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Chao Zhang,
Prabha N Ibrahim,
Jiazhong Zhang,
Elizabeth A Burton,
Gaston Habets,
Ying Zhang,
Ben Powell, Brian L West,
Bernice Matusow,
Garson Tsang, [......],
Clarence R Hurt,
Dean R Artis,
Guoxian Wu,
Marika Nespi,
Wayne Spevak,
Paul Lin,
Keith Nolop,
Peter Hirth,
Greg H Tesch,
Gideon Bollag
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ABSTRACT: Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.
Proceedings of the National Academy of Sciences 03/2013; · 9.68 Impact Factor
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Debyani Chakravarty,
Elmer Santos,
Mabel Ryder,
Jeffrey A Knauf,
Xiao-Hui Liao, Brian L West,
Gideon Bollag,
Richard Kolesnick,
Tin Htwe Thin,
Neal Rosen,
Pat Zanzonico,
Steven M Larson,
Samuel Refetoff,
Ronald Ghossein,
James A Fagin
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ABSTRACT: Advanced human thyroid cancers, particularly those that are refractory to treatment with radioiodine (RAI), have a high prevalence of BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. However, the degree to which these cancers are dependent on BRAF expression is still unclear. To address this question, we generated mice expressing one of the most commonly detected BRAF mutations in human papillary thyroid carcinomas (BRAF(V600E)) in thyroid follicular cells in a doxycycline-inducible (dox-inducible) manner. Upon dox induction of BRAF(V600E), the mice developed highly penetrant and poorly differentiated thyroid tumors. Discontinuation of dox extinguished BRAF(V600E) expression and reestablished thyroid follicular architecture and normal thyroid histology. Switching on BRAF(V600E) rapidly induced hypothyroidism and virtually abolished thyroid-specific gene expression and RAI incorporation, all of which were restored to near basal levels upon discontinuation of dox. Treatment of mice with these cancers with small molecule inhibitors of either MEK or mutant BRAF reduced their proliferative index and partially restored thyroid-specific gene expression. Strikingly, treatment with the MAPK pathway inhibitors rendered the tumor cells susceptible to a therapeutic dose of RAI. Our data show that thyroid tumors carrying BRAF(V600E) mutations are exquisitely dependent on the oncoprotein for viability and that genetic or pharmacological inhibition of its expression or activity is associated with tumor regression and restoration of RAI uptake in vivo in mice. These findings have potentially significant clinical ramifications.
The Journal of clinical investigation 11/2011; 121(12):4700-11. · 15.39 Impact Factor
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David G DeNardo,
Donal J Brennan,
Elton Rexhepaj,
Brian Ruffell,
Stephen L Shiao,
Stephen F Madden,
William M Gallagher,
Nikhil Wadhwani,
Scott D Keil,
Sharfaa A Junaid,
Hope S Rugo,
E Shelley Hwang,
Karin Jirström, Brian L West,
Lisa M Coussens
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ABSTRACT: Immune-regulated pathways influence multiple aspects of cancer development. In this article we demonstrate that both macrophage abundance and T-cell abundance in breast cancer represent prognostic indicators for recurrence-free and overall survival. We provide evidence that response to chemotherapy is in part regulated by these leukocytes; cytotoxic therapies induce mammary epithelial cells to produce monocyte/macrophage recruitment factors, including colony stimulating factor 1 (CSF1) and interleukin-34, which together enhance CSF1 receptor (CSF1R)-dependent macrophage infiltration. Blockade of macrophage recruitment with CSF1R-signaling antagonists, in combination with paclitaxel, improved survival of mammary tumor-bearing mice by slowing primary tumor development and reducing pulmonary metastasis. These improved aspects of mammary carcinogenesis were accompanied by decreased vessel density and appearance of antitumor immune programs fostering tumor suppression in a CD8+ T-cell-dependent manner. These data provide a rationale for targeting macrophage recruitment/response pathways, notably CSF1R, in combination with cytotoxic therapy, and identification of a breast cancer population likely to benefit from this novel therapeutic approach. SIGNIFICANCE: These findings reveal that response to chemotherapy is in part regulated by the tumor immune microenvironment and that common cytotoxic drugs induce neoplastic cells to produce monocyte/macrophage recruitment factors, which in turn enhance macrophage infiltration into mammary adenocarcinomas. Blockade of pathways mediating macrophage recruitment, in combination with chemotherapy, significantly decreases primary tumor progression, reduces metastasis, and improves survival by CD8+ T-cell-dependent mechanisms, thus indicating that the immune microenvironment of tumors can be reprogrammed to instead foster antitumor immunity and improve response to cytotoxic therapy.
