Mapeamento genético laboral: prospecção axiológica, bioética e legislação brasileira / Occupational genetic mapping: axiological prospecting, bioethics and Brazilian legislation / Mapeo genético laboral: prospección axiológica, bioética y legislación brasileña

Resumo Objetiva-se comparar o estado da legislação brasileira sobre mapeamento genético com o de legislações internacionais visando dimensionar a realidade normativa do país quanto às tendências sociais de reconhecimento das diferenças e a abertura jurídica prospectiva, com foco na área laboral. Trata-se de revisão de literatura e pesquisa documental sobre o diálogo entre bioética, medicina do trabalho e genética, que têm a dignidade humana como ponto em comum. Concluiu-se que se tende a admitir o mapeamento genético de trabalhadores para pesquisa e prevenção do adoecimento, inferindo-se, dado seu referencial comum e de acordo com a perspectiva culturalista do Código Civil, que essa possibilidade se estende à identificação genética de habilidades do trabalhador para o exercício de atividades.

Abstract This work aims to verify the status of Brazilian legislation on genetic mapping, focusing on the occupational sphere, in comparison to international legislation, to assess the country's normative reality regarding social trends related to the recognition of differences and prospective legal opening. This is a review of literature and documents regarding the dialogue between bioethics, occupational medicine and genetics, taking into account that they have human dignity as a common ground. It was concluded that there is a tendency to accept the genetic mapping of workers for research and prevention of illness. Given their common reference and in accordance with the culturalist perspective of the Civil Code, it is inferred that this possibility extends to the genetic identification of workers' skills for the exercise of their duties.

Resumen El objetivo de este trabajo es comparar el estado de la legislación brasileña sobre mapeo genético en relación con el de las legislaciones internacionales, buscando dimensionar la realidad normativa del país ante las tendencias sociales de reconocimiento de las diferencias y la apertura jurídica prospectiva, con foco en el área laboral. Se trata de una revisión de la literatura y de una investigación documental sobre el diálogo entre bioética, medicina del trabajo y genética, considerando que tienen a la dignidad humana como punto en común. Se concluyó que se tiende a admitir el mapeo genético de los trabajadores para la investigación y prevención de enfermedades, infiriéndose, dada su referencia común y de acuerdo con la perspectiva culturalista del Código Civil, que esta posibilidad se extiende a la identificación genética de habilidades del trabajador para para el ejercicio de actividades.

Structure-based virtual screening and discovery of New PPARδ/γ dual agonist and PPARδ and γ agonists.

Peroxisome proliferator-activated receptors (PPARs) are involved in the control of carbohydrate and lipid metabolism and are considered important targets to treat diabetes mellitus and metabolic syndrome. The available PPAR ligands have several side effects leading to health risks justifying the search for new bioactive ligands to activate the PPAR subtypes, in special PPARδ, the less studied PPAR isoform. Here, we used a structure-based virtual screening protocol in order to find out new PPAR ligands. From a lead-like subset of purchasable compounds, we identified 5 compounds with potential PPAR affinity and, from preliminary in vitro assays, 4 of them showed promising biological activity. Therefore, from our in silico and in vitro protocols, new PPAR ligands are potential candidates to treat metabolic diseases.

Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists.

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/ß-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the ß-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the ß-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

Helix 12 dynamics and thyroid hormone receptor activity: experimental and molecular dynamics studies of Ile280 mutants.

Nuclear hormone receptors (NRs) form a family of transcription factors that mediate cellular responses initiated by hormone binding. It is generally recognized that the structure and dynamics of the C-terminal helix 12 (H12) of NRs' ligand binding domain (LBD) are fundamental to the recognition of coactivators and corepressors that modulate receptor function. Here we study the role of three mutations in the I280 residue of H12 of thyroid hormone receptors using site-directed mutagenesis, functional assays, and molecular dynamics simulations. Although residues at position 280 do not interact with coactivators or with the ligand, we show that its mutations can selectively block coactivator and corepressor binding, and affect hormone binding affinity differently. Molecular dynamics simulations suggest that ligand affinity is reduced by indirectly displacing the ligand in the binding pocket, facilitating water penetration and ligand destabilization. Mutations I280R and I280K link H12 to the LBD by forming salt bridges with E457 in H12, stabilizing H12 in a conformation that blocks both corepressor and coactivator recruitment. The I280M mutation, in turn, blocks corepressor binding, but appears to enhance coactivator affinity, suggesting stabilization of H12 in agonist conformation.

