Biomedical subjects

T S Scanlan

Publications and source records attributed to T S Scanlan.

24 records · Page 2Linked to original sources

PubMed1997

Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites.

The transactivation properties of the two estrogen receptors, ERalpha and ERbeta, were examined with different ligands in the context of an estrogen response element and an AP1 element. ERalpha and ERbeta were shown to signal in opposite ways when complexed with the natural hormone estradiol from an AP1 site: with ERalpha, 17beta-estradiol activated transcription, whereas with ERbeta, 17beta-estradiol inhibited transcription. Moreover, the antiestrogens tamoxifen, raloxifene, and Imperial Chemical Industries 164384 were potent transcriptional activators with ERbeta at an AP1 site. Thus, the two ERs signal in different ways depending on ligand and response element. This suggests that ERalpha and ERbeta may play different roles in gene regulation.

Animals↗

PubMed1997

The structural and functional basis of antibody catalysis.

Ten years have passed since the initial reports that antibodies could be programmed to have enzymatic activity by immunization with a transition-site analog. Much of the research over the last decade has focused on defining the scope and generality of antibody catalysis; however, during the past two years the first few crystal structures of catalytic antibody transition-state analogs have been reported. This review analyzes four such structures of catalytic antibodies that catalyze markedly different reactions, including ester hydrolysis, sulfide oxidation, and a pericyclic rearrangement. Structure-function relations for these catalysts are discussed and compared to the structure and function of natural enzymes, as well as the chemistry that occurs in solution.

Antibodies, Catalytic↗

PubMed1995

Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH99-HisH35 (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G11 compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.

Amino Acid Sequence↗

PubMed1995

Screening and characterization of estrogenic activity from a hydroxystilbene library.

BACKGROUND: Compounds that either inhibit or induce an estrogen response in vivo are important as potential drugs and biochemical tools. Non-steroidal stilbene analogs such as tamoxifen are known to function as both estrogen agonists and antagonists depending upon the analog structure. This family of compounds is amenable to parallel-manifold synthesis because stilbene analogs are easily synthesized using a single-step olefination reaction. RESULTS: We have prepared a small 23-component hydroxystilbene library using a solid phase synthesis approach. The library was screened for estrogenic and antiestrogenic activity using a cell-based bioassay that measures estrogen receptor-mediated transcription of a reporter gene. Three of the analogs proved to have dose-dependent estrogenic activity with EC50 values between 5 microM and 15 microM. Further characterization of the hydroxystilbene-mediated estrogenic activity suggests that the agonist activity results from direct binding to the steroid site on the estrogen receptor with IC50 values of 1-10 microM. CONCLUSIONS: The results of this study show that classic olefination chemistry can be adapted to a solid-phase format for parallel synthesis of analog libraries. Although yields varied for the individual analogs, sufficient quantity of pure material was obtained directly from the resin for structural characterization and biological evaluation. This study further validates solid-phase organic synthesis as a useful approach for rapid parallel-manifold library synthesis to augment both lead compound discovery and optimization.

Binding, Competitive↗

PubMed1995

Evolution in action.

The alpha/beta barrel enzyme phosphotriesterase from soil bacteria appears to have evolved the ability to hydrolyze the insecticide paraoxon at the diffusion limit in only a few decades. A newly-identified open reading frame from Escherichia coli may offer a clue to its origins.

Amino Acid Sequence↗

PubMed1994

Crystal structure of a catalytic antibody with a serine protease active site.

The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.

Amino Acid Sequence↗