Cancer discovery. 06/2011; 1(1):54-67.
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Gideon Bollag,
Peter Hirth,
James Tsai,
Jiazhong Zhang,
Prabha N Ibrahim,
Hanna Cho,
Wayne Spevak,
Chao Zhang,
Ying Zhang,
Gaston Habets, [......],
Igor Puzanov,
Kevin B Kim,
Antoni Ribas,
Grant A McArthur,
Jeffrey A Sosman,
Paul B Chapman,
Keith T Flaherty,
Xiaowei Xu,
Katherine L Nathanson,
Keith Nolop
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ABSTRACT: B-RAF is the most frequently mutated protein kinase in human cancers. The finding that oncogenic mutations in BRAF are common in melanoma, followed by the demonstration that these tumours are dependent on the RAF/MEK/ERK pathway, offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients. Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity. Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts. Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032 (ref. 5). In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment. This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumour regressions. At higher drug exposures afforded by a new amorphous drug formulation, greater than 80% inhibition of ERK phosphorylation in the tumours of patients correlated with clinical response. Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily. These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.
Nature 09/2010; 467(7315):596-9. · 36.28 Impact Factor
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Dean R Artis,
Jack J Lin,
Chao Zhang,
Weiru Wang,
Upasana Mehra,
Mylene Perreault,
David Erbe,
Heike I Krupka,
Bruce P England,
James Arnold, [......],
Gaston Habets, Brian L West,
Kam Y J Zhang,
Michael V Milburn,
George P Vlasuk,
K Peter Hirth,
Keith Nolop,
Gideon Bollag,
Prabha N Ibrahim,
James F Tobin
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ABSTRACT: In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARalpha, PPARgamma, and PPARdelta. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARgamma, to circumvent the clinically observed side effects of full PPARgamma agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARgamma. Compared with full PPARgamma-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).
Proceedings of the National Academy of Sciences 01/2009; 106(1):262-7. · 9.68 Impact Factor
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James Tsai,
John T Lee,
Weiru Wang,
Jiazhong Zhang,
Hanna Cho,
Shumeye Mamo,
Ryan Bremer,
Sam Gillette,
Jun Kong,
Nikolas K Haass, [......],
Kam Y J Zhang, Brian L West,
Ben Powell,
Gaston Habets,
Chao Zhang,
Prabha N Ibrahim,
Peter Hirth,
Dean R Artis,
Meenhard Herlyn,
Gideon Bollag
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ABSTRACT: BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.
Proceedings of the National Academy of Sciences 03/2008; 105(8):3041-6. · 9.68 Impact Factor
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James W. Apriletti,
Ralff CJ Ribeiro,
Richard L. Wagner,
Weijun Feng,
Paul Webb,
Peter J. Kushner, Brian L. West,
Stefan Nilsson,
Thomas S. Scanlan,
Robert J. Fletterick,
John D. Baxter
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ABSTRACT: SUMMARY1. Thyroid hormone receptors (TR) are expressed from two separate genes (α and β) 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) TRα and the human (h) TRβ bound to several different ligands. We have also developed ligands that bind selectively to the TRβ, which may provide ways to explore the differential functions of TRα compared with TRβ isoforms.4. The LBD is comprised mostly of α-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.
Clinical and Experimental Pharmacology and Physiology 06/2007; 25(S1):S2 - S11. · 1.85 Impact Factor
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Weiru Wang,
Adhirai Marimuthu,
James Tsai,
Abhinav Kumar,
Heike I Krupka,
Chao Zhang,
Ben Powell,
Yoshihisa Suzuki,
Hoa Nguyen,
Maryam Tabrizizad,
Catherine Luu, Brian L West
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ABSTRACT: Protein kinases are a large family of cell signaling mediators undergoing intensive research to identify inhibitors or modulators useful for medicine. As one strategy, small-molecule compounds that bind the active site with high affinity can be used to inhibit the enzyme activity. X-ray crystallography is a powerful method to reveal the structures of the kinase active sites, and thus aid in the design of high-affinity, selective inhibitors. However, a limitation still exists in the ability to produce purified kinases in amounts sufficient for crystallography. Furthermore, kinases exist in different conformation states as part of their normal regulation, and the ability to prepare crystals of kinases in these various states also remains a limitation. In this study, the c-Abl, c-Src, and c-Met kinases are produced in high yields in Escherichia coli by using a bicistronic vector encoding the PTP1B tyrosine phosphatase. A 100-fold lower dose of the inhibitor, Imatinib, was observed to inhibit the unphosphorylated form of c-Abl kinase prepared by using this vector, compared to the phosphorylated form produced without PTP1B, consistent with the known selectivity of this inhibitor for the unactivated conformation of the enzyme. Unphosphorylated c-Met kinase produced with this vector was used to obtain the crystal structure, at 2.15-A resolution, of the autoinhibited form of the kinase domain, revealing an intricate network of interactions involving c-Met residues documented previously to cause dysregulation when mutated in several cancers.
