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T S Scanlan

Publications and source records attributed to T S Scanlan.

34 records · Page 2Linked to original sources
PubMed1999

Nuclear-receptor ligands and ligand-binding domains.

The determination of several structures of nuclear receptor ligand binding domains (LBD) has led to new insights into the mechanism of action of this very important class of receptors. This review describes and compares the different LBD structures and their relationship to the function of the nuclear receptors. The role of the ligand in the LBD structures and the implications of ligand structure on receptor activity are also discussed. Structural information regarding interactions between the LBD and coactivator proteins and the potential role of these interactions in ligand agonism and antagonism is reviewed. Different pathways for nuclear receptor signaling and the use of new ligands to investigate these pathways are also described.

Ligands↗
PubMed1998

A comparison of the crystallographic structures of two catalytic antibodies with esterase activity.

The crystallographic structure of the Fab fragment of the catalytic antibody, 29G11, complexed with an (S)-norleucine phenyl phosphonate transition state analog was determined at 2.2 A resolution. The antibody catalyzes the hydrolysis of norleucine phenyl ester with (S)-enantioselectivity. The shape and charge complementarity of the binding pocket for the hapten account for the preferential binding of the (S)-enantiomer of the substrate. The structure is compared to that of the more catalytically efficient antibody, 17E8, induced by the same hapten transition state analog. 29G11 has different residues from 17E8 at eight positions in the heavy chain, including four substitutions in the hapten-binding pocket: A33V, S95G, S99R and Y100AN, and four substitutions at positions remote from the catalytic site, I28T, R40K, V65G and F91L. The two antibodies show large differences in the orientations of their variable and constant domains, reflected by a 32 degrees difference in their elbow angles. The VL and VH domains in the two antibodies differ by a rotation of 8.8 degrees. The hapten binds in similar orientations and locations in 29G11 and 17E8, which appear to have catalytic groups in common, though the changes in the association of the variable domains affect the precise positioning of residues in the hapten-binding pocket.

Antibodies↗
PubMed1998

Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family.

Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase, a hydrolytic, bacterial enzyme with an unusual substrate specificity for synthetic organophosphate triesters and phosphorofluoridates, which are common constituents of chemical warfare agents and agricultural pesticides. No natural substrate has been identified for phosphotriesterase, and it has been suggested that the enzyme may have evolved the ability to hydrolyze synthetic compounds in bacteria under selective pressure to meet nutritional needs. PHP, which has 28% sequence identity with phosphotriesterase, may belong to the family of proteins from which phosphotriesterase evolved. Here we report the cloning, expression, initial characterization, and high-resolution X-ray crystallographic structure of PHP. Biochemical analysis shows that PHP is monomeric and binds two zinc ions per monomer. Unlike phosphotriesterase, PHP does not catalyze the hydrolysis of nonspecific phosphotriesters. The structure, similar to that of phosphotriesterase, consists of a long, elliptical alpha/beta barrel and has a binuclear zinc center in a cleft at the carboxy end of the barrel at the location of the presumptive active site.

Amino Acid Sequence↗
PubMed1998

X-ray crystallographic and functional studies of thyroid hormone receptor.

We have solved several X-ray crystallographic structures of TR ligand-binding domains (LBDs), including the rat (r) TR alpha and the human (h) TR beta bound to diverse ligands. The TR-LBD folding, comprised mostly of alpha-helices, is likely to be general for the superfamily. The ligand, buried in the receptor, forms part of its hydrophobic core. Tight fitting of ligand into the receptor explains its high affinity for the TR, although the structure suggests that ligands with even higher affinities might be generated. The kinetics of 3,5,3'-triiodo-L-thyronine (T3) and 3,5,3',5'-tetraiodo-L-thyronine (T4) binding suggest that folding around the ligand, rather than receptor opening, is rate-limiting for high affinity binding. TR beta mutations in patients with resistance to T3 cluster around the ligand; these different locations could differentially affect on other receptor functions and explain the syndrome's clinical diversity. Guided by the structure, mutations have been placed on the TR surface to define interactions with other proteins. They suggest that a similar surface in the LBD is utilized for homo- or heterodimerization on direct repeats and inverted palindromes but not on palindromes. Coactivator proteins that mediate TR transcriptional activation bind to a small surface comprised of residues on four helices with a well-defined hydrophobic cleft, which may be a target for pharmaceuticals. The coactivator-binding surface appears to form upon ligand-binding by the folding of helix 12 into the scaffold formed by helices 3, 4 and 5. The analysis of most currently used antagonists suggest that although they probably fit into the ligand-binding pocket, they possess a group that may alter proper folding of the receptor, with disruption of the coactivator-binding surface (the 'extension model').

Animals↗
PubMed1998

An efficient substitution reaction for the preparation of thyroid hormone analogues.