Síndrome de resistência ao hormônio tireoidiano e dominância negativa: revisão de literatura / Thyroid resistance hormone syndrome and negative dominance: a review of literature

A síndrome de resistência ao hormônio tireoidiano é doença genética, caracterizada pela resposta reduzida dos tecidos-alvo ao hormônio tireoidiano. É autossômica dominante, causada, na maioria das vezes, por mutações na isoforma beta do receptor de hormônio tireoidiano (TR?). Os pacientes com a síndrome apresentam níveis séricos elevados de tiroxina livre (T4) e tri-iodotironina livre (T3) associados a feedback negativo anormal na regulação do hormônio estimulador da tireoide (TSH) e do hormônio liberador de TSH (TRH). Por essa razão, os níveis do hormônioestimulador estão pouco aumentados ou inapropriadamente normais. O diagnóstico final é feito pelo sequenciamento do gene da isoforma beta do receptor de hormônio tireoidiano. Os indivíduos afetados são, na maioriadas vezes, heterozigotos com mutações no gene TR?. Na fisiopatogenia da doença, tem sido descrito que o gene mutado inibe a função do gene normal, em um fenômeno conhecido como dominância negativa que, por sua vez, ocorre nos genes regulados negativamente e positivamente pelo hormônio tireoidiano. O mecanismo molecular da dominância negativa envolve: (1) competição da ligação do receptor mutante ao DNA, (2) dimerização do receptor mutante com o receptor do retinoide X ou com o receptor normal e (3) interação do receptor mutado com correguladores,o que acarreta em falha da regulação transcricional pelo hormônio tireoidiano. A intensidade da síndrome de resistência ao hormônio tireoidiano depende, então, em parte, do nível de expressão do receptor mutado e do tipo de mutação.

Thyroid resistance hormone syndrome is a genetic disease characterized by a reduced responsiveness of target tissues to thyroid hormone. Such patients show persistent elevation of circulating free thyroxine (T4) and free triiodothyronine (T3) levels associated with nonsuppressed serum thyrotropin (TSH). Inheritance is autosomal dominant and mutations inthe TR? gene have been identified in the majority patients with the syndrome. Most patients are heterozygous, with only one mutated TR? gene. In the pathogenesis of the disease, it has been described that the TR? mutant impairs the normal function of TR wild type (TRwt), a phenomenon described as a dominant-negative effect, which occurs in positive and negative TH regulated genes. The mutant receptor diminishes TRwt function by competing with normal receptor for DNA binding, by dimerizing with retinoid X receptor or normal receptor, and also by interacting with coregulators and disrupting the transcription activity modulated by thyroid hormone.

Differential effects of TR ligands on hormone dissociation rates: evidence for multiple ligand entry/exit pathways.

Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself. This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here, we show that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation (T(1/2)<2h) of radiolabeled T(3) vs. T(3) (T(1/2) approximately 5-7h). We cannot discern relationships between this effect and ligand size, activity or affinity for TRbeta. One ligand, GC-24, binds the TR LBC and (weakly) to the TRbeta-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T(3) dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T(3) release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T(3) release and that this effect enhances hormone dissociation.

Molecular basis for dimer formation of TRbeta variant D355R.

Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.

Complex actions of thyroid hormone receptor antagonist NH-3 on gene promoters in different cell lines.

It is desirable to obtain new antagonists for thyroid hormone receptors (TRs) and other nuclear receptors (NRs). We previously used X-ray structural models of TR ligand binding domains (LBDs) to design compounds, such as NH-3, that impair coactivator binding to activation function 2 (AF-2) and block thyroid hormone (triiodothyronine, T(3)) actions. However, TRs bind DNA and are transcriptionally active without ligand. Thus, NH-3 could modulate TR activity via effects on other coregulator interaction surfaces, such as activation function (AF-1) and corepressor binding sites. Here, we find that NH-3 blocks TR-LBD interactions with coactivators and corepressors and also inhibits activities of AF-1 and AF-2 in transfections. While NH-3 lacks detectable agonist activity at T(3)-activated genes in GC pituitary cells it nevertheless activates spot 14 (S14) in HTC liver cells with the latter effect accompanied by enhanced histone H4 acetylation and coactivator recruitment at the S14 promoter. Surprisingly, T(3) promotes corepressor recruitment to target promoters. NH-3 effects vary; we observe transient recruitment of N-CoR to S14 in GC cells and dismissal and rebinding of N-CoR to the same promoter in HTC cells. We propose that NH-3 will generally behave as an antagonist by blocking AF-1 and AF-2 but that complex effects on coregulator recruitment may result in partial/mixed agonist effects that are independent of blockade of T(3) binding in some contexts. These properties could ultimately be utilized in drug design and development of new selective TR modulators.

Structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic beta-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor beta (TRbeta), in a high-throughput screen. Initial evidence suggested that the aromatic beta-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRbeta surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRbeta with the inhibitor HPPE at 2.3-A resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRbeta and its coactivator. Several lines of evidence, including experiments with TRbeta mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRbeta, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) beta-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRbeta. DHPPA represents a novel class of potent TRbeta antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Thyroid hormone response element organization dictates the composition of active receptor.

Thyroid hormone (triiodothyronine, T(3)) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T(3) response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TR beta activates transcription more efficiently than TR alpha at an inverted palindrome (F2), and this correlates with preferential TR beta homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TR beta homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TR beta mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TR beta homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TR beta requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR.TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo.

A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function.

The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.

Negative regulation of superoxide dismutase-1 promoter by thyroid hormone.

The role of thyroid hormone [L-3,5,3'-triiodothyronine (T3)] and the thyroid hormone receptor (TR) in regulating growth, development, and metabolic homeostasis is well established. It is also emerging that T3 is associated with oxidative stress through the regulation of the activity of superoxide dismutase-1 (SOD-1), a key enzyme in the metabolism of oxygen free radicals. We found that T3 reverses the activation of the SOD-1 promoter caused by the free radical generators paraquat and phorbol 12-myristate 13-acetate through the direct repression of the SOD-1 promoter by liganded TR. Conversely, the SOD-1 promoter is significantly stimulated by unliganded TRs. This regulation requires the DNA-binding domain of the TR, which is recruited to an inhibitory element between -157 and +17 of the SOD-1 promoter. TR mutations, which abolish recruitment of coactivator proteins, block repression of the SOD-1 promoter. Conversely, a mutation that inhibits corepressor binding to the TR prevents activation. Together, our findings suggest a mechanism of negative regulation in which TR binds to the SOD-1 promoter but coactivator and corepressor binding surfaces have an inverted function. This effect may be important in T3 induction of oxidative stress in thyroid hormone excess.

Discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators.

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Rheological properties of fast skeletal myosin rod and light meromyosin from walleye pollack and white croaker: contribution of myosin fragments to thermal gel formation.

Myosin rod and light meromyosin (LMM) of walleye pollack and white croaker were examined for their rheological properties by measuring dynamic viscoelastic parameters. Rods from walleye pollack and white croaker increased their storage moduli (G') in the ranges of 29-43 degrees C and 31-38 degrees C, respectively, in temperature sweep analysis. Walleye pollack LMM showed no peak of G' upon heating, whereas the white croaker counterpart exhibited a single sharp peak of G' at 35 degrees C. Loss modulus (G") showed similar temperature-dependent changes for the two fish species as the case of G', irrespective of rod and LMM, although G" values were lower than those of G'. Thus, rheological properties of rod and LMM were different between walleye pollack and white croaker. Taken together with data previously reported for myosin, it was considered that both myosin rods from walleye pollack and white croaker are attributed to thermal gel formation of myosin in a low-temperature range, though in a species-specific manner.

Rearrangements in thyroid hormone receptor charge clusters that stabilize bound 3,5',5-triiodo-L-thyronine and inhibit homodimer formation.

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.

Conformational adaptation of nuclear receptor ligand binding domains to agonists: potential for novel approaches to ligand design.

Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5' extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5' iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRbeta structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3' benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.

Thyroxine-thyroid hormone receptor interactions.

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.

Ligand selectivity by seeking hydrophobicity in thyroid hormone receptor.

Selective therapeutics for nuclear receptors would revolutionize treatment for endocrine disease. Specific control of nuclear receptor activity is challenging because the internal cavities that bind hormones can be virtually identical. Only one highly selective hormone analog is known for the thyroid receptor, GC-24, an agonist for human thyroid hormone receptor beta. The compound differs from natural hormone in benzyl, substituting for an iodine atom in the 3' position. The benzyl is too large to fit into the enclosed pocket of the receptor. The crystal structure of human thyroid hormone receptor beta at 2.8-A resolution with GC-24 bound explains its agonist activity and unique isoform specificity. The benzyl of GC-24 is accommodated through shifts of 3-4 A in two helices. These helices are required for binding hormone and positioning the critical helix 12 at the C terminus. Despite these changes, the complex associates with coactivator as tightly as human thyroid hormone receptor bound to thyroid hormone and is fully active. Our data suggest that increased specificity of ligand recognition derives from creating a new hydrophobic cluster with ligand and protein components.