Proceedings of the National Academy of Sciences 04/2006; 103(10):3563-8. · 9.68 Impact Factor
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Weiru Wang,
Chao Zhang,
Adhirai Marimuthu,
Heike I Krupka,
Maryam Tabrizizad,
Rafe Shelloe,
Upasana Mehra,
Kevin Eng,
Hoa Nguyen,
Calvin Settachatgul,
Ben Powell,
Michael V Milburn, Brian L West
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ABSTRACT: Steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1) belong to the fushi tarazu factor 1 subfamily of nuclear receptors. SF-1 is an essential factor for sex determination during development and regulates adrenal and gonadal steroidogenesis in the adult, whereas LRH-1 is a critical factor for development of endodermal tissues and regulates cholesterol and bile acid homeostasis. Regulatory ligands are unknown for SF-1 and LRH-1. A reported mouse LRH-1 structure revealed an empty pocket in a region commonly occupied by ligands in the structures of other nuclear receptors, and pocket-filling mutations did not alter the constitutive activity observed. Here we report the crystal structures of the putative ligand-binding domains of human SF-1 at 2.1-A resolution and human LRH-1 at 2.5-A resolution. Both structures bind a coactivator-derived peptide at the canonical activation-function surface, thus adopting the transcriptionally activating conformation. In human LRH-1, coactivator peptide binding also occurs to a second site. We discovered in both structures a phospholipid molecule bound in a pocket of the putative ligand-binding domain. MS analysis of the protein samples used for crystallization indicated that the two proteins associate with a range of phospholipids. Mutations of the pocket-lining residues reduced the transcriptional activities of SF-1 and LRH-1 in mammalian cell transfection assays without affecting their expression levels. These results suggest that human SF-1 and LRH-1 may be ligand-binding receptors, although it remains to be seen if phospholipids or possibly other molecules regulate SF-1 or LRH-1 under physiological conditions.
Proceedings of the National Academy of Sciences 06/2005; 102(21):7505-10. · 9.68 Impact Factor
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Kam Y J Zhang,
Graeme L Card,
Yoshihisa Suzuki,
D Richard Artis,
Daniel Fong,
Sam Gillette,
Davin Hsieh,
Joshua Neiman, Brian L West,
Chao Zhang,
Michael V Milburn,
Sung-Hou Kim,
Joseph Schlessinger,
Gideon Bollag
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ABSTRACT: Phosphodiesterases (PDEs) comprise a family of enzymes that modulate the immune response, inflammation, and memory, among many other functions. There are three types of PDEs: cAMP-specific, cGMP-specific, and dual-specific. Here we describe the mechanism of nucleotide selectivity on the basis of high-resolution co-crystal structures of the cAMP-specific PDE4B and PDE4D with AMP, the cGMP-specific PDE5A with GMP, and the apo-structure of the dual-specific PDE1B. These structures show that an invariant glutamine functions as the key specificity determinant by a "glutamine switch" mechanism for recognizing the purine moiety in cAMP or cGMP. The surrounding residues anchor the glutamine residue in different orientations for cAMP and for cGMP. The PDE1B structure shows that in dual-specific PDEs a key histidine residue may enable the invariant glutamine to toggle between cAMP and cGMP. The structural understanding of nucleotide binding enables the design of new PDE inhibitors that may treat diseases in which cyclic nucleotides play a critical role.