The substitution of the sterically hindered carbon of the potent thyroid hormone agonist, GC-1, was effected by a reaction based on the solvolysis of the benzylic hydroxyl group. The reaction was found to proceed in high yield with a variety of nucleophiles including alcohols, thiols, allyl silanes and electron-rich aromatic compounds, providing a convenient route to the synthesis of new thyroid hormone analogues.

Benzophenones↗
PubMed1998

A high-affinity subtype-selective agonist ligand for the thyroid hormone receptor.

BACKGROUND: Thyroid hormones regulate many different physiological processes in different tissues in vertebrates. Most of the actions of thyroid hormones are mediated by the thyroid hormone receptor (TR), which is a member of the nuclear receptor superfamily of ligand-activated transcription regulators. There are two different genes that encode two different TRs, TR alpha and TR beta, and these two TRs are often co-expressed at different levels in different tissues. Most thyroid hormones do not discriminate between the two TRs and bind both with similar affinities. RESULTS: We have designed and synthesized a thyroid hormone analog that has high affinity for the TRs and is selective in both binding and activation functions for TR beta over TR alpha. The compound, GC-1, was initially designed to solve synthetic problems that limit thyroid hormone analog preparation, and contains several structural changes with respect to the natural hormone 3,5,3'-triiodo-L-thyronine (T3). These changes include replacement of the three iodines with methyl and isopropyl groups, replacement of the biaryl ether linkage with a methylene linkage, and replacement of the amino-acid sidechain with an oxyacetic-acid sidechain. CONCLUSIONS: The results of this study show that GC-1 is a member of a new class of thyromimetic compounds that are more synthetically accessible than traditional thyromimetics and have potentially useful receptor binding and activation properties. The TR beta selectivity of GC-1 is particularly interesting and suggests that GC-1 might be a useful in vivo probe for studying the physiological roles of the different thyroid hormone receptor isoforms.

Animals↗
PubMed1998

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (alpha and beta) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins. 2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator. 3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligand-binding domains (LBD) of both the rat (r) TR alpha and the human (h) TR beta bound to several different ligands. We have also developed ligands that bind selectively to the TR beta, which may provide ways to explore the differential functions of TR alpha compared with TR beta isoforms. 4. The LBD is comprised mostly of alpha-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonist-induced conformation changes in the receptor. 5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket. 6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo- and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding of coactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.

Animals↗
PubMed1997

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding.

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. "Humanized" 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH --> Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.

Animals↗
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↗
PubMedDate not supplied

Selective modulation of thyroid hormone receptor action.

Thyroid hormones have some actions that might be useful therapeutically, but others that are deleterious. Potential therapeutically useful actions include those to induce weight loss and lower plasma cholesterol levels. Potential deleterious actions are those on the heart to induce tachycardia and arrhythmia, on bone to decrease mineral density, and on muscle to induce wasting. There have been successes in selectively modulating the actions of other classes of hormones through various means, including the use of pharmaceuticals that have enhanced affinities for certain receptor isoforms. Thus, there is reason to pursue selective modulation of thyroid hormone receptor (TR) function, and several agents have been shown to have some beta-selective, hepatic selective and/or cardiac sparring activities, although development of these was largely not based on detailed understanding of mechanisms for the specificity. The possibility of selectively targeting the TRbeta was suggested by the findings that there are alpha- and beta-TR forms and that the TRalpha-forms may preferentially regulate the heart rate, whereas many other actions of these hormones are mediated by the TRbeta. We determined X-ray crystal structures of the TRalpha and TRbeta ligand-binding domains (LBDs) complexed with the thyroid hormone analog 3,5,3'-triiodithyroacetic acid (Triac). The data suggested that a single amino acid difference in the ligand-binding cavities of the two receptors could affect hydrogen bonding in the receptor region, where the ligand's 1-position substituent fits and might be exploited to generate beta-selective ligands. The compound GC-1, with oxoacetate in the 1-position instead of acetate as in Triac, exhibited TRbeta-selective binding and actions in cultured cells. An X-ray crystal structure of the GC-1-TRbeta LBD complex suggests that the oxoacetate does participate in a network of hydrogen bonding in the TR LBD polar pocket. GC-1 displayed actions in tadpoles that were TRbeta-selective. When administered to mice, GC-1 was as effective in lowering plasma cholesterol levels as T(3), and was more effective than T(3) in lowering plasma triglyceride levels. At these doses, GC-1 did not increase the heart rate. GC-1 was also less active than T(3) in modulating activities of several other cardiac parameters, and especially a cardiac pacemaker channel such as HCN-2, which may participate in regulation of the heart rate. GC-1 showed intermediate activity in suppressing plasma thyroid stimulating hormone (TSH) levels. The tissue/plasma ratio for GC-1 in heart was also less than for the liver. These data suggest that compounds can be generated that are TR-selective and that compounds with this property and/or that exhibit selective uptake, might have clinical utility as selective TR modulators.

Animals↗