Search PubMed⌕ Search

Biomedical subjects

Daniel F Wyss

Publications and source records attributed to Daniel F Wyss.

11 recordsLinked to original sources
PubMed2003

Flexible lid to the p53-binding domain of human Mdm2: implications for p53 regulation.

The stabilization of p53 against Mdm2-mediated degradation is an important event in DNA damage response. Initial models of p53 stabilization focused on posttranslational modification of p53 that would disrupt the p53-Mdm2 interaction. The N-terminal regions of both p53 and Mdm2 are modified in vivo in response to cellular stress, suggesting that modifications to Mdm2 also may affect the p53-Mdm2 interaction. Our NMR studies of apo-Mdm2 have found that, in addition to Mdm2 residues 25-109 that form the well ordered p53-binding domain that was observed in the p52-Mdm2 complex, Mdm2 residues 16-24 form a lid that closes over the p53-binding site. The Mdm2 lid, which is strictly conserved in mammals, may help to stabilize apo-Mdm2. It also competes weakly with peptidic and nonpeptidic antagonists. Modifications to the Mdm2 lid may disrupt p53-Mdm2 binding leading to p53 stabilization. Mdm2 and Mdm4 possess nearly identical p53-binding domains but different lids suggesting that lid modifications may select for p53 binding.

Amino Acid Sequence↗
PubMed2003

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2.

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.

Alanine↗
PubMed2002

Spatial localization of ligand binding sites from electron current density surfaces calculated from NMR chemical shift perturbations.

Rapid, accurate structure determination of protein-ligand complexes is an essential component in structure-based drug design. We have developed a method that uses NMR protein chemical shift perturbations to spatially localize a ligand when it is complexed with a protein. Chemical shift perturbations on the protein arise primarily from the close proximity of electron current density from the ligand. In our approach the location of the center of the electron current density for a ligand aromatic ring was approximated by a point-dipole, and dot densities were used to represent ligand positions that are allowed by the experimental data. The dot density is increased in the region of space that is consistent for the most data. A surface can be formed in regions of the highest dot density that correlates to the center of the ligand aromatic ring. These surfaces allow for the rapid evaluation of ligand binding, which is demonstrated on a model system and on real data from HCV NS3 protease and HCV NS3 helicase, where the location of ligand binding can be compared to that obtained from difference electron density from X-ray crystallography.

Binding Sites↗
PubMed2002

Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications.

The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.

Animals↗
PubMed2002

Structures of protein-protein complexes are docked using only NMR restraints from residual dipolar coupling and chemical shift perturbations.

NMR structures of protein-protein and protein-ligand complexes rely heavily on intermolecular NOEs. Recent work has shown that if no significant conformational changes occur upon complex formation residual dipolar coupling can replace most of the NOE restraints in protein-protein complexes, while restraints derived from chemical shift perturbations can largely replace intermolecular NOEs in protein-ligand structures. By combining restraints from chemical shift perturbations with orientation restraints derived from measurements of residual dipolar couplings, we show that the structure of the EIN-HPr complex can be calculated without NOE restraints. The final structure, built from the crystal structures of EIN and HPr in their uncomplexed form and docked only with NMR restraints, places HPr within 2.5 A of the position determined from the mean NMR structure of the complex.

Bacterial Proteins↗
PubMed2002

Structure, biology, and therapeutic implications of pegylated interferon alpha-2b.

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.

Amino Acid Sequence↗
PubMed2002

NMR-based approaches for lead discovery.

NMR methods have long been used for studying molecular interactions. In the last few years, various NMR approaches have been developed to aid lead discovery. These involve different NMR screening methods to identify initial compounds, which often bind only weakly (in the micro- to millimolar range) to the drug target. Intelligent and focused follow-up strategies enable the development of these compounds into potent, submicromolar drug-like inhibitors for use as leads in drug discovery projects. NMR can be used as both a remarkably reliable screening tool and a structural tool; thus, this technique has unique opportunities for lead discovery.

Animals↗