Molecular Cell 08/2004; 15(2):279-86. · 14.18 Impact Factor
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ABSTRACT: Resistance to hormones is commonly due to mutations in genes encoding receptors. Resistance to thyroid hormone is due mostly to mutations of the beta-form of the human (h) thyroid hormone receptor (hTRbeta). We determined x-ray crystal structures of two hTRbeta ligand-binding domains (LBDs), Ala 317 Thr and Arg 316 His. Amino acids 316 and 317 form part of the hormone-binding pocket. The methyl of Ala 317, contacting iodine, sculpts the T3 hormone-binding pocket. Arg 316 is not in direct contact with T3 and has an unknown role in function. Remarkably, the Arg forms part of an unusual buried polar cluster in hTRbeta. Although the identity of the amino acids changes, the polar cluster appears in all nuclear receptors. In spite of the differing roles of 316 and 317, both resistance to thyroid hormone mutants display decreased T3 affinity and weakened transcriptional activation. The two mutants differ in that the Arg 316 His receptor does not form TR-TR homodimers on DNA. 3,5,3'-Triiodothyroacetic acid is bound to both receptors. Thr 317 repositions 3,5,3'-triiodothyroacetic acid distending the face of the receptor that binds coregulators. Arg 316 forms two hydrogen bonds with helix 1. Both are lost with mutation to His displacing helix 1 of the LBD and disordering the loop after helix 1. The stability of the helix 1, deriving in part from the buried polar cluster, is important for hormone binding and formation of TR dimers. The observation that the Arg 316 His mutation affects these functions implies a role for helix 1 in linking hormone binding to the DNA-binding domain-LBD configuration.
Molecular Endocrinology 05/2003; 17(4):643-52. · 4.54 Impact Factor
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ABSTRACT: Resistance to thyroid hormone (RTH) syndrome is associated with mutations in the human thyroid hormone receptor-beta (hTRbeta), many of which show marked reduction in hormone binding. Here, we investigated the structural consequences of two RTH mutants (A234T and R243Q), residing in the flexible N-terminal portion of the ligand binding domain (LBD), which exhibit modestly reduced hormone binding with impaired release of corepressor. X-ray crystallography analyses revealed that these two RTH mutants modulate the position of this flexible region by either altering the movement of helix 1 (A234T) or disrupting a salt bridge (R243Q). The subsequent increased flexibility and mobility in regions after the two sites of mutation coincided with a disorganized LBD. Consistent with this finding, the ability of these mutant N-terminal regions (234-260) to recruit the remaining LBD was decreased in a ligand-dependent helix assembly assay. Collectively, these data suggest that structural information imparted by the flexible segment in the N-terminal LBD is critical for overall stability of the LBD. Thus, these structural analyses provide mechanistic insight into the etiology of RTH disease in human TRbeta mutants that exhibit hormone binding with decreased ligand-dependent corepressor release.
Molecular Endocrinology 02/2003; 17(1):107-16. · 4.54 Impact Factor
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Paul Webb,
Ngoc-Ha Nguyen,
Grazia Chiellini,
Hikari A I Yoshihara,
Suzana T Cunha Lima,
James W Apriletti,
Ralff C J Ribeiro,
Adhirai Marimuthu, Brian L West,
Patrick Goede,
Karin Mellstrom,
Stefan Nilsson,
Peter J Kushner,
Robert J Fletterick,
Thomas S Scanlan,
John D Baxter
[show abstract]
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ABSTRACT: It is desirable to obtain TR antagonists for treatment of hyperthyroidism and other conditions. We have designed TR antagonists from first principles based on TR crystal structures. Since agonist ligands are buried in the fold of the TR ligand binding domain (LBD), we reasoned that ligands that resemble agonists with large extensions should bind the LBD, but would prevent its folding into an active conformation. In particular, we predicted that extensions at the 5' aryl position of ligand should reposition helix (H) 12, which forms part of the co-activator binding surface, and thereby inhibit TR activity. We have found that some synthetic ligands with 5' aryl ring extensions behave as antagonists (DIBRT, NH-3), or partial antagonists (GC-14, NH-4). Moreover, one compound (NH-3) represents the first potent TR antagonist with nanomolar affinity that also inhibits TR action in an animal model. However, the properties of the ligands also reveal unexpected aspects of TR behavior. While nuclear receptor antagonists generally promote binding of co-repressors, NH-3 blocks co-activator binding and also prevents co-repressor binding. More surprisingly, many compounds with extensions behave as full or partial agonists. We present hypotheses to explain both behaviors in terms of dynamic equilibrium of H12 position.
The Journal of Steroid Biochemistry and Molecular Biology 01/2003; 83(1-5):59-73. · 3.05 Impact Factor
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John D Baxter,
Patrick Goede,
James W Apriletti, Brian L West,
Weijun Feng,
Karin Mellstrom,
Robert J Fletterick,
Richard L Wagner,
Peter J Kushner,
Ralff C J Ribeiro,
Paul Webb,
Thomas S Scanlan,
Stefan Nilsson
[show abstract]
[hide abstract]
ABSTRACT: Antagonists have been developed for several nuclear receptors but not for others, including TRs. TR antagonists may have significant clinical utility for treating hormone excess states and other conditions. A structure derived "extension hypothesis" was applied to synthesize a TR antagonist. The principal design feature was to attach an extension group to a TR agonist whose structure would perturb formation of the TR coactivator-binding surface. The compound, 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid, has no (TRalpha) or very weak partial (TRbeta) TR agonist activity and blocks TR binding of T3, formation of the coactivator-binding surface, and both a positive T3 response on a thyroid hormone response element and a negative T3 response on the TSHbeta promoter in cultured cells. The results suggest that 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid is a TR antagonist for thyroid hormone response element-mediated responses, this approach can be used more generally to generate nuclear receptor antagonists, and this compound or analogues may have medical and research utility.
Endocrinology 03/2002; 143(2):517-24. · 4.46 Impact Factor
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ABSTRACT: Residues of the TR that are critical for binding the nuclear receptor corepressor (N-CoR) were identified by testing more than 100 separate mutations of the full-length human TRbeta that scan the surface of its ligand binding domain. The primary inferred interaction surface overlaps the surface described for binding of p160 coactivators, but differs by extending to a novel site underneath which helix 12 rests in the liganded TR, rather than including residues of helix 12. Nonconservative mutations of this surface diminished binding similarly to three isolated N-CoR receptor interaction domains (RIDs), but conservative mutations affected binding variably, consistent with a role for this surface in RID selectivity. The commonality of this surface in binding N-CoR was confirmed for the RXRs and ERs. Deletion of helix 12 increased N-CoR binding by the TR modestly, and by the RXR and ER to a much greater extent, indicating a competition between this helix and the corepressor that regulates the extent of corepressor binding by nuclear receptors. When helix 12 was deleted, N-CoR binding by the ER was stimulated by tamoxifen, and binding by the TR was stimulated by Triac, indicating that helix 12 is not the only feature that regulates corepressor binding. Two additional mutationsensitive surfaces were found alongside helix 1, near the previously described CoR box, and above helix 11, nearby but separate from residues that help link receptor in dimers. Based on effects of selected mutations on T(3) and coactivator binding, and on results of combined mutations of the three sites on corepressor binding, we propose that the second and third surfaces stabilize TR unliganded conformation(s) required for efficient N-CoR binding. In transfection assays mutations of all three surfaces impaired the corepressor-mediated functions of unliganded TR repression or activation. These detailed mapping results suggest approaches for selective modulation of corepressor interaction that include the shape of the molecular binding surface, the competitive occupancy by helix 12, pharmacological stimulation, and specific conformational stabilization.
Molecular Endocrinology 03/2002; 16(2):271-86. · 4.54 Impact Factor
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Paul Webb,
Ngoc-Ha Nguyen,
Grazia Chiellini,
Hikari A.I. Yoshihara,
Suzana T. Cunha Lima,
James W. Apriletti,
Ralff C.J. Ribeiro,
Adhirai Marimuthu, Brian L. West,
Patrick Goede,
Karin Mellstrom,
Stefan Nilsson,
Peter J. Kushner,
Robert J. Fletterick,
Thomas S. Scanlan,
John D. Baxter
[show abstract]
[hide abstract]
ABSTRACT: It is desirable to obtain TR antagonists for treatment of hyperthyroidism and other conditions. We have designed TR antagonists from first principles based on TR crystal structures. Since agonist ligands are buried in the fold of the TR ligand binding domain (LBD), we reasoned that ligands that resemble agonists with large extensions should bind the LBD, but would prevent its folding into an active conformation. In particular, we predicted that extensions at the 5′ aryl position of ligand should reposition helix (H) 12, which forms part of the co-activator binding surface, and thereby inhibit TR activity. We have found that some synthetic ligands with 5′ aryl ring extensions behave as antagonists (DIBRT, NH-3), or partial antagonists (GC-14, NH-4). Moreover, one compound (NH-3) represents the first potent TR antagonist with nanomolar affinity that also inhibits TR action in an animal model. However, the properties of the ligands also reveal unexpected aspects of TR behavior. While nuclear receptor antagonists generally promote binding of co-repressors, NH-3 blocks co-activator binding and also prevents co-repressor binding. More surprisingly, many compounds with extensions behave as full or partial agonists. We present hypotheses to explain both behaviors in terms of dynamic equilibrium of H12 position.
The Journal of Steroid Biochemistry and Molecular Biology.
