. This analysis suggests that small delta delta/delta T values need not correlate with H-bonding for conformational mixtures. In ET-1 the greatest motional averaging occurs from Ser2 through Ser5 (not in the C-terminus) and may be so extensive as to approximate a flexible random coil population as high as 30%. The C-terminus shows less rapid and less extensive conformational averaging, but no definitive structures for individual conformers could be derived in the absence of stereospecific constraints. The pharmacological implications of the consensus structural features are discussed.

Conformation of endothelin in aqueous ethylene glycol determined by 1H-NMR and molecular dynamics simulations.

The solution conformation of a 21-residue vasoconstrictor peptide endothelin-1 (ET-1) in water-ethylene glycol has been determined by two-dimensional 1H-NMR spectroscopy and constrained molecular dynamics simulations. The N-terminus (residues 1-4) appears to undergo conformational averaging and no single structure consistent with the NMR constraints could be found for this region. Residues 5-8 form a turn, and residues 9-16 exist in a helical conformation. A flexible 'hinge' between residues 8-9 allows various orientations of the turn relative to the helix. Another 'hinge' at residue 17 connects the extended C-terminus to the bicyclic core region (residues 1-15). Residues important for binding and biological activity form a contiguous surface on one side of the helix, with the two disulfides extending from the other side of the helix.

Computer-aided conformational analysis based on NOESY signal intensities.

The basis for the development of a suite of programs that allow the user to determine motional features (the correlation time and the significance of segmental motion) and the optimum conditions for future experiments from a NOESY signal matrix is presented. This automated evaluation of NOESY data serves as the initial step of an iterative conformational analysis which uses the molecular model manipulation capabilities of modern graphics workstations. Incorporated in these programs is NOESYSIM, a calculation subroutine which uses a set of molecular coordinates (and a correlation time estimate) together with user entered experimental parameters (acquisition time, sweep width, mixing time and cycle repetition time) to generate an accurately calculated NOESY signal matrix reflecting those conditions and the specified conformational model. Conformational refinement then consists of iterative comparisons of the experimental signal matrix with a series (or systematically sampled set) of model coordinates corresponding to a dynamics' course, driven-minimization or torsional grid search. These procedures and developments are illustrated with examples including: solution conformations of prostanoids; studies of the folding preferences and media-dependent changes in conformation for peptide hormones; and the structure elucidation of a novel undecapeptide macrolide antibiotic (lysobactin). For larger molecules, even constrained grid searches have too high a dimensionality and one must resort to distance-constraint based minimizations. A novel procedure for deriving more accurate distance constraints (corrected for secondary NOEs) is detailed and a new strategy for conformation elucidation, based on this procedure, is outlined.

Pyridine coenzyme analogues. Synthesis and characterization of alpha- and beta-nicotinamide arabinoside adenine dinucleotides.

The synthesis and characterization of a new pyridine coenzyme analogue containing a nicotinamide arabinonucleotide moiety are reported. The redox potentials are -339 mV for beta-oxidized nicotinamide arabinoside adenine dinucleotide and -319 mV for alpha-oxidized nicotinamide adenine dinucleotide, and the lambda max is 346 and 338 nm for beta- and alpha-reduced nicotinamide arabinoside adenine dinucleotides (araNADH), respectively. Anomerization of the reduced analogues leads to a 5:1 ratio of alpha-araNADH to beta-araNADH at 90 degrees C. These results establish that the relative configuration of the 2'-hydroxyl to the base is the primary determinant for the configuration-dependent changes in lambda max, the redox potential of the pyridine nucleotides, and the preferred anomeric configuration of the reduced coenzymes. Comparison of the 1H and 31P NMR spectral data of the analogues with those for the ribo coenzymes is reported and the conformational analysis discussed. The coenzyme properties of the arabino analogues have been evaluated with yeast and horse liver alcohol dehydrogenases. Both the alpha- and beta-anomers are found to serve as coenzymes, and the stereochemistry of hydride transfer is identical for both anomers.

Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.

Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin.

In vivo sodium-23 magnetic resonance surface coil imaging: observing experimental cerebral ischemia in the rat.

Sodium-23 magnetic resonance imaging can be used to detect and assess experimental cerebral ischemia in the rat. An imaging technique utilizing a surface coil is described to produce sodium magnetic resonance images of good quality and resolution within 10 min. A novel method of hemispheric occlusion showed edema in the right brain of the rat head within 3 hr after injury. The edema was especially pronounced by 12 hr with effects in the right brain, eye and surrounding muscle